Scalable expansion and harvesting of hiPSCs using dissolvable

microcarriers

André Lopes Rodrigues

Thesis to obtain the Master of Science Degree in

Biotechnology

Supervisors: Professor Dr. Maria Margarida Fonseca Rodrigues Diogo

Doctor Carlos André Vitorino Rodrigues

Examination Committee

Chairperson: Prof. Dr. Marília Clemente Velez Mateus Supervisor: Doctor Carlos André Vitorino Rodrigues Member of the Committee: Doctor Ana Margarida Pires Fernandes Platzgummer

October 2017

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Acknowledgments

First, I would like to thank Professor Joaquim Cabral for the opportunity that I was given

to develop this work at SCERG. Thank you for all the funding that made the progress of this work

possible.

To my supervisor Professor Margarida Diogo, I am extremely grateful for accepting me

as a student and for being always available for any questions or problems that I would have. To

Dr Carlos Rodrigues, thank you for being the best supervisor that one could ever asked for. For

all the patient and tolerance, for all the precious thoughts on Stem cell bioprocessing and most

importantly for always listening to what I had to say about the ongoing work, thank you. I would

also like to thank Corning, Inc® for providing the core material for this work, and for answering all

of the questions regarding the properties of the dissolvable microcarriers.

To Dr. Tiago Fernandes and Dr. Ana Fernandes thank you for the input that both of you

made for this work, namely with the flow cytometry and metabolic analysis, respectively.

To my lab co-workers, I would like to thank Ana Rita, Teresinha and Diogo, for all the

precious lessons on how to be a productive scientist, for all the time spent in the lab, whether it

was the most graceful or not, and for every second lost on teaching me and listening to what I

had to say. Thank you for always treating me as fellow junior associate, thank you for the laughs

and gifts that I’ve received from you. To the three of you, no matter what, I will always regard you

as my eternal “colegas” who became my you-know-what-cousins.

To my dearest lab partners Kikas, Mariana Pina, Leonor, Diogo, Cláudia, Carina, Mariana

and every member of SCERG thank you for making this journey a lot easier, your good mood and

positive attitude made this past year a lot more fun.

To Jão and Catizinha, who started this journey with me. We have stood together even in

the moments where everything seemed lost. Thank you for the moments spent in the lab, for

those days in cold Amsterdam, for the bits and pieces that we’ve put together that made this year

a happy moment. A special thanks to my partner in crime Jão, who without whom, I could not

present this work today. I also would like to thank the most amazing people that made the

Biotechnology Master’s class of 2015.

To Nini, Ana, Hugo, Chini and Pêga, thank you for the being my friends, for being there

when I needed and when I desired. It has been a pleasure and I am proud to call you my dearest

friends. After all, the Colégio Moderno gave me something better than a good education, a good

friendship. To Joana, thank you for every glass wine and every place we’ve visited this past year,

thank you for listening to me, and I am grateful that I’ve met you during those cold days in the

Netherlands. To all of you, I regard our friendship with the outmost respect.

Last but not the least, my family. My parents, my brother, my grandparents and my

godmother. Everything that I am, I owe it to you. To my parents who worked day and night to

provide everything for me and for my brother, thank you is not enough to express my gratitude to

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you. What would I have done without you? I have the best education that I could possibly asked

for, I have your love and support to guide me through life and despite my stubbornness, I treasure

every moment spent with you. To my brother, who I am lucky enough to call my mother and

father’s partner, thank you for your patience and sense of humor. To my godmother and her

family, thank you for the fun moments with all of you. To my grandparents who taught me the

values of hard work and perseverance, I can only hope that I could make you proud with what I

have accomplished so far. Thank you for everything that you’ve built and conquered that made

everything possible. I am very lucky to have you in my life. THANK YOU.

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Abstract

Polystyrene microcarriers (PSM) have been exploited as 3D-platform for culturing

hiPSCs. These micro-spherical beads can be further incorporated into xeno-free

suspension cultures to achieve clinically-relevant cell numbers. However, equal

importance should be given to the downstream processing, which is subject of cell losses

and reduced viability and requires the integration of a filtration step after cell expansion.

To overcome these issues, in this thesis, xeno-free microcarriers made of a digestible

polymer based on polygalacturonic acid were used for hiPSC expansion and to prompt

an efficient cell harvesting. Moreover, these dissolvable microcarriers (DM) are coated

with a xeno-free substrate, Synthemax-II (SII), to promote the expansion of hiPSCs

under GMP-compliance. After the static screening of culturing media and substrates, the

fully defined combination of mTeSR™1 and SII-coated DM was scaled-up to a dynamic

culture system, using a spinner-flask. A maximum fold increase of 3.76 after 5 days of

expansion was achieved by inoculating 55,000 cells/cm2 of microcarrier surface area and

using 25 rpm, which generates a final cell density of 9.39x105 cells/mL. These results

were found to be reproducible with another hiPS cell line. Afterwards, this system was

transposed to a xeno-free platform by the replacement of mTeSR™1 for TeSR™2. In

both cases, the downstream processing of the expanded hiPSCs was performed by the

digestion of the DM-SII beads within the spinner flask. The 97%-harvesting yield of DM-

SII was considerably higher than what was obtained by the filtration of PSM cultured

cells. After cell harvesting, replated hiPSCs maintained their undifferentiated state,

exhibiting the expression of pluripotency-associated markers. Moreover, their

differentiation capability was confirmed by their spontaneous differentiation through

embryoid body formation and direct differentiation towards neural progenitors and

cardiomyocytes.

Keywords

Human Induced Pluripotent Stem Cells; Dissolvable Microcarriers; Spinner-Flask; Cell

Harvesting

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Resumo

Os microcarrers de plástico (PSM) têm sido explorados como uma plataforma 3D para cultivar

células estaminais pluripotentes induzidas (hiPSCs). Estas estruturas podem ser incorporadas

em culturas de suspensão de forma a obter um número relevante de células para aplicações

clínicas. No entanto, os diversos estudos publicados focam-se apenas na expansão de células,

dando pouco enfase à recolha das mesmas, que muitas das vezes está sujeito a perda de células

e a uma redução da viabilidade. Para evitar este problema, o tema desta tese propõe o uso de

um polímero digerível para uma recolha celular eficiente. Este polímero de ácido

poligalacturónico pode ser revestido por um substrato xeno-free, o Synthemax-II, para promover

a adesão e expansão de hiPSCs de acordo com a boas práticas laboratoriais (GMP). Após

seleção em condições estáticas, os microcarriers digeríveis revestido por SII (DM-SII) em

combinação com o meio de cultura mTeSR™1 foram incorporados num sistema dinâmico,

recorrendo a um spinner-flask. Uma densidade de 55000 células/cm2 de área superficial de

microcarriers foi inoculada no reator com uma agitação de 25 rpm. Após 5 dias de expansão

dinâmica foi atingida uma densidade celular de 9.39x105 células/mL. Estes resultados foram

reproduzidos com outra linha células de hiPSCs. Posteriormente, este sistema foi transposto

para uma plataforma xeno-free pela substituição de mTeSR™1 para TeSR™2. A recolha celular

foi feita pela digestão dos DM-SII dentro do spinner-flask. O rendimento deste processo foi de

97%, o que é consideravelmente maios do que o obtido pela filtração de células cultivadas com

PSM. Após a recolha de células, as hiPSCs expandidas mantiveram seu estado indiferenciado,

apresentando marcadores associados à sua pluripotência. Além disso, a sua capacidade de

diferenciação foi confirmada pela sua diferenciação espontânea em Embroyoid Bodies e pela

sua diferenciação direta para progenitores neurais e cardíacos.

Palavras-chave

Células estaminais pluripotentes induzidas de humanos; Microcarriers digeríveis;

Spinner-Flasks; Recolha Celular.

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List of Figures

Figure 1 – Biomedical applications of hiPSCs generated by somatic cell reprogramming. Adult

somatic cells, like fibroblasts, can be reprogrammed towards a more primitive state, giving rise

to iPSCs. These cells may be differentiated into different lineages of the three germ layers.

Several applications for these cells may include disease modeling, to understand the molecular

mechanism underlying such phenotype, drug screening, to develop and determine what drugs

are best suited to treat such disease, and cellular therapies, using the differentiated progeny. 4

Figure 2 – Evolution of the Substrates used for culturing hPSCs. The development of culturing

substrates moves from the use of xenogeneic elements, such as mouse embryonic fibroblasts,

towards the use of synthetic polymers, like Synthemax-II. ........................................................ 10

Figure 3 – 3D-platforms for hPSCs culture and expansion, which include cell agregates,

microcarriers and microencapsulation of the cells. .................................................................... 11

Figure 4 - Schematic representation of bead digestion. Cells adhere to a peptide- or protein-

coated ionically cross-linked polysaccharide microcarrier. Chelation of divalent cations by EDTA

destabilizes the polymer crosslinking and exposes the polymer chains to degradation by a

protease-free enzyme. ................................................................................................................ 14

Figure 5 – Representation of the procedure for hiPSCs expansion under static conditions and

cell harvesting using both PSM and DM coated with different substrates. ............................... 24

Figure 6 - Schematic representation of the hiPSCs expansion and harvesting under dynamic

conditions. Cells were cultured for 7 days, at which time they were harvested accordingly to the

type of microcarriers used. ......................................................................................................... 26

Figure 7 – Expansion of hiPSCs under static conditions using both polystyrene (PSM) and

dissolvable microcarriers (DM). (A) From left to right: Cell adhesion yield and cell fold increase

for all the tested combinations of microcarriers, coatings and culture media. (B) From left to

right: Bright-field microscopy images from day 1 and day 5 of the previous combinations.

Maximum intensity projection of confocal microscopy images of the pluripotency markers for

the expanded cells. The nuclei were counterstained with DAPI. Scale bar: 132µm. Abbreviatures:

vitronectin-coated polystyrene microcarriers and E8®medium (PSM-VTN E8); Matrigel-coated

dissolvable microcarriers and mTeSR™1 medium (DM-Mat mT1); Synthemax-II coated

dissolvable microcarriers and mTeSR™1 medium (DM-SII mT1); Synthemax-II coated dissolvable

microcarriers and TeSR™2 (DM-SII T2). ...................................................................................... 34

Figure 8 – Expansion of TCLab hiPSCs under dynamic conditions using Synthemax-II dissolvable

microcarriers with mTeSR™1. (A) Total number of cells over 7 days of expansion. Results are

presented as the mean average of n=4 experiments. The error bars represent the standard error

of mean (SEM) (B) Graphic representation of the adhesion yield and fold increase attained on

the first day and throughout the culture, respectively. This is the outcome of the mean of n=4

experiments, with the error bar standing for the Standard Error of Mean (SEM). (C) Bright-field

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microscopy images of the cells attached to the beads on day 1 and on day 7, respectively. (D)

Maximum confocal intensity projection of the immunocytochemistry analysis for expression of

intracellular OCT4 and extracellular TRA-1-60 pluripotency markers. (E) Viability test of cells

attached to the microcarriers cultured on day 1 and day 7 of the dynamic expansion. Green is

the calcein metabolized by the living cells, whereas the dead cells (red) were stained by the

ethidium homodimer. ................................................................................................................. 36

Figure 9 – Characterization assay of the TCLAB cell line after expansion under dynamic conditions

combining the use of DM-SII and mTeSR™1. (A) Confocal microscopy images of

immunocytochemistry for the pluripotency markers: SOX2, TRA-1-60 (Scale bar: 132 µm) and

OCT4 (Scale bar: 66 µm) The nuclei were counterstained with DAPI. (B) Flow cytometry analysis

of the hiPSCs harvested after 7 days of expansion in the spinner flask. Cells were stained for Oct4

and SOX2 intracellular markers and TRA-1-60 and SSEA-4 cell surface markers. The error bars

represent the SEM of n=4 experiments. (C) Quantitative RT-PCR analysis of the pluripotency and

differentiation genes of hiPSCs after seven days of culture. mRNA was isolated at the beginning

and at end of the culture. (D) Immunostaining showing the formation of cells expressing TUJ1

(ectoderm), SOX17 (endoderm) and α-SMA (mesoderm) after the EB formation and

spontaneous differentiation assay with hiPSC cultured in spinner-flask. Scale bar: 100µm for

TUJ1 and 50 µm for SOX17 and α-SMA. (E) Confocal microscopy images for immunostaining for

SOX2 and ZO-1. The nuclei were counterstained with DAPI. Scale bar: 33 µm. ......................... 38

Figure 10 –Expansion of GIBCO hiPSC line under dynamic conditions using Synthemax-II

dissolvable microcarriers with mTeSR™1. (A) Total number of cells over 7 days of expansion. This

graphical representation is the mean average of n=4 experiments. The error bars represent the

standard error of mean (SEM) (B) Graphic representation of the adhesion yield and cell fold

increase attained on the first day and throughout the culture, respectively. This is the outcome

of the mean of n=4 experiments, with the error bar standing for the Standard Error of Mean

(SEM). (C) Bright-field microscopy images of the cells attached to the beads on day 1 and on day

7, respectively. Scale bar: 100 µm. (D) Maximum intensity projection, obtained with confocal

microscopy, for immunocytochemistry of OCT4 (Scale bar: 94 µm) and TRA-1-60 (Scale bar: 66

µm) pluripotency markers. .......................................................................................................... 39

Figure 11 – Characterization assay of the GIBCO cell line after expansion under dynamic

conditions resorting to DM-SII and mTeSR™1. (A) Immunostaining for the OCT4 and SOX2

intracellular markers and TRA-1-60 and SSEA-4 extracellular markers for cells replated after

dynamic expansion. The nuclei were counterstained with DAPI. Scale bar: 100 µm. (B) Flow

cytometry analysis of the hiPSCs harvested after 7 days of expansion in the spinner flask. Cells

were stained for Oct4 and SOX2 intracellular marker and TRA-1-60 and SSEA-4 extracellular

marker. The error represents the SEM of n=4 experiments. (C) Immunostaining showing the

formation of cells expressing TUJ1 (ectoderm), SOX17 (endoderm) and α-SMA (mesoderm) after

the EB formation and spontaneous differentiation assay with hiPSC cultured in spinner-flask. The

nuclei were counterstained with DAPI. Scale bar: 100µm. ......................................................... 40

Figure 12 – Metabolic profile of TCLAB cell line during expansion under dynamic conditions with

DM-SII and mTeSR™1. The culture media was daily changed for newly mTeSR™1 culture media.

The results are the mean of n=4 experiments, with the error bars standing for the standard error

of mean (SEM) (A) Concentration of glucose (mM) over seven days of expansion. (B) Specific rate

of glucose consumption over seven days of expansion (µM.cell-1.day-1). (C) Concentration of

lactate (mM) over 7 days of expansion. (D) Specific production rate of lactate per day over seven

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days of expansion (µM.cell-1.day-1). (E) Apparent yield of lactate produced from glucose over

seven days of expansion. ............................................................................................................ 42

Figure 13 Expansion of TCLab hiPSC line under dynamic conditions using Synthemax-II

dissolvable microcarriers with TeSR™2. (A) Total number of cells over 7 days of expansion. This

graphical representation is the mean average of n=4 experiments. The error bars represent the

standard error of mean (SEM) (B) Graphic representation of the adhesion yield and fold increase

attained on the first day and throughout the culture, respectively. This is the outcome of the

mean of n=4 experiments, with the error bar standing for the Standard Error of Mean (SEM). (C)

Bright-field microscopy images of the cells attached to the beads on day 1 and on day 7,

respectively. (D) Maximum intensity projection obtained with confocal microscopy of the

immunocytochemistry results for intracellular OCT4 and extracellular SSEA-4 pluripotency

markers. (E) Representative images from cell-viability assays (Calcein, live cells in green;

Ethidium homodimer, dead cells in red). Scale bar: 100 µm. ..................................................... 44

Figure 14 – Characterization assay for the TCLAB cell line propagated onto DM-SII and TeSR™2.

(A) Confocal microscopy images of the immunocytochemistry for SOX2, OCT4 intracellular

markers and SSEA-4 extracellular marker. The nuclei were counterstained with DAPI. Scale bar:

94 µm (B) Flow cytometry analysis of the hiPSCs harvested after 7 days of expansion in the

spinner flask. Cells were stained for Oct4 and SOX2 intracellular marker and TRA-1-60 and SSEA-

4 extracellular marker. The error represents the SEM of n=4 experiments. (C) Quantitative RT-

PCR analysis of the pluripotency and differentiation genes of hiPSCs after seven days of culture.

mRNA was isolated at the beginning and end of the culture. The error bars represent the SEM

of n=4 experiments for the pluripotency genes, where the differentiation genes were the

outcome of n=3 experiments. (D) Immunostaining showing the formation of cells expressing

TUJ1 (ectoderm), SOX17 (endoderm) and α-SMA (mesoderm) after the EB formation and

spontaneous differentiation assay with hiPSC cultured in spinner-flask. The nuclei were

counterstained with DAPI. Scale bar: 100µm (E) Confocal microscopy images for the

immunostaining of cells expressing SOX2 and apical ZO-1 neural progenitor markers. The nuclei

were counterstained with DAPI. Scale bar: 33 µm. (F) Immunostaining of cells expressing cTnT

cardiac marker of hiPSCs differentiated into cardiac marker, after seven days of dynamic

expansion. The nuclei were counterstained with DAPI. Scale bar: 50 µm. ................................ 46

Figure 15 – Graphical representation of the Harvesting yield for different hiPSCs lines cultured

on dissolvable and polystyrene microcarriers. The protocol developed by Nienow et al. [94] was

adapted for the harvesting of the expanded cells. The results for the harvesting yield resorting

to polystyrene microcarriers are the outcome of a single experiment, whereas for DM is the

mean of n=7 experiments. The error bar represents the standard error of mean .................... 49

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List of Tables Table 1 – Schematic representation of the conditions tested in static and dynamic conditions.

The TCLab cell line was used for the expansion under static conditions using both types of

microcarriers (PSM and DM) with possible media and coating combinations. The same cell line

was then used for the expansion under dynamic conditions using PSM-VTN in E8® media, and

DM-SII in mTeSR™1 and TeSR™2. ............................................................................................... 21

Table 2 - Overview of the primary and secondary antibodies used to perform

immunocytochemistry of the intracellular pluripotency markers. Both types of antibodies were

diluted in staining solution. ......................................................................................................... 28

Table 3 – Overview of the primary and secondary antibodies used to perform

immunocytochemistry of the extracellular pluripotency markers. Both antibodies were diluted

in 3% (v/v) of Bovine Serum albumin. ......................................................................................... 28

Table 4 – Antibodies and respective dilutions for the immunocytochemistry analysis of cells from

the three-germ layers derived from the embryoid body assays. SOX 17, TUJ1 and α-SMA were

used for the analysis of endoderm, ectoderm and mesoderm, respectively. ............................ 31

Table 5 – Growth kinetic parameters analyzed for the expansion of two hiPSC lines, using DM-

SII. The results are the mean average of n=4 experiments, with the error representing the SEM.

..................................................................................................................................................... 48

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List of Abbreviations

2D Two-dimensional

3D Three-dimensional

AKT Protein kinase B

ALS Amyotrophic lateral sclerosis

BSA Bovine Serum Albumin

CAMs Cell adhesion molecules

CD Cluster of differentiation

CTnT Cardiac troponin T

DAPI 4',6-diamidino-2-phenylindole

DAS-NG diffusion assisted synthesis-grown nanocrystalline graphene

DM Dissolvable Microcarriers

DMEM/F12 Dulbecco’s modified eagle medium

DM-Mat Matrigel-coated Dissolvable microcarriers

DM-SII Synthemax-II dissolvable microcarriers

DMSO dimethylsulfoxide

DNA Deoxyribonucleic acid

DOPA/PSF polymerized catecholamines dopamine

E8 Essential 8 ®

EB Embryoid Body

ECM Extracellular matrix

EDTA Ethylenediaminetetraacetic acid

ERK Extracellular signal-redugulated kinases

ESCs Embryonic Stem cells

FBS Fetal Bovine Serum

FGF Fibroblast growth factor

GMP Good Manufacturing practices

hESCs Human Embryonic stem cells

HIF Hypoxia-inducible factors

hiPSCs Human induced pluripotent stem cells

HTS High-throughput

ICM Inner cell mass

IGF Insulin growth factor

iPSC Induced pluripotent stem cells

Klf Kruppel-like factors

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KO-SR Knock-Out Serum Replacement

LIF Leukemia inhibitor factor

Mat Matrigel

MEFs Mouse Embryonic Fibroblasts

MEK/ERK Ras-Raf-MEK-ERK pathway

mEpiSCs Mouse epiblast stem cells

mESCs Mouse Embryonic Stem cells

MLC Myosin light chain

mRNA messenger RNA

MSC Mesenchymal stem cell

mT1 mTeSR™1

MYHs Myosin II heavy chains

NaHCO3 Sodium Bicarbonate

O2 Oxygen

OCT4 Octomer-biding transcription factor 4

PBS Phosphate-Buffered Saline

PenStrep Penicillin and Streptomycin

PFA Parformaldehyde

PGA Polygalacturonic acid

PI3K Phisphatidylinositol 3-Kinase

PMEDASH Poly[2-(methacryloyloxy) ethyl dimethyl-(3-sulfopropyl) ammonium

hydroxide]

PSCs Pluripotent Stem cells

PSM Polystyrene microcarriers

PSM-VTN Vitronectin-coated polystyrene microcarriers

qRT-PCR Quantitative Real-time PCR

RGD Arginine – Glycine - Aspartate

RNA Ribonucleic acid

ROCK Rho-associated kinase

RT Room Temperature

SII Synthemax-II

SMA Spinal Muscular atrophy

SMA Smooth muscle actin

SOX2 Sex determining region Y)-box 2

S-R Synthemax-R

SSEA4 Stage specific embryonic antigen

T2 TeSR™2

TGFβ 1 Transforming growth factor β1

TRA Tumor rejection antigen

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TUJ1 Neuron-specific class III beta-tubulin

ULA Ultra-low attachment plates

VTN Vitronectin

Wnt Wingless-type MMTV integration site family members signaling

pathway

ZO-1 Tight junction protein-1

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Table of Contents

1. Introduction .............................................................................................................. 1

1.1 Pluripotent stem cells.................................................................................................... 1

1.1.1 Human embryonic stem cells ................................................................................... 2

1.1.2 Human induced pluripotent stem cells .................................................................... 2

1.2 Human pluripotency network ....................................................................................... 5

1.2.1 Transcription factors – OCT4/SOX2/Klf4/NANOG .................................................... 5

1.2.2 Main signalling pathways involved in pluripotency maintenance ........................... 6

1.3 Microenvironment of pluripotency – the niche ............................................................ 6

1.3.1 The importance of Extracellular matrix (ECM), cell interactions and biophysical

stimuli 7

1.4 Stem cell Bioprocessing towards xeno-free culture methods ...................................... 8

1.4.1 2D-platforms ............................................................................................................. 8

1.4.2 3D platforms ........................................................................................................... 10

1.5 Bioreactors .................................................................................................................. 14

2. Aim of study ............................................................................................................ 17

3. Materials and Methods .......................................................................................... 19

3.1 Cell lines ...................................................................................................................... 19

3.2 Culture media for hiPSCs expansion ........................................................................... 19

3.2.1 mTeSR™1 ................................................................................................................ 19

3.2.2 TeSR™2 ................................................................................................................... 19

3.2.3 Essential 8 ™ ........................................................................................................... 20

3.3 hiPSCs thawing and cryopreservation ......................................................................... 20

3.3.1 Thawing hiPSCs ....................................................................................................... 20

3.3.2 Cryopreservation of hiPSCs .................................................................................... 20

3.4 Expansion of hiPSCs ..................................................................................................... 20

3.4.1 Static Culture Systems ............................................................................................ 21

3.4.2 Inoculation of microcarriers and hiPSCs expansion ............................................... 22

3.4.3 Cell Harvesting ........................................................................................................ 23

3.5 Dynamic culture systems ............................................................................................ 24

3.5.1 Inoculation and expansion in dynamic conditions ................................................. 24

3.5.2 Cell harvesting ........................................................................................................ 25

3.5.3 Growth kinetics ....................................................................................................... 26

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3.5.4 Metabolic profile .................................................................................................... 26

3.6 Characterization of hiPSCs .......................................................................................... 27

3.6.1 Immunocytochemistry and confocal microscopy ................................................... 27

3.6.2 Flow cytometry ....................................................................................................... 28

3.6.3 Quantitative Real-time PCR analysis....................................................................... 29

3.6.4 hiPSCs differentiation ............................................................................................. 30

3.7 Statistical analysis........................................................................................................ 31

4. Results and Discussion ............................................................................................ 33

4.1 Expansion of hiPSCs under static conditions using both polystyrene and dissolvable

microcarriers ........................................................................................................... 33

4.2 Expansion of hiPSCs under dynamic conditions with DM-SII and mTeSR™1 culture

media and cell characterization .............................................................................. 35

4.2.1 Metabolic profile of TCLab expansion using DM-SII and mTeSR1 media. .............. 42

4.3 Expansion and characterization of TCLab cell line under xeno-free conditions. ........ 43

4.4 Growth kinetics and Cell Harvesting. .......................................................................... 48

5. Conclusion .............................................................................................................. 51

6. Future Work ............................................................................................................ 53

7. Literature ................................................................................................................ 54

1

1. Introduction

The defining properties of a stem cell comprise the ability for long-term self-renewal, which

enables the maintenance of an undifferentiated state, as well as the capacity to generate a specialized

progeny [1, 2].

According to their differentiation potential (i.e. potency), stem cell may be classified as toti-,

pluri-, multi-, and unipotent. Primarily, the zygote is an example of a totipotent stem cell. These cells

originate a functional organism, including the extra-embryonic tissues that support fetal development

[3]. Pluripotent stem cells (PSCs) share common traits with the previously mentioned. In this category,

cells present the ability to adopt any cellular fate from the three germ layers (ecto-, meso- and endoderm)

but not the trophoblast lineage, which gives rise to the extraembryonic tissues [4]. As stem cells

differentiate into more specialized cell types, they are restricting their potency achieving a multipotent

state. These cells are deemed to replace cells in the organ lineage they are derived from. Lastly,

unipotent stem cells refer to cells with the ability to differentiate into only one specific cell type [1, 3].

Parallelly, stem cells may also be considered into different categories based on their tissue of

origin. For instances, Embryonic stem cells (ESCs) are collected from the inner cell mass (ICM) of the

blastocyst [3]. Also within the pluripotent stem cell type, human induced pluripotent stem cells (hiPSCs)

may be obtained by reprogramming somatic cells towards a more primitive state [5]. Nonetheless, stem

cells may also be found after embryonic development. These adult stem cells are undifferentiated cells

with some degree of specialization. They reside in specific microenvironments (i.e. niches) where they

serve the purpose of maintaining a steady state of self-renewal for tissues whenever the replacement

of specific cell type is required. Hematopoietic stem cells are the most studied case of such cells, being

responsible for replacing all blood cell types during adulthood [3].

1.1 Pluripotent stem cells

Pluripotency is defined by the capability to give rise to cells from the three germ layers. PSCs

also express high telomerase activity, which is translated by an indefinite self-renewal capacity.

Moreover, these cells can be maintained in culture without differentiating due to a symmetric cell division

mechanism [6].

In recent years, a ground state of pluripotency has been defined by the distinguished definition

between naïve and primed pluripotency. These states recapitulate several developmental properties of

the early and late-stage human epiblast, providing further insights of mechanisms underlying human

development [7].

The main differences rely on the signaling pathways that maintain such states. For instances,

primed pluripotency refers to post-implantation embryonic state configurations, where the self-renewing

capabilities are maintained through the Fibroblast Growth Factor 2 (FGF2) and transforming growth

2

factor-β1 (TGFβ 1)/Activin A signaling pathways [4]. Moreover, these cells rarely contribute to chimeric

embryos after blastocyst injection [6]. As an example of primed PSCs, there are hESCs and hiPSCs,

which resemble to mouse epiblast stem cells (mEpiSCs), as they also present a more flattened colony

appearance and inactivation of one female X chromosome [8].

Regarding naïve PSCs, they are defined by leukemia inhibitory factor (LIF)-dependent

signaling, that generates daughter cells capable of re-entering embryogenesis in pre-implantation

embryos. Mouse ESCs (mESCs) are an example of cells having naïve pluripotency [2, 6]. Concerning

hPSCs, the naïve sate may be obtained through primed-to-naïve resetting. This procedure may be

performed by exogenous transgene expression, or in a transgene-independent manner by using naïve

human stem cell media (NHSM) [4, 7, 9]. Advantages regarding the establishment of pure naïve hPSCs

comprise the high single cell cloning efficiency, facile genome editing and higher developmental

potential for the generation of more mature cells [2].

Nevertheless, current protocols for this transition result in a heterogenous cell mixture, of which

a small portion is likely to be naïve. Consequently, methods for the identification of a naïve sate have

been developed based on surface cell proteins, such as CD7 and CD77 [7].

1.1.1 Human embryonic stem cells

The different cell types that constitute the adult body are derived from the sequential

differentiation of the ICM cell population. If these cells are cultured in conditions which promote

symmetric cell division and potency, we obtain Embryonic Stem Cells (ESCs) [2]. Despite being derived

from the ICM, this does not mean that ESCs are the in vitro equivalent of what happens in vivo, as no

pluripotent cell in the intact embryo undergoes long-term self-renewal [6].

The first protocol reporting the isolation of mESCs dates back to 1981 [10], but it was not until

1998 that Thomson et al. reported the advent of human embryonic stem cells (hESCs). In this paper,

the three derived cell lines expressed high levels of telomerase activity and the expression of a set of

pluripotency surface markers, including the stage-specific embryonic antigen SSEA4 and the tumor

rejection antigen TRA-1-60. Pluripotency was also confirmed by the teratoma formation in

immunocompromised mice [11].

Furthermore, Thomson et al. clearly highlighted the use of hECS as a universal source of

different tissue-specific precursors, for tissue repair, disease modelling and drug screening [11].

However, in addition to the ethical issues of embryo destruction for the hESCs derivation, there are

major drawbacks if clinical use is to be considered. These include the possibility of teratoma formation

and the immune rejection by the host [3, 11].

1.1.2 Human induced pluripotent stem cells

The generation of patient-specific pluripotent stem cells, known as human induced pluripotent

stem cells (hiPSCs), was a crucial step to overcome the ethical and immunologic issues underlying the

use of hESCs.

3

In 2006, Takahashi et al generated the first embryonic state-like cells from mouse fibroblasts by

introducing four genes (Oct3/4, Sox2, c-Myc and Klf4) under ESCs culture conditions [12]. A year later,

the same approach was reproduced in human fibroblasts and afterwards improved by the removal of

the c-Myc tumorigenic factor [5, 13]. Parallelly, Yu et al used a different set of reprograming factors that

do not include oncogenic factors (Sox2, Oct4, Nanog and LIN28) to generate hiPSCs [14].

The protocols developed by Yamanaka’s and Thomson’s groups rendered similar outcomes to

hESCs when considering properties such as proliferation, differentiation potential and teratoma

formation. Moreover, pluripotency of these cell lines was also confirmed by the expression of hESC-

markers namely SSEA-4, TRA-1-60, SOX2 and OCT4 [5, 13, 14].

Since Yamanaka’s breakthrough, hiPSCs have been subject of intense studies namely to

discover new strategies of reprogramming somatic cells towards a more primitive state. Reprogramming

methods are of paramount importance if clinical use is to be considered. However, a protocol that better

balances safety, efficiency and standardization is yet to be achieved.

Chronologically speaking, the first generated hiPSCs relied on the use of retroviral vectors. The

high efficiency and reproducibility of this method do not preclude the risk of insertional mutagenesis of

viral genetic material into the host genome [5, 15]. Therefore, non-integrative methods were developed

to enable the transient expression of reprogramming factors. Among these, adenovirus and sendai virus-

based vectors were presented as viable option with complete absence of viral integration in cell lines

with higher passages [15, 16] . Other non-integrative methods comprise the use of episomal vectors

[15, 17]. However, the foreign DNA is still a major concern in terms of regulatory issues for human safety.

Further improvement in vector- and DNA-free reprogramming methods were made resorting to mRNA,

direct delivery of proteins and the use of small molecules [18, 19].

1.1.2.1 Biomedical Applications of hiPSCs

Human biological development is achieved based on mechanisms of selective gene expression

that control cell proliferation and cell specialization [1]. Understanding this process at a cellular level

prompts the development of disease models, which are then used to discover new therapeutics. Current

systems rely on the use of animal models such as mice and non-human primates. However, intrinsic

characteristics of such species (i.e. genomic and physiologic) hampers the recapitulation of human

disease phenotypes [20].

Human iPSCs may overcome problems related with animal models, making them suitable

candidates for in vitro disease modelling and drug screening. Furthermore, these biological assets lend

themselves to future cell-based therapies for regenerative medicine purposes [21]. The use of hiPSCs

for the development of human disease models is built on their ability to adopt any cellular fate through

differentiation. As an example, neurodegenerative diseases have been studied using hiPSCs, namely

Parkinson’s disease, amyotrophic lateral sclerosis (ALS) and Spinal muscular atrophy (SMA). Other

examples include heart diseases, such as hypertrophic cardiomyopathy, and Duchenne’s Muscular

Dystrophy [22]. The next generation of disease models will be based on the combination of different

cell types in more complex and organized cellular structures, the organoids, and also by culturing

different types of cells in microfluidic devices according to the “body on a chip” concept. In the case of

4

organoids, these are defined as in vitro 3D clusters derived from stem cells by processes of self-

organization and exhibiting similar organ functionality, hence its use for disease modelling [23, 24].

Drug discovery is also one of the main applications of hiPSCs. Once the disease phenotypes

are established there are two main strategies to identify potential drugs: the candidate drug approach

and the High-throughput screening (HTS). The former promotes the validation and confirmation of

existing drugs in hiPSCs-derived models, with the purpose of identifying the most potent therapy.

Moreover, it makes possible for drugs to be immediately tested on a new disease model, and thus to

rapidly provide initial results before clinical trials. On the other hand, HTS does not require previous

knowledge to initiate drug discovery, as it enables the testing of over a million compounds. However,

HTS has the disadvantages of requiring a phenotype that can be automatically measured and quantified

which is difficult in more complex diseases. Furthermore, a large number of cells are required, which

may be difficult to obtain due to low differentiation yields [20].

Figure 1 – Biomedical applications of hiPSCs generated by somatic cell reprogramming. Adult somatic cells, like fibroblasts, can be reprogrammed towards a more primitive state, giving rise to iPSCs. These cells may be differentiated into different lineages of the three germ layers. Several applications for these cells may include disease modeling, to understand the molecular mechanism underlying such phenotype, drug screening, to develop and determine what drugs are best suited to treat such disease, and cellular therapies, using the differentiated progeny.

The ability to generate patient-specific cells opens a wide range of possible applications in

regenerative medicine, including cell therapies. In 2014, the first hiPSC-based trial was held in Japan

for the treatment of macular degeneration. In this clinical trial, hiPSCs were generated from patient-

specific fibroblasts and differentiated into retinal pigment epithelial cells that were then transplanted into

the patient eyes [25]. Although hiPSCs overcome some major ethical concerns, there are some technical

problems to generate clinical-grade lines, namely epigenetic abnormalities and the risk of tumor

formation [26].

Furthermore, there is an interest on using gene-editing tool, like CRISPR-Cas9 to correct the

mutations causing a certain disease. With this approach, autologous cells from an affected patient would

first be harvest and reprogrammed into hiPSCs. Gene modification would then be performed and cells

subsequently would be expanded and differentiated into the target cell line [27].

5

1.2 Human pluripotency network

The possible applications of hiPSCs rely on the properties that make them unique assets.

Therefore, to better exploit their potentialities, it is important to understand what intrinsic networks lie

beneath hPSCs.

The differentiation potential and self-renewal capability of these cells are controlled by a set of

transcription factors at the heart of pluripotency: OCT4, SOX2 and NANOG. These factors are ultimately

regulated by signaling pathways like TGF-β/activin/nodal, FGF and canonical Wnt signaling, that will be

reviewed latter on.

1.2.1 Transcription factors – OCT4/SOX2/Klf4/NANOG

The two Yamanaka’s factors OCT4 and SOX2, alongside with NANOG, work in an

interdependent network that regulates the expression of genes involved in self-renewal while

simultaneously repressing genes that mediate differentiation.

The octamer-binding transcription factor 4 (OCT4) is encoded by the POUF5F1 gene and it

binds to the octamer POU motif 5’-ATGCAAAT-3’, recognizing DNA regulatory regions. The activity of

OCT4 has also been reported as fundamental for keeping expression of pluripotency and silencing

genes involved in differentiation [3, 28]. Furthermore, OCT4 was shown to be essential for early

development, as OCT4 null embryos do not pass the ICM formation stage [29].

SOX2 belongs to the SRY-related gene family, which consists of transcription factors with well-

established HMG DNA-binding domains responsible for potency maintenance. Like POUF5F1, SOX2

expression is vital for embryonic development and it is initiated at the morula stage [29]. During the early

stages of neural lineage commitment, SOX2 promotes the neuroectodermal fate by suppressing

mesodermal regulator genes, such as BRACHYURY (T)[30].

These two major transcription factors are believed to work together at numerous levels. The first

reported evidence was the regulation of FGF4 gene that contains two binding sites, for OCT4 and SOX2,

known as the HMG/POU cassette. Additionally, the NANOG gene was found to possess regulatory

regions able to bind to these two factors [29].

Lastly, NANOG encodes for homeodomain proteins that maintain hESCs identity. It is essential

for early embryonic development, as forced expression of NANOG is sufficient to drive cytokine-

independent self-renewal of hESCs. Disruption of NANOG expression causes loss of pluripotency as

neural commitment is induced. Moreover, NANOG null embryos do not develop beyond implantation

[31, 32]. Besides OCT4 and SOX2, NANOG is regulated by the Kruppel-like factor 4 (Klf4), also known

to be part of the Yamanaka’s four transcription factors. Klf4 comes from a family of conserved zinc finger

transcription factors and plays an important role in cell proliferation and inhibition of differentiation and

apoptosis [33, 34].

6

1.2.2 Main signalling pathways involved in pluripotency maintenance

As it was formerly mentioned, signaling pathways control the expression of intrinsic factors that

define hPSCs.

One of these conserved pathways is mediated via fibroblast growth factors – FGF signaling.

FGF ligands bind to tyrosine kinase receptors, resulting in the activation of intracellular signaling, namely

the PI3K-AKT and MERK/ERK pathways [35]. The former starts with the phosphorylation of the

Phosphatidylinositol 3-kinase (PIK3), which in turn activates the v-Akt (AKT), mediating the inhibition of

apoptosis and stimulation of cell proliferation. Parallelly, MEK/ERK signaling is also activated, which

controls survival and differentiation. Inhibition of FGF signaling results in decline of NANOG homeobox

expression in hESCs, and therefore compromising its survival [36]. In hPSCs media, the FGF2

supplementation prevents spontaneous differentiation. This FGF ligand also promotes hPSCs

proliferation through stimulation of the NANOG gene, and prevents neural induction [37].

The transforming growth factor β (TGF- β) is another signaling pathway governing stem cell

fate. It is composed by two subgroups: the TGF- β/Actvin/Nodal ligands, that are responsible for

pluripotency maintenance, and the bone morphogenic proteins (BMPs), that promote differentiation.

After ligand stimulation, the receptors will phosphorylate Smad proteins and make a ternary complex

with Co-Smad, resulting in the formation of Smad4. The latter will accumulate in the nucleus where it

exerts its function upon target genes, including NANOG. The difference between the two subgroups lies

on the receptors and Smad proteins being phosphorylated. For instances, the TGF-β/Activin/Nodal

ligands stimulate phosphorylation of SMAD2/3 proteins via ALK4/5/7 receptors, whereas the BMPs

ligands bind to the ALK1/2/3/6 receptors that phosphorylate SMAD1/5/8 proteins [38].

Finally, the canonical Wnt/β signaling pathway plays a major role in defining stem cell fate. This

pathway promotes β catenin accumulation and further translocation into the nucleus through the

inhibition of glycogen synthase kinase 3 (GSK3). In the nucleus, β-catenin associates with T cell factor

(TCF) and activates the transcription of pluripotency genes, such as Sox2, Nanog and Oct4 [39].

Moreover, the prowess of Wnt signaling in controlling stem cell fate has been demonstrated through the

generation of mesoderm differentiation protocols using hiPSCs [40].

1.3 Microenvironment of pluripotency – the niche

Regardless of whether stem cells are pluripotent in vitro or multipotent in the adult body, stem

cell fate is regulated by a combination of intrinsic and extrinsic mechanisms. The former was briefly

described in section 1.2., whereas the latter comprises signals provided by the local microenvironment.

Therefore, to support growth of pluripotent stem cells in vitro, culture methods should mimic this

microenvironment or niche [41].

The term niche was originally used by Schofield to define certain factors that influence

hematopoietic stem cell fate [42]. In the case of hPSCs, namely hESCs, the niche only exists transiently

during embryonic development. Nevertheless, the current culture systems for the maintenance of the

pluripotency of these cells are designed to mimic the extracellular matrix (ECM), cell-to-cell interactions,

oxygen tension and soluble factors, that promote hPSCs’ expansion [3, 41].

7

1.3.1 The importance of Extracellular matrix (ECM), cell interactions and biophysical stimuli

The ECM is a complex and dynamic structure that provides the scaffold wherein cells are

located. Moreover, it is composed by the several growth factors, namely FGF, TGF-β and WNTs, which

regulate several cellular processes. Among these processes are growth, cell migration, differentiation,

morphogenesis and homeostasis. The ECM composition is tissue-dependent, comprising structural

proteins like collagen, fibronectin, elastin and laminin, as well as proteoglycans. These components

contain various interacting sites which make them capable of binding to each other, forming a complex

three-dimensional network [43, 44].

Briefly, regarding the ECM composition, collagens are the main structural proteins of the ECM

and are classified in both fibrillar (collagens I–III, V and XI) and non-fibrillar forms. Collagen fibrils provide

tensile strength to the ECM, limiting the stretchiness of tissues. Laminins, elastin and fibronectin are

part of the glycoproteins class, being involved in ECM-cell interactions by acting as ligands for cell

surface receptors, such as integrins [45]. After receiving extracellular stimuli, integrins trigger signaling

pathways within the cell as they are directly connected to the cytoskeleton [46]

Another important feature of anchorage-dependent hPSCs is cell-to-cell interactions. Integrins

and E-cadherins are cell adhesion molecules (CAMs) that contribute to cell-ECM and cell-cell

interactions, respectively. Upon disruption of integrin mediated cell-ECM adhesion, E-cadherin

interactions are insufficient to promote cell survival, which in turn leads to single cell apoptosis (anoikis).

These events occur because E-cadherin and integrins are linked to actin-myosin motors, known as

stress fibers. These complexes contain non-muscular myosin connected to actin filaments by the myosin

II heavy chain (MYHs). The disruption of E-cadherin will induce the phosphorylation of the myosin II light

chain (MLC) by Rho-associated kinase (ROCK), resulting in the hypercontraction of stress fibers.

Therefore, if cells are removed from these interactions, the balance is broken, and stress fibers are free

to contract, ultimately leading to cell death [47, 48].

As it was formerly mentioned, hPSCs must be cultured in conditions that better promote their

proliferative capacities, hence the necessity of counteracting the anoikis mechanism. The use of ROCK

inhibitors, such as Y-27632, improves the colony formation upon cell dissociation, as it was reported by

Watanabe et al [49].

Biophysical signals are also an important aspect that composes the hPSC niche. Stem cell

differentiation can be regulated by the external manipulation of the substrate rigidity, topography or

geometry of ECM patterning. Moreover, biophysical forces have proven to be sufficient in directing stem

cell fate, having as examples flow-induced shear stress and hydrostatic pressure [50, 51]. This

parameter is essential when considering the efficient scale-up of hiPSCs culture system in bioreactor-

based approaches (see section 1.4.3) [3, 50, 51]. Likewise, oxygen has been proved to regulated stem

cell fate. For instances, reprograming efficiencies of somatic cells is improved by low oxygen tension

conditions, known as hypoxia [52]. Moreover, spontaneous differentiation of hPSCs is also reduced in

such conditions [53].

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1.4 Stem cell Bioprocessing towards xeno-free culture methods

Human PSCs are an asset for cellular therapy as they can adopt any cellular fate through

differentiation. However, its use in regenerative medicine is mainly hampered by the lack of

bioprocessing methods that can generate a large number of hiPSCs in an economically feasible manner

and in compliance with good manufacturing practices (GMP). For instance, it is estimated that 1-2x109

myocytes are needed to treat a myocardial infarction, therefore, hPSCs must be efficiently expanded

and differentiated into the target cell type [54]. Additionally, the differentiated progeny must be purified,

which involves the development of downstream processing. As an example, magnetic activated cell

sorting has been efficiently used to negatively select the unwanted hPSCs from the differentiated neural

progenitors [55]. Without further ado, this section will review the methods used for the scalable

expansion of clinical-grade hiPSCs.

1.4.1 2D-platforms

1.4.1.1 Culture media and soluble factors

One of the requirements for clinical applications of hPSCs is the development of a xeno-free,

chemically defined medium. Nevertheless, there are additional parameters that must be considered

since they influence cell expansion. These include the pH, oxygen tension, temperature and the

presence of metabolic by-products, namely lactate and ammonia.

The first generation of growth medium for hPSCs comprised the use of fetal bovine serum (FBS)

and undefined components [11]. Over time, it was established the use of a more consistent and

chemically defined medium, avoiding xenogeneic elements. For instances, Vallier et al. used a

chemically defined medium with FGF-2, Activin A and Nodal to propagate hESCs, showing that these

components were sufficient to block the differentiation process [56]. Additionally, Thomson and his

colleagues developed mTeSR™1 defined medium containing FGF-2, TGF-β and γ-aminobutyric acid

(GABA), which are known to be involved in the pluripotency-signaling maintenance [57]. However,

mTeSR™1 culture medium contains bovine serum albumin (BSA) fragment V, an animal product that

has complex albumin and lipid components not fully defined [58].

Chen et al reported a further refinement of the culture media used for hPSCs that totally

suppressed the use of BSA in the mTSeR™1 formula. When considering the necessity of the medium

components in this medium, they found that BSA was not necessary in the absence of β-

mercaptoethanol. After further filtration, they narrow the formula to eight components (L-ascorbic acid,

selenium, transferrin, NaHCO3, insulin FGF2, TGF-β and DMEM/F12), reducing both manufacturing

cost and batch-to-batch variability [59]. This Essential 8® medium (E8) combined with EDTA passaging,

was shown to be suitable for culturing a broad range of hiPSC and hESC lines, particularly to improve

episomal vector-based reprogramming efficiencies as well as experimental consistency [59].

9

1.4.1.2 Substrates for culturing hiPSCs

As it was previously stated, stem cell fate is determined by extracellular cues, including the

interaction between the ECM and other cells. Therefore, it is necessary to mimic these interactions for

an efficient and scalable expansion.

The first culture system developed for hPSCs cells had mouse embryonic fibroblasts (MEFs) as

a feeder-layer, which secreted growth factors that promoted self-renewal, and extracellular matrix

molecules (ECM) that sustain hESCs adhesion. In addition, the culture medium was supplemented with

fetal bovine serum (FBS) as a source of soluble factors, such as essential vitamins and lipids [11].

Despite the results obtained, this method has problems associated to it, as feeder-cells may be a source

of pathogens for hPSCs. Furthermore, the reproducibility of the results is hampered by the batch to

batch variability of undefined components, such as the FBS [58].

Therefore, efforts to use hPSCs in clinical application have been pointed towards the

development of a xeno-free system, that better balances the reconstruction of the niche with the

chemically defined nature of the medium, free of contaminants and with GMP-compliance.

The development of feeder-free cultures was firstly based on the use of Matrigel™, a basement

membrane substrate with ECM components retrieved from the Engelbreth-Holm-Swarm mouse

sarcoma. Such elements include laminin, entactin, collagen IV and several growth factors, like insulin

growth factor (IGF), TGF-β and bFGF [60, 61]. Despite being a feeder free- methodology, the use of

Matrigel strikes the same problems previously mentioned, as its animal origin compromises the desired

reproducibility, due to batch-to-batch variability [60].

To ensure the safety of clinical-grade hPSCs, there is an increasing interest for substitutes of

Matrigel. Therefore, the use of recombinant proteins has proven to be an alternative. For instances, Xu

et al. first reported the use of laminin as an efficient substrate for hESCs expansion, in comparison to

feeder-cells and Matrigel [61]. On the other hand, Rodin et al. demonstrated that the use of laminin-511

would enable the expansion of hESC and hiPSCs in xeno-free conditions, without the loss of

pluripotency [62]. Additionally, the same authors would further present results on the proliferation of

hESCs in a laminin-521/E-cadherin matrix, without the addition of anoikis inhibitors [63].

Other ECM-proteins have been shown to support hESCs expansion. When developing the E8®

medium, Chen et al. used vitronectin as a xeno-free substrate in combination with the use of ROCK

inhibitor [59].

More recently, the development of xeno-free substrates is moving towards the use of synthetic

surfaces over biological substrates, mainly due to the advantages in terms of a defined composition and

low degradability. As an example, the diffusion assisted synthesis-grown nanocrystalline graphene

(DAS-NG) was applied for culturing hPSCs, as DAS-NG holds topological features suitable for cell

adhesion. These include its nanoroughness, oxygen containing functional groups and hydrophilicity.

Moreover, this method allows to synthetize nanographene directly onto the desired substrate at large-

scale, with complete cell adhesion in ECM-depleted surfaces, after 24 hours of culture [64].

Additionally, polysulfone membranes coated with polymerized catecholamines dopamine

(DOPA/PSF) were reported as suitable for hPSCs expansion in defined conditions, with cell adhesion

10

mediated via integrins. Furthermore, cells were cultured up to 10 passages without the loss of

pluripotency, presenting a normal karyotype [65].

Other synthetic substrates for culturing hPSCs include the use of poly[2-(methacryloyloxy) ethyl

dimethyl-(3-sulfopropyl) ammonium hydroxide] (PMEDASH) and Peptide-acrylate surfaces, the latter

commercially sold as Synthemax ®(Corning).

The PMEDASH substrate was first used for culturing hESCs in fully defined synthetic polymer

coatings. However, efficient expansion in defined media was only observed in StemPro media as cells

cultured in mTeSR™1 could not be passed [66]. Further refinement of such polymer was made through

the manipulation of its architecture, which resulted in the long-term expansion of hESCs [67].

Regarding the Synthemax-R (S-R), this substrate is composed by human recombinant

vitronectin, covalently bound to an acrylate moiety. The work developed by Melkoumian et al.

demonstrated that a short peptide sequence of vitronectin was sufficient to support long-term self-

renewal of hESCs in defined conditions. Moreover, the authors enhanced the reduced risk of

contamination with animal-derived pathogens and demonstrated that cells cultured on S-R could be

cryopreserved and thawed back onto the same polymer for further expansion and differentiation. This

property is of the essence for the creation of cell banks [68]. Additionally, S-R was shown to support

differentiation of hESCs towards different cell types [69, 70].

Synthemax-II (S-II) is a derivative from the S-R that includes the RGD-containing sequence from

human vitronectin ECM protein, promoting cell adhesion. Moreover, it is a GMP-compliant polymer and

self-adsorbs onto tissue culturing plastics and glass surfaces, which is an important feature when

considering the scale-up production of clinical-grade hPSCs. Synthemax-II differs from S-R on its

coating chemistry and peptide density, as the peptide on S-II is less dense than S-R [71].

Figure 2 – Evolution of the Substrates used for culturing hPSCs. The development of culturing substrates moves from the use of xenogeneic elements, such as mouse embryonic fibroblasts, towards the use of synthetic polymers, like Synthemax-II.

1.4.2 3D platforms

Despite the advances on 2D-culture systems, these platforms are unable to achieve high cell

densities of clinical grade hPSCs. Moreover, the conventional systems are inadequate in resembling

the niche, namely tissue-specific architecture and its biomechanical cues derived from cell-ECM

interactions. From the industrial point of view, the establishment of suspension culture simplified the

transition of hPSCs from 2D-monolayers to 3D-culturing approaches. The latter include the use of

microcarriers for cell immobilization, the formation of cell aggregates and cell encapsulation within

hydrogel polymers [72].

11

Figure 3 – 3D-platforms for hPSCs culture and expansion, which include cell agregates, microcarriers and microencapsulation of the cells.

1.4.2.1 Aggregates

Cell aggregates are defined as spheroids that better mimic the niche, enhancing the

differentiation and functional capabilities of hPSCs. This suspension culture technique can be easily

implemented in stirred bioreactors, with the advantage of not requiring seeding/adhesion time.

Furthermore, aggregates can be maintained in dynamic bioreactor configurations over long periods of

time [73]. Nevertheless, such strategy presents some limitations namely the tendency towards the

formation of large-size aggregates, which impose limitations on oxygen and nutrient diffusion. This will

further affect viability, proliferation and differentiation propensity of hPSCs in vitro. For instance, hESCs

are less prone to spontaneous differentiation in hypoxia (2-5% of O2) without reducing the proliferation.

However, lower oxygen tension tends to predispose stem cell fate to commit into specific lineages, such

as chondrocytes and endothelial cells, as the hypoxia-inducible transcription factor (HIF) interacts with

pluripotency genes [74]. Therefore, the heterogeneity and spatial disorganization of the aggregate will

lead to an inefficient differentiation into specific cell types. To control the aggregate size, Bauwens et al.

developed a microwell-based aggregation system to track cardiac differentiation of single cell

suspensions. As a result, the maximum expression of cardiac troponin T (cTnT) was observed on day

16, of aggregates initiated with 1000 cells [75].

For long-term culture, one way to control the aggregate size is through enzymatic dissociation.

Moreover, under dynamic conditions, aggregate size may be regulated through physical cues, such as

shear stress. However, transition towards the use of aggregates in large-scale bioprocesses has been

challenging, since such culture regimes may induce enhanced turbulent energy dissipation which

exposes cells to higher and less controllable shear stress [76].

1.4.2.2 Microencapsulation

Cell encapsulation is presented as an alternative method to minimize the aforementioned

drawbacks. Confined hPSCs are protected from shear stress and excessive agglomeration, which to

some extent enhances diffusion of oxygen and nutrients. For example, Serra et al. reported the use of

alginate for further encapsulation of hESCs as single-cells, aggregates and when immobilized on

microcarriers. In the first case, it was observed a drastically decrease in cell viability even after ROCKi

treatment. However, when encapsulated as aggregates or immobilized on microcarriers, hESCs

experienced an improvement on cell expansion and viability, comparing with non-encapsulated cultures.

12

These results demonstrate that microencapsulation is a very efficient strategy for long-term culture as it

provides a shear-stress free environment [77].

One of the several challenges of microencapsulation comprises diffusional limitations within the

polymer gel. Moreover, cell viability may be reduced by the downstream processing of the cultured cells

as mechanic and enzymatic treatment are used to harvest the hPSCs. Therefore, thermos-responsible

polymers are an attractive culture method as an efficient cell harvesting is possible due to a temperature

variation. Other advantages include the chemically defined nature of the polymer as well as its low

toxicity [78].

On the other hand, cell encapsulation has proven to be an efficient way of directing hPSCs

towards specific lineages. For example, fibrinogen hydrogel has been used for hiPSCs differentiation

into cardiomyocytes [79].

1.4.2.3 Microcarriers

Microcarriers were first used to overcome the limited surface area of traditional 2D culture

systems. In this system cells grow attached to a small solid particle, which offers a significant increase

in the surface area to culture volume ratio over 2D-platforms. The characteristics of the microcarriers

should promote an efficient cell adhesion and further robust cell proliferation/differentiation. Moreover,

it should be suitable for applications in dynamic systems and allow an efficient and technically simple

cell harvesting [80].

Regarding its core material, microcarriers can be divided into three main categories: nonporous

and microporous, whose matrix is composed of materials like polystyrene and dextran, respectively, and

macroporous microcarriers, which can be made of cellulose or gelatin. The latter allows the cells to

potentially enter inside of the microcarriers, which increases the surface area and protects cells from

shear stress when incorporated in bioreactor systems [81].

To promote cell adhesion, the surface of the microcarrier should be functionalized with any of

the aforementioned substrates, like ECM proteins or synthetic polymers. For instance, Oh et al. used

Matrigel coated-microcarriers to promote hESCs proliferation. Furthermore, it was demonstrated that

this system achieved higher cell densities under dynamic conditions (3.5x10¨6cells/mL), when compared

to static expansion using the same microcarriers (1.5x106cells/mL). Growth kinetics also showed a

higher doubling time when compared to 2D systems [82]. Additionally, Bardy et al. was able to achieve

high cell densities (1.3x106cells/mL) for further neural differentiation by resorting to a similar strategy

[83]. Despite the results showing the potential use of ECM-coated microcarriers in large scale

expansion, such platforms relied on the use of xenogeneic elements to promote cell proliferation, which

hampers its use in clinical settings.

To overcome such drawbacks, there are studies reporting the use of animal or human ECM-

derived proteins. Chen et al. used purified mouse laminin as a microcarrier coating to expand hESCs in

spinner-flasks, achieving cell densities close to 1.4x106 cells/ml. Moreover, cultured cells retain their

pluripotency and the ability to differentiate into the three germ layers via embryoid body configurations

[84]. Other examples comprise the use of microcarriers coated with vitronectin purified from the human

plasma [85]. Nevertheless, these purified proteins still suffer from batch-to-batch variability. Therefore,

13

the use of recombinant DNA technology is an alternative method to obtain human ECM-proteins, which

are then used to coat microcarriers. As an example, Badenes et al recently developed a xeno-free

platform to expand hiPSCs, by coating polystyrene microcarriers with human recombinant vitronectin.

In this study, the highest cell density achieved (1.4x106cells/ml) was similar to what was reported by

Bardy et al, with the advantage of being a xeno-free system. Moreover, results obtained by Badenes et

al raise the possibility to integrate a differentiation step in the bioprocess, which will be advantageous in

the medical scenario [86].

Microcarriers can be further employed in fully-defined systems using synthetic polymers as a

coating surface. Taking this into consideration, Fan et al. reported a combination of peptide conjugated-

microcarriers coated with a synthetic polymer to expand hiPSCs, under dynamic conditions. The results

were similar to what was previously described, with cell densities close to 1.55x106cells/ml [87]. Other

examples include the use of Synthemax-II-coated polystyrene microcarriers [88].

1.4.2.3.1 Downstream processing – Dissolvable microcarriers

The previous studies show that xeno-free systems resorting to microcarriers are feasible. Up

until now, the question of how the expanded cells are efficiently harvested is yet to be answered. The

use of xeno-free polystyrene microcarriers requires a filtration unit integrated in the bioprocess diagram.

This coupled with an enzymatic treatment should enable the downstream processing of the expanded

cells. However, it contributes to a decrease in cell viability due to the plasma membrane degradation.

Fan et al. specified the use of biodegradable microcarriers as an advantageous strategy, since it

eliminates steps for downstream separation of cells from beads, thus reducing the overall cost. For

instance, lactic/glycolic acid-based biomaterials featuring chemical groups to attach CAMs, may be used

to engineer such microcarriers [87]. Other strategies comprise the use of thermo-responsive polymer-

grafted microcarriers [89].

Another alternative towards a more standardized and efficient downstream processing is the

use of dissolvable scaffolds as microcarriers. Therefore, Corning, Inc® developed dissolvable

polygalacturonic acid (PGA) polymer chains cross-linked via calcium ions (Ca2+). Cell harvesting is

achieved by adding EDTA, pectinase and a standard cell culture protease, such as Accutase. The EDTA

will chelate calcium ions and destabilize the polymer crosslinking, whereas the pectinase and protease

will degrade the PGA polymer and ECM network, respectively. These microcarriers can be further

functionalized with ECM proteins or synthetic peptides like Synthemax-II, which enables the expansion

of hiPSCs in xeno-free configurations. The use of such microcarriers was never reported for hiPSCs

expansion. Nevertheless, preliminary results on the expansion of MSCs show that these matrices

sustain cell growth up to 40 days, with a 10,000-accumulative fold increase. Moreover, the harvesting

yield was higher than 85%, with almost 100% of cell viability [90].

Extrapolating these results to the expansion of hiPSCs, these dissolvable microcarriers should

present an alternative and cost-effective manner of expanding clinical-grade hiPSCs, when employed

in bioreactors suspension cultures.

14

Figure 4 - Schematic representation of bead digestion. Cells adhere to a peptide- or protein-coated ionically cross-linked polysaccharide microcarrier. Chelation of divalent cations by EDTA destabilizes the polymer crosslinking and exposes the polymer chains to degradation by a protease-free enzyme.

1.5 Bioreactors

The envisioned clinical and industrial application of hPSCs will depend on the constant and

controlled production of relevant cell numbers. The current protocols are only able to generate cells in

a laboratory scale, which hampers its use in regenerative medicine. Additionally, the development of

large-scale bioprocesses must be GMP-compliant, therefore, the use bioreactors intents to standardize

and automate the hPSCs manufacturing process. These systems facilitate the development of dynamic

suspension culture, thus overcoming the issues of typical static conditions. Bioreactors are defined as

scale-up processes by the increase in the overall manufacturing scale. They are equipped with

monitoring technologies which allow the control of the culture parameters through a feedback-based

mechanism, namely temperature and pH. Moreover, it avoids the formation of gradients ensuring the

homogeneous distribution of nutrients, gases and metabolites [91].

Bioreactor configurations comprise the use of stirred-suspension vessels, wave-bioreactors,

roller bottles, among others. Stirred suspension bioreactors (SBs) are widely used for the production of

recombinant proteins expressed by mammalian cells. In this type of bioreactors, cells can be cultured

as 3D-platforms using microcarriers, cell aggregates and cell encapsulation Another advantage is the

homogenous distribution of cells nutrients and gases. Additionally, automated SBs allow the adaptation

of feeding strategies to promote higher cell densities. For instances, Kropp et al. achieved close to

3.01x106 cells/mL when culturing cells as suspended aggregates. At the perfusion process endpoint,

the medium consumption was almost 43% less when compared to conventional 2D culture [92].

Alternatively, Silva et al. developed a microcarrier-based 3D-platform to culture hESCs in spinner-flasks.

Interestingly, this platform was transposed to a computer-controlled SB which achieved similar

outcomes [88]. When developing a bioreactor-based platform for the expansion of hPSCs, several

parameters must be considered, namely agitation. Regarding the microcarriers, Nienow et al. specified

the performance of such systems relied on the shear stress arising from the agitation that will detach

cells from the microcarriers, or damage the cells whilst still on the scaffold [93]. In stirred vessels, electric

power is used to drive the impellers which consumption per unit volume depends on several parameters,

namely the type of fluid, the stirrer speed, the impeller shape and size, the tank geometry and the fluid

viscosity and density. The Reynolds number (Rei) and the power number (Np) are used to correlate

these variables. The relationship between Rei and Np has been determined experimentally for the

impeller configuration and the tanks geometry. Additionally, this relationship is dependent upon the flow

15

regime in the tank which can be laminar, turbulent or a transition regime [94]. In the case of stirred-tank

bioreactors operating in a turbulent flow, the power number is independent of the Reynolds number. As

such, the power consumption can be determined through the following equation:

𝑃 = 𝑁𝑝′ 𝜌𝑁𝑖

3𝐷𝑖5

Where 𝜌 is the density of the fluid, 𝑁𝑖is the stirrer speed and 𝐷𝑖 is the diameter of the impeller.

Regarding the power number 𝑁𝑝′ , this is a constant value in the turbulent regime which depends upon

the impeller type being used (Table 1).

Table 1 – Values of the constant in Equation (1.1) for the stirred-tank geometry. Adapted from Doran, M.Pauline (2013).

Impeller type 𝑵𝒑′ (Rei =105)

Rushton turbine 5.0

Pitched-blade turbine 1.3

Marine-propeller 0.35

Anchor 0.35

Helical ribbon 0.35

The kinetic energy being generated from the turbulence flow is directed into regions of rotational

flow called eddies, which size can be determined through the Kolmogorov’s scale or scale of turbulence

(λk). This scale can be further calculated knowing the viscosity of the bulk (v) and the maximum

turbulence energy dissipation rate per mass unit ϵ, accordingly to the following equation:

𝜆𝑘 = (𝑣3

𝜖)

1/4

One approach to determine ϵ is to assume an equal dissipation of energy through the entire

tank volume. However, this method can underestimate the damages caused by eddies. Therefore, it

can be assumed that the power is dissipated mostly in the impeller zone and thus, ϵ can be calculated

through the following equation:

𝜖 =𝑃

𝜌𝐷𝑖3

Where 𝐷𝑖 is the impeller diameter and 𝐷𝑖3 corresponds to the volume around the impeller where

most of the energy is dissipated. Therefore, the Kologomorov’s theory to fluid dynamic stress provides

hydrodynamic cues to establish an optimize agitation to promote cell growth. As such, to avoid cellular

detachment from the microcarriers, the bioreactors must be operated so that the turbulence scale

remains greater than the biological entity being cultured [94].

On the other hand, wave bioreactors may be considered as an option. This type of configuration

comprises a disposable bag, partly filled with media and inoculated with cells, with the remainder inflated

with air. The hydrodynamic motions occur due to a rocking platform on which the bag is deposited. This

agitation provides an efficient and continuous nutrient and gas mixing, without high shear stress levels.

16

Moreover, wave bioreactors are GMP-compliant as the culture bags are pre-sterilized and designed for

single-use to reduce the risk of contamination. Additionally, it can be operated under different batch,

fed-batch and continuous modes, the latter recurring to special filters for cell retention. However, the

disadvantages of these system comprise the difficulty in operating with microcarriers, sampling and

controlling the culture conditions within, as well as the expensive scaling-up [95].

17

2. Aim of study

In the field of regenerative medicine, one of the major innovations was the development of

hiPSCs technology. However, a reprogramming method that better balances safety and efficiency is yet

to be achieved. Moreover, cell-based therapies and other biomedical applications using hiPSCs are

hampered by the lack of reproducible methods for the xeno-free and large-scale expansion of clinically-

relevant cell numbers. Several studies use microcarriers to support hiPSC culture, as these can be

further incorporated into suspension cultures. Despite the establishment of xeno-free protocols for

hiPSCs expansion resorting to this 3D-platform, little focus has been given to the harvesting procedures.

For instance, the use of polystyrene microcarriers represents a challenge in terms of cell harvesting

since it requires the integration of a filtration unit following the bioreactor. Furthermore, the mechanical

action needed to separate the cells from the beads would often present cell losses and reduced viability.

The use of biodegradable microcarriers would prompt a reduction in downstream processing

steps, and thus reducing the overall cost. For that purpose, Corning Inc. developed a new type of

microcarriers envisaging the scalability of the harvesting process. These dissolvable microcarriers are

made of a polygalacturonic acid polymer that can be further coated with any appropriate substrate for

the expansion of hiPSCs, namely Synthemax-II. The latter is a xeno-free and chemically defined human

vitronectin-based peptide containing the RGD-sequence. This substrate has been proven to support the

expansion of hiPSCs on 2D-cultures.

The aim of this study is to exploit the use of these dissolvable microcarriers for the xeno-free

expansion and harvesting of hiPSCs. Initially, in a ultra-low attachment plate (ULA), it was performed a

static screening using both dissolvable and polystyrene microcarriers. The beads were coated with

different substrates, namely Matrigel and Vitronectinto assess the cell adhesion yield, fold increase and

the maintenance of pluripotency. From the results obtained in the static expansion, two conditions were

chosen to be scaled-up. The dynamic cultures were performed in a spinner-flask, with a working volume

of 30 ml. Within this context, it was intended to determine certain parameters such as cell adhesion yield

(%), fold increase, specific growth rate (µ, day-1) and the doubling time (t2, days) of the expansion

method. Most importantly, the harvesting yield (%) was also assessed after the dynamic expansion of

hiPSCs performed in dissolvable microcarriers by using a method which enables to harvest the cells

within the spinner-flask. The expansion and recovery of cells using vitronectin-coated polystyrene

microcarriers was then compared with the ones obtained with the dissolvable microcarriers.

After the harvesting procedure, the phenotype of the expanded hiPSCs was characterized by

immunocytochemistry, flow cytometry and qRT-PCR assays. The differentiation capabilities were

assessed through the formation of Embryoid Bodies. Furthermore, it was also performed the direct

differentiation towards neural progenitors and cardiomyocytes of replated hiPSCs

18

19

3. Materials and Methods

3.1 Cell lines

In this work, two cell lines were used to test the expansion of hiPSCs using Synthemax-II

dissolvable microcarriers (Corning®) in xeno-free defined conditions. Therefore, it was used the WT-

F00.1A.13 (TCLab – Terapias Celulares para Aplicação Médica, Unipessoal Lda.) and the Gibco cell

line (Life Technologies™). The former cell line was reprogrammed from fibroblasts obtained from a skin

biopsy of an adult female. By using a retroviral system, the hiPSCs have been generated through ectopic

expression of a defined set of reprogramming factors, Oct4, Sox2, Klf4 and c-Myc. The latter was

derived from CD34+ cells of healthy donors.

From now on, the WT – F00.1A.13 cell line will be designated as TCLab cell line.

3.2 Culture media for hiPSCs expansion

3.2.1 mTeSR™1

The mTeSR™1 medium is a defined, feeder-free maintenance medium for hESCs and hiPSCs.

It is commercially available by STEMCELL™ Technologies and is based on the publication of Dr. James

Thomson’s lab [57]. The medium is composed of a DMEM/F12 base supplemented with albumin,

vitamins, antioxidants, trace minerals, specific lipids and cloned growth factors. The preparation of

complete mTeSR™1 medium is made by thawing the 5X supplement at 15-25ºC or overnight at 2-8ºC.

100mL were added to 400mL of basal medium and mix thoroughly. If prepared aseptically, complete

mTeSR™1 is ready for use, but the medium can also be filtered using a 0.2µm low-protein binding filter.

The media is also supplemented with Penicillin/Streptomycin (PenStrep, Gibco®) in a dilution of 1:200

(v/v).

3.2.2 TeSR™2

The TeSR™2 is closely related to mTeSR™1 in terms of composition with the advantage of

being free of xenogenic components. The preparation of complete TeSR™2 medium is made by thawing

the 5X and 250X supplements at 15-25ºC, or overnight at 2-8ºC. The medium is made by adding. 100mL

and 2mL of each supplement, respectively, to 400mL of basal medium. Once thawed use immediately

or aliquot and store at -20ºC for up to 3 months. If prepared aseptically, complete mTeSR™1 is ready

for use, but the medium can also be filtered using a 0.2µm low-protein binding filter. The media is also

supplemented with PenStrep in a proportion of 1:200 (v/v).

20

3.2.3 Essential 8 ™

The Essential 8™ media (Life Technologies ™) is a xeno-free and feeder-free medium. It

contains eight essential components for growth and expansion of hPSCs, namely the basal medium

DMEM/F12 supplemented with insulin, transferrin, selenium, L-ascorbic acid, FGF2 and TGFβ, as well

as NaHCO3 for pH adjustment [59]. The E8™ medium is prepared according to the manufacturer’s

instructions by thawing the supplement at 15-25ºC or overnight at 2-8ºC. The media is also

supplemented with Penicillin/Streptomycin (PenStrep by Gibco®) in a proportion of 1:200 (v/v).

3.3 hiPSCs thawing and cryopreservation

3.3.1 Thawing hiPSCs

The cryovials (Thermo Scientific ™) containing the hiPSCs are cryopreserved in liquid nitrogen.

Before initiating the thawing procedure, a 15mL falcon with 4mL of culture medium is pre-warmed in a

37ºC water bath. After retrieving the desired vial, 1mL from the previously warmed washing medium is

used to resuspend the cells within the cryovial. The content of the vial is afterwards transferred to the

falcon tube with the washing medium, which is then centrifuged for 3 min at 1,500xg. The supernatant

is removed, and the pellet is resuspended with the culture medium that is further used in the experiment.

The content is uniformly distributed in 9.6 cm2 culture-wells pre-coated with Matrigel®. The plate is then

placed in an incubator (Memmert) at 37ºC, 5% of CO2 and 20% of O2.

3.3.2 Cryopreservation of hiPSCs

Cells are cryopreserved in cryovials (Thermo Scientific ™) with at least 1x106 cells per 250µL.

First, the culture medium is removed from the culture well and the well is washed with EDTA

(Invitrogen™). Afterwards, 1mL of EDTA is used to incubate the cells for 5 minutes. To recover the cells,

the EDTA is removed and cells are flushed from the well using 1mL of washing medium. The cell

suspension is then transferred to on 15 mL falcon, which is centrifuged for 3 minutes at 1,500xg. After

centrifugation, the supernatant is removed, and the pellet is resuspended with freezing medium. The

latter is composed of KO-SR with 10% (v/v) of dimethylsulfoxide (DMSO, Gibco®). The cell suspension

is collected for the cryovial and placed overnight at -80ºC before being transferred to liquid nitrogen.

3.4 Expansion of hiPSCs

The expansion of hiPSCs may be done by two alternative systems (2D vs 3D) previously

discussed on section 1.4. In this work, it was assessed the possible use of dissolvable microcarriers

(Corning®) on 3D-culture systems to expand hiPSCs, as well as its possible advantages for cell

harvesting, over the use of polystyrene microcarriers (PSM, SoloHill®). First, cells were grown as 2D-

monolayer cultures to have enough cells for the static and dynamic expansion using microcarriers. The

static experiment corresponds to a screening of possible combinations of media and coated-

21

microcarriers for the expansion of hiPSCs (Table 1). Afterwards, the Synthemax-II dissolvable

microcarriers (DM-SII, Corning®) were tested in combination with mTeSR™1 and TeSR™2 for the

expansion of the TCLAB cell line under dynamic conditions. The cell harvesting yield was then compared

to the cell harvesting yield using vitronectin-coated PSM (PSM-VTN). The DM-SII were then tested for

the expansion Gibco® cell line in mTeSR™1 culture media.

Table 2 – Schematic representation of the conditions tested in static and dynamic conditions. The TCLab cell line was used for the expansion under static conditions using both types of microcarriers (PSM and DM) with possible media and coating combinations. The same cell line was then used for the expansion under dynamic conditions using PSM-VTN in E8® media, and DM-SII in mTeSR™1 and TeSR™2.

Microcarrier Coating Culture media

Cell line

Static expansion Dynamic expansion

Polystyrene Vitronectin E8™ TCLAB TCLAB

Dissolvable

Matrigel mTeSR™1 TCLAB -

Synthemax-II mTeSR™1 TCLAB TCLAB/Gibco

TeSR™2 TCLAB TCLAB

3.4.1 Static Culture Systems

3.4.1.1 Monolayer culture system

3.4.1.1.1 Matrigel ™ Coating

Cells were grown in multi-well tissue culture plates (Falcon®) which were previously coated with

Matrigel ™ (Corning®). This substrate is a gelatinous protein mixture extracted from the Engelbreth-

Holm-Swan mouse sarcoma containing ECM proteins like collagen IV, laminin and entactin. Matrigel ™

is stored in aliquots at -20ºC. First, an aliquot is thawed on ice at 15-25ºC, or overnight at 4ºC.

Afterwards, the Matrigel™ was diluted on a falcon containing Dulbecco’s Modified Eagle’s

Medium/Nutrient Mixture F-12 (DMEM/F12, Gibco®), in a proportion of 1:100 (v/v). Afterwards, 1mL of

this solution was placed in each well of a 6-well plate, and incubated at 15-25ºC for immediate use,

otherwise it was stored at 4ºC for up to two weeks.

3.4.1.1.2 EDTA cell passaging and cell replating

Cells were grown in Matrigel-coated plates and passed when they achieved 80% of confluence.

For this, EDTA was used as an enzyme-free method to detach hiPSCs colonies in small clumps. First

the culture medium was removed, followed by a rapid wash with EDTA (0.5 mM) in each well. After this

procedure, the wells were incubated with 1mL of EDTA (0.5 mM) for 5 minutes. The cells were then

22

flushed with the culture medium into a falcon. After counting the cells, these were replated at a cell

density of 50,000 cells/cm2, in Matrigel™ coated plates. For hiPSCs, the culture media must be daily

replaced by fresh media. For cells cultured on 6-well plates (9.5 cm2/well), 1.5 mL of fresh media is

added after the exhausted media is aspirated.

3.4.1.2 Microcarrier culture system

For the static expansion of hiPSCs using microcarriers, cells were incubated with the

microcarriers in a 24-well ultra-low attachment culture plates (ULA, 3 cm2/well, Corning®). In this

system, polystyrene and dissolvable microcarriers were coated with the substrates of interest (Table 1)

to assess the fold increase and adhesion yield.

3.4.1.2.1 Polystyrene microcarriers

The SoloHill® polystyrene microcarriers (PSM) have a surface area of 360cm2 per gram.

Therefore, 8.3 mg of PSM were needed per well. Prior to utilization, the necessary amount of PSM is

weighted inside a falcon and sterilized in Ethanol 70%(v/v) for a period no less than 1 hour. After

sterilization, the PSM were washed three times with Phosphate buffered saline solution (PBS, Gibco®).

Coating of PSM was performed for 2h at 15-25ºC with Vitronectin (VTN) in sterile PBS, using 0.5µg/cm2.

3.4.1.2.2 Dissolvable microcarriers

The dissolvable microcarriers (DM) provided by Corning® have a surface area of 5,000 cm2 per

gram. Therefore, 0.6 mg were needed per well. In this experiment, two types of microcarriers were used

to expand hiPSCs: Synthemax-II coated-DM (DM-SII) and uncoated-DM. For the DM-SII, there was no

need to perform the coating procedure whereas the uncoated-DM were incubated with a Matrigel

solution (see section 3.4.2.2.1). The necessary amount of microcarriers was weighted inside a glass

bottle, and afterwards hydrated for 1h, by adding 150mL of sterile water (Corning®) per gram of DM.

This procedure was made under agitation. After hydration, the DM were allowed to settle, and the water

replaced by culture medium.

3.4.1.2.2.1 Matrigel Coating Matrigel™ coating was performed on uncoated-DM by diluting the substrate in a proportion of

1:30(v/v) of culture medium. The microcarriers were then incubated in the Matrigel solution for 2 hours

at room temperature (RT) and under agitation.

3.4.2 Inoculation of microcarriers and hiPSCs expansion

Human iPSCs were recovered from 2D-monolayer cultures using the EDTA cell passaging

procedure described in section 3.4.1.1.2. However, the culture medium used for flushing the cells was

supplemented with 1:1000 (v/v) of ROCK inhibitor Y-27632 (ROCKi, STEMCELL Technologies®), to

enhance cell survival. After cell counting, the VTN-coated PSM, Matrigel-coated DM and DM-SII were

inoculated at a cell density of 55,000 cells/cm2, which corresponded to 1.65x105 cells/well. The

23

expansion lasted for five days and cells were harvested and counted on day 1 and day 5 to assess the

adhesion yield and fold increase, respectively.

3.4.3 Cell Harvesting

For polystyrene microcarriers, the culture medium was removed and a washing step with PBS

was performed. Afterwards, 500µL of Accutase were added per well, and the plate was incubated at

37ºC for 5 minutes. Each well was flushed with 1mL of Washing Medium to detach cells from each other

and to stop the enzymatic reaction. Afterwards, the content of the well was filtrated into a falcon, using

a 100µm strainer (Corning®). The falcon was then centrifuged for 5 minutes at 1,500xg, and the

supernatant was discarded before resuspending the cells in washing medium.

For dissolvable microcarriers, the harvesting process consists on digesting the polymer. The

culture medium was removed and each well washed with PBS. Afterwards, 500µL of harvesting solution

diluted in Accutase (1:5(v/v)) were added to each well. The plate was then incubated at 37ºC for 7

minutes. Each well was flushed with 1mL of washing medium to detach cells from each other as well as

to stop the enzymatic dissociation. The cellular suspension was transferred into a falcon and centrifuged

for 3 minutes at 1,500xg. Cells were then resuspended in washing medium.

After the harvesting process, the cells were counted in a Neubauer chamber under the

microscope. To distinguish the dead cells from the living, the cellular suspension was diluted in Trypan

Blue (TB) in a proportion of 1:2(v/v), and 10µL of the resulting suspension were loaded onto the

Neubauer chamber. Therefore, cells have been counted by using the following equation:

𝐶𝑒𝑙𝑙𝑠. 𝑚𝐿−1 =𝑛𝑜.𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑

𝑛𝑜.𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑∗ 𝑇𝐵𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 ∗ 104

Equation 3.1

The adhesion yield is calculated by the following equation:

𝐴𝑑ℎ𝑒𝑠𝑖𝑜𝑛 𝑌𝑖𝑒𝑙𝑑 =𝑛𝑜. 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑜𝑛 𝑑𝑎𝑦1

𝑛𝑜. 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑜𝑛 𝑑𝑎𝑦0× 100%

Equation 3.2

The fold increase is calculated by the following equation:

𝐹𝑜𝑙𝑑 𝑖𝑛𝑐𝑟𝑒𝑎𝑠𝑒 =𝑛𝑜. 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑜𝑛 𝑑𝑎𝑦5

𝑛𝑜. 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑜𝑛 𝑑𝑎𝑦1

Equation 3.3

24

Figure 5 – Representation of the procedure for hiPSCs expansion under static conditions and cell harvesting using both PSM and DM coated with different substrates.

3.5 Dynamic culture systems

For hiPSC culture on PSM-VTN coated microcarriers, cells were expanded in E8 medium with

a concentration of 20 g/L of microcarriers. For 30mL of useful volume, 600mg of PSM were needed.

Since these microcarriers have a surface are of 360cm2/g, the surface area of the total microcarriers

corresponded to 216 cm2. This value was then used to estimate the weight of dissolvable microcarriers

needed to culture hiPSCs. Therefore, considering that the DM have a surface area of 5000cm2/g, it was

needed a total of 43mg for the same useful volume.

Regarding the cell density, 55,000 cells/cm2 were used to inoculate the spinners for both types

of microcarriers, which translates as 12x106 cells for 216 cm2 of surface area.

3.5.1 Inoculation and expansion in dynamic conditions

The inner surface of the spinner-flasks was siliconized with SIGMACOTE® (SigmaAldrich)

overnight, according to the manufacturer’s instructions. Afterwards, the interior of the spinner-flasks was

washed with distillate water before sterilization in the autoclave. Before inoculation, the sterile spinner

flask was washed using culture media and both types of microcarriers were prepared according to

section 3.4.1.2.

On day 0, the spinner-flasks were inoculated using a cell density of 55,000 cells/cm2 which were

previously expanded as 2D-monolayer cultures and harvested using EDTA (section 3.4.1.1.2). The

working volume on this day corresponds to 15mL containing the previously mentioned cell and

microcarriers density cultured in medium supplemented with 1:1000(v/v) of ROCKi and 1:200(v/v) of

PenStrep. Afterwards, hiPSCs are expanded for 7 days in 30mL with daily culture medium replacement.

From day 1 to day 7, the culture medium used was supplemented only with 1:200(v/v). of PenStrep.

Moreover, the number of cells was monitored every day by retrieving two samples of 500µL of cell

suspension and quantifying by direct cell counting (Eq.3.1).

Regarding the agitation protocol, on day 0 there was no agitation. On day 1, it was performed

and intermittent agitation with 3min at 25 rpm every 2 hours. From day 2 until day 7, it was employed a

continuous agitation at 25 rpm.

25

Cell viability assays were performed using the LIVE/DEAD® viability/cytotoxicity Kit (Thermo

Fisher Scientific). This was performed upon a sample of 500µL retrieved from the spinner-flask.

3.5.2 Cell harvesting

Cell harvesting was performed by adapting a method previously described in the literature for

harvesting MSC, cultured on microcarriers, inside of the spinner flask [93]. However, for PSM the

harvesting method relied on a filtration step, whereas for DM the cells are harvested upon the digestion

of the polymer. On day 7 and before initiating the harvesting procedure, cells were counted in the same

manner previously described in section 3.4.1.2.4. After the harvesting process, the cells were also

counted to quantify the harvesting yield, which is assessed through the following equation:

𝜂𝐻𝑎𝑟𝑣𝑒𝑠𝑡𝑖𝑛𝑔(%) =𝑛𝑜. 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑜𝑛 𝑑𝑎𝑦 7

𝑛𝑜. 𝑜𝑓 ℎ𝑎𝑟𝑣𝑒𝑠𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠× 100%

Equation 3.4

3.5.2.1 Filtration

After the dynamic expansion on PSM, the cells attached to the beads were allowed to settle at

the bottom of the vessel and the culture medium was carefully removed to avoid any cell loss.

Afterwards, 15mL of Accutase were added to the spinner-flask, which was incubated at 37ºC and

agitated at 100 rpm for 5min. Subsequently, 30 mL of culture medium were added to stop the enzymatic

reaction. The total volume was filtered through a 100µm strainer into to a falcon, which is then

centrifuged for 3min at 1,500xg. The supernatant was discarded, and cells are resuspended in culture

medium supplemented with 1:1000 (v/v) of ROCKi. Afterwards cells were counted, and the harvesting

yield is assessed through the previous equation (Eq. 3.4).

3.5.2.2 Digestion

After the dynamic expansion on DM, the cells attached to the beads were allowed to settle at

the bottom of the vessel and the culture medium was carefully removed to avoid any cell loss.

Afterwards, 15mL of 1:5 (v/v) of harvesting solution diluted in Accutase were added to the spinner-flask,

which was incubated at 37ºC and agitated at 100 rpm for 5min. Subsequently, 30 mL of culture medium

were added to stop the enzymatic reaction. The total volume was transferred to a falcon, which is then

centrifuged for 3min at 1,500xg. The supernatant is discarded, and cells were resuspended in culture

medium supplemented with 1:1000 (v/v) of ROCKi. Afterwards cells were counted, and the harvesting

yield is assessed through the previous equation 3.4.

3.5.2.2.1 Harvest solution

The harvest solution is prepared according to the manufacturer’s instructions by adding EDTA

(stock solution 0.5M, pH 8) to the protease solution, ensuring a final pectinase concentration of 100

U/mL and EDTA concentration of 10mM.

26

Figure 6 - Schematic representation of the hiPSCs expansion and harvesting under dynamic conditions. Cells were cultured for 7 days, at which time they were harvested accordingly to the type of microcarriers used.

3.5.3 Growth kinetics

As it was previously mentioned, cells were counted every day during expansion under dynamic

conditions. The adhesion yield was assessed through equation 3.2 and the fold increase through

equation 3.3. To calculate the apparent specific growth rate µ (day-1), the growth was considered

exponential and Ln X was plotted against time t, where X is the number of cells. According to the

following equation:

𝑙𝑛 𝑋 = 𝑙𝑛 𝑋0 + µ𝑡 Equation 3.5

The doubling time was calculated according to the following equation

𝑡2 =𝑙𝑛2

µ

Equation 3.6

3.5.4 Metabolic profile

For the analysis of the concentration of nutrients and metabolites, the exhausted medium was

recovered from the culture at each culture media change. The samples were centrifuged for 10 min at

1500xg to remove dead cells, debris and microcarriers. The supernatants were collected and frozen

until further analysis. Fresh medium was also analyzed, and the results used for the determination of

27

consumption and production rates. Glucose, lactate, and glutamine concentrations were measured

using a multi-parameter analyzer (YSI 7100MBS, Yellow Springs Instruments). The specific metabolic

rates (qMet, nmol/(cell.day)) were calculated for each day the culture medium was changed. For that

purpose, the following equation was used:

𝑞𝑚 = ∆𝑀𝑒𝑡(∆𝑡. ∆𝑋𝑣)⁄ Equation 3.5

Where, ∆Met is the variation in metabolite concentration during the time period ∆𝑡 and ∆𝑋𝑣 the

logarithmic average of viable cell number during the same period. The apparent yield of lactate from

glucose (Y´qLac/qGlu) was also calculated as the ratio between qlactate and qglucose.

3.6 Characterization of hiPSCs

After the expansion of hiPSCs in DM-SII, cells were replated onto Matrigel™-coated plates for

further characterization of their properties. Therefore, several techniques were used to assess the

pluripotency of cells expanded in DM-SII, including immunocytochemistry coupled with confocal

microscopy, flow cytometry and Real-time PCR. The replated hiPSCs were also differentiated into

cardiomyocytes and neural progenitors. Furthermore, cells were spontaneously differentiated into cells

of the three embryonic germ layers by the Embryoid body (EB’s) formation protocol.

3.6.1 Immunocytochemistry and confocal microscopy

3.6.1.1 Intracellular markers

The replated hiPSCs were washed with PBS and cells were fixed with 4% (v/v) PFA during 30

min. Then, cells were incubated with blocking solution (10% FBS and 1%Triton, in PBS) overnight or

left during 60 min at room temperature. Primary antibodies were diluted in staining solution (5% NGS

and 0.1% Triton, in PBS), added to the wells and left at 4ºC overnight. Secondary antibodies were also

diluted in staining solution and left to incubate with cells during 1h in the dark, at room temperature. The

cells were washed with PBS in order to remove the excess of secondary antibody and were left with

4’,6-diamidino-2-phenylindole (DAPI), the fluorescent stain that binds to DNA, (diluted 1:10000 in

NaHCO3; Sigma) during 2 min at room temperature. Finally, cells were washed to remove any DAPI

crystals and left with PBS for further observation under fluorescence optical microscope (Leica

Microsystems CMS GmbH, model DMI3000 B) or confocal microscopy (Zeiss LSM 710). For confocal

observation, the lamellas were taken and by using Mowiol, a mounting medium, they were assembled

on blades. The data was retrieved and analyzed with Fiji software (for imageJ).

28

Table 3 - Overview of the primary and secondary antibodies used to perform immunocytochemistry of the intracellular pluripotency markers. Both types of antibodies were diluted in staining solution.

Marker Primary antibody Secondary antibody

Antibody Dilution Antibody Dilution

OCT4 Mouse IgG (Invitrogen) 1:150 Goat anti-mouse IgM (Invitrogen)

1:500

SOX2 Mouse IgG (R&D Systems) 1:200 Goat anti-mouse IgG

(Invitrogen) 1:500

3.6.1.2 Cell surface markers

For the cell surface markers cells were washed with PBS and fixed with 4% (v/v) PFA during 30

min. Then cells were incubated with Bovine serum albumin (BSA, 3% (v/v) diluted in PBS) for 1h at RT.

Primary antibodies were diluted in the same concentration of BSA and incubated for 45 min at RT. The

secondary antibodies were diluted in BSA and incubated in the dark for 45 min at RT. The cells were

washed with PBS to remove the excess of secondary antibody and were left with 4’,6-diamidino-2-

phenylindole (DAPI), the fluorescent stain that binds to DNA, (diluted 1:10000 in NaHCO3; Sigma)

during 2 min at room temperature. Finally, cells were washed to remove any DAPI crystals and left with

PBS for further observation under fluorescence optical microscope (Leica Microsystems CMS GmbH,

model DMI3000 B) or confocal microscopy (Zeiss LSM 710). For confocal microscopy observation, the

lamellas were taken and by using mowiol, a mounting medium, they were assembled on blades. The

data was retrieved and analyzed with Fiji software (for imageJ).

Table 4 – Overview of the primary and secondary antibodies used to perform immunocytochemistry of the extracellular pluripotency markers. Both antibodies were diluted in 3% (v/v) of Bovine Serum albumin.

Marker Primary antibody Secondary antibody

Antibody Dilution Antibody Dilution

Tra-1-60 Mouse IgM (Miltenyi Biotech) 1:100 Goat anti-mouse IgM (Invitrogen)

1:500

SSEA-4 Mouse IgG (Miltenyi Biotech) 1:100 Goat anti-mouse IgG (Invitrogen)

1:500

3.6.2 Flow cytometry

After the expansion of hiPSCs on dissolvable microcarriers and under dynamic conditions, the

expression of pluripotency markers was also assessed by flow cytometry, namely for OCT4, SOX2,

TRA-1-60 and SSEA4 expression. Harvested cells were already singularized due to the harvesting

procedure, therefore, samples were retrieved to a falcon tube and centrifuged for 3 min at 1,500xg. The

supernatant was discarded, and the pellet was resuspended with 2% PFA in PBS. The samples were

stored at 4ºC.

29

3.6.2.1 Intracellular markers

First, Eppendorf’s were coated with 3% (v/v) BSA for 15 min prior to utilization at RT. Meanwhile,

samples kept in 2% PFA solution were centrifuged for 5 min at 1,500 xg, washed twice with of 3%(v/v)

NGS solution, and centrifuged for 5 min at 1,500 xg, each time. The supernatant was discarded, and

cells were resuspended in 500 μL of 3% (v/v) NGS solution for each analysis to be performed with that

same sample. BSA solution is removed from the Eppendorf vials, and 500 μL of cell suspension were

added to each one. Vials were centrifuged for 3 min at 1,500 xg, supernatant was removed, and the

pellet was resuspended in saponin diluted in 3% (v/v) NGS in a proportion of 1:1 (v/v). After incubation,

the vials are centrifuged for 3 min at 1,500 xg, the supernatant was removed, and the pellet was

resuspended in NGS solution and incubated for 15 min at room temperature. After incubation, the vials

were centrifuged for 3 min at 1,500 rpm. The supernatant was removed and the pellet resuspended in

the appropriate primary antibody solutions – Anti-OCT4 (mouse IgG, Merck Millipore) antibody is used

in a 1:300 proportion – and incubated for 1.5 h in the dark.

After incubation, vials were centrifuged for 3 min at 1000 rpm, pellet was washed twice with 1%

NGS solution, and centrifuged for 3 min at 1,500 xg, each time. The supernatant was removed, all

samples were resuspended in 300 μL of secondary antibody solution – Alexa 488 anti-mouse IgG

antibody (Invitrogen) in a 1:300 (v/v) proportion –, and incubated for 45 min in the dark. After incubation,

vials are centrifuged for 3 min at 1000 rpm, pellet is washed twice with 1% NGS solution, and centrifuged

for 3 min at 1000 rpm, each time. Supernatant is removed, the pellet is resuspended in 500 μL of PBS

solution, cell suspension is transferred to FACS vials and analyzed in a FACScalibur (Becton Dickinson)

flow cytometer, using CellQuest™ software (Becton Dickinson) for data acquisition.

3.6.2.2 Extracellular staining

After cells were fixed with PFA 4%, the suspension is centrifuged for 3 min. at 1,500xg. The

supernatant was discarded, and the pellet was resuspended in FACS buffer (4% foetal bovine serum

(FBS; Sigma Aldrich) in PBS). Afterwards, 100µL of cell suspension was transferred to a FACS vial and

10µL of phycoerythrin (PE) conjugated antibody solution- SSEA-4 (Miltenyi Biotec), TRA-1-60 (Miltenyi

Biotec) – are added to each vial and incubated for 15 min at RT, in the dark. After incubation, 2 mL of

PBS solution are added to each vial, which is then centrifuged for 3 min at 1,500xg; supernatant was

discarded and 2 mL of PBS are added to each vial, which are again centrifuged and the supernatant

discarded. PBS was used to resuspend the pellet and the cell suspension obtained is then analysed in

a FACScalibur (Becton Dickinson) flow cytometer, using CellQuest™ software (Becton Dickinson) for

data acquisition.

3.6.3 Quantitative Real-time PCR analysis

Total RNA from cell samples of selected time points was extracted using Invitrogen™

PureLink™ RNA Mini Kit (Thermo Fisher Scientific) following the provided instructions. RNA was treated

with Invitrogen™ TURBO DNA-free™ for total DNA digestion and then it was quantified using a

nanodrop. 1 µg of RNA was converted into cDNA with Applied Biosystems™ High Capacity cDNA

30

Reverse Transcription Kit (Thermo Fisher Scientific) also following the provided instructions. PCR

reactions were run using 12.5 ng of cDNA and 250µM of Applied Biosystems™ Taqman™ Gene

Expression Assays (Thermo Fisher Scientific), along with Applied Biosystems™ Taqman™ Gene

Expression Master Mix (Thermo Fisher Scientific). Reactions were run in triplicate using Applied

Biosystems™ ViiA™ 7 Real-Time PCR Systems (Thermo Fisher Scientific) and data were analysed

using Applied Biosystems™ QuantStudio™ Real-Time PCR Software. The analysis was performed

using the ΔΔCt method and values were normalized against the expression of the housekeeping gene

glyceraldehyde-3-phosohate dehydrogenase (GAPDH). For the qRT-PCR analysis, the following genes

were assessed: OCT4, NANOG, PAX6, SOX17 and T.

3.6.4 hiPSCs differentiation

3.6.4.1 Neural induction

After expansion, cells were harvested and replated onto Matrigel coated plated. When

confluence was near 90-100%, the neural induction was performed by using the N2B27 medium. From

day 0 to day 11, the medium was changed daily and supplemented with small molecules SB-431542

(SB, StemMACS™) and LDN-193189 (LDN, StemMACS™) in a proportion of 1:1000 (v/v). On day 12,

cells are replated onto laminin-coated plates which were previously coated with poly-ornithine. The

N2B27 media is used for this procedure. From day 12 to day 13, the media is changed daily without

adding any of the previous molecules. On day 14, N2B27 media is supplemented with 1:500 (v/v) of

bFGF until day 16, which corresponds to the last day. On this day, cells were fixed with 4% PFA for

further immunocytochemistry analysis. The formulated medium consists of 50%(v/v) of N2 medium and

B27 medium. N2 medium was DMEM/F12 (1:1) + Glutamax (Gibco®) supplemented with 1% (v/v) N-2

Supplement (Gibco®), 1.6 g/L of glucose (Sigma), 1% (v/v) PenStrep and 20 μg/mL Insulin (Sigma).

B27 medium was formulated with Neurobasal® Medium (Gibco®) supplemented with 2% (v/v) of B-27®

Supplement (Gibco®), 2 mM of L-glutamine (Gibco®) and 0.5% (v/v) of PenStrep. During 12 days of

neural commitment, the previous described medium formulation was supplemented with 10 μM of SB

and 100 nM of LDN.

3.6.4.2 Cardiac induction

For hiPSCs differentiation into cardiomyocytes, the harvested cells were replated onto Matrigel-

coated 12 well-plates at a cell density of 4x105 cells/well. Culture medium was changed daily until a

confluence of 90% was achieved. Gibco™ Roswell Park Memorial Institute (RPMI) 1640 medium (by

Thermo Fisher Scientific) was used as basal medium. From day 0 to day 6, cells were cultured in RPMI

supplemented with Gibco™ B-27™ minus insulin (Thermo Fisher Scientific), the first basal medium, and

from day 7 until the end of differentiation, cells were cultured in the second basal medium B-27™

(Thermo Fisher Scientific). Basal medium was always supplemented 1:200 (v/v) with PenStrep (Thermo

Fisher Scientific). At day 0 of differentiation, after mTeSR™1 culture medium removal, cells were

cultured in the first basal medium supplemented with the GSK3 inhibitor CHIR99021 (Stemgent) at a

final concentration of 6 µM. After 24 hours, medium was changed to the first basal medium. At day 3,

31

cells were cultured in the first basal medium supplemented with Wnt inhibitor IWP-4 (Stemgent) at a

final concentration of 5 µM. At day 5, medium was changed to the first basal medium. At day 7, medium

was changed to the second basal medium. Thereafter, medium was changed every 3 days until cell

harvest.

3.6.4.3 Embryoid body formation

After cell harvesting, hiPSCs are replated onto Matrigel-coated plates for further Embryoid Body

formation. After 80%-confluence, cells are recovered resorting to EDTA cell passaging, Afterwards,

1x106 cells/well are plated on a ULA 6-well plate, and cultured on the respective culture media from

which they are derived from the dynamic conditions. On the first day, the culture medium is

supplemented with 1:200 (v/v) PenStrep and 1:1000 (v/v) of ROCKi. After 24h, 80% of the culture media

volume is replaced with fresh medium without ROCKi. After 2 days of cell expansion, 80% of the

exhausted medium is replaced by EB differentiation medium every other day for 28 days. The EB

differentiation medium was composed by 77% (v/v) of KO-DMEM, 20% (v/v) FBS, 1% (v/v) non-essential

amino acids, 1%(v/v) PenStrep, 1%(v/v) L-glutamine. On the 29th day of differentiation, the EBs are

collected to a falcon and dissociated by the action of Accutase for 5 min at 37ºC. To stop the reaction,

2x of the Accutase volume of EB medium was added to the falcon, which is then centrifuged at 1,500xg

for 3min. After centrifugation, the supernatant is discarded, and cells are resuspended in EB medium

and replated on laminin-coated plates. The medium is replaced every other day for fresh EB medium

up to 7 days, after which the replated cells are fixed with 4%(v/v) PFA. Immunocytochemistry was

performed to confirm the presence of the three-germ layer-derived progeny. The antibodies used for

this analysis can be seen in table 4.

Table 5 – Antibodies and respective dilutions for the immunocytochemistry analysis of cells from the three-germ layers derived from the embryoid body assays. SOX 17, TUJ1 and α-SMA were used for the analysis of endoderm, ectoderm and mesoderm, respectively.

Marker Germ layer Primary antibody Secondary antibody

Antibody Dilution Antibody Dilution

TUJ1 Ectoderm Mouse IgG (Covance) 1:1000 Goat anti-mouse IgG Alexa 564 1:500

α-SMA Mesoderm Mouse IgG (Dako) 1:1000 Goat anti-mouse IgG Alexa 564 1:500

SOX17 Endoderm Mouse IgG (R&D Systems) 1:500 Goat anti-mouse IgG Alexa 564 1:500

3.7 Statistical analysis

Statistical analysis was performed using GraphPad Prism 6 software. When appropriate,

statistical analysis was performed using two-tailed nonparametric tests, as Mann-Whitney test for

independent samples. The software ranks the values from low to high and then the P-value was

computed, which depends on the discrepancy between the mean ranks of two data groups. Error bars

represent the standard error of the mean (SEM). When statistical analysis was applied, at least two

independent samples were evaluated. A p-value less than 0.05 was considered statistically significant.

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33

4. Results and Discussion

4.1 Expansion of hiPSCs under static conditions using both polystyrene and dissolvable microcarriers

The use of Synthemax-II-coated dissolvable microcarriers (DM-SII) has not yet been reported

for the expansion of hiPSCs. Initially, a screening under static conditions was performed to assess cell

adhesion and fold increase of hiPSCs onto this type of microcarriers. Therefore, different combinations

of coatings and culture media were tested to evaluate the previous parameters (Table 1). The system

developed by Badenes et al. was used as a control system of hiPSC expansion under xeno-free

conditions [86].

Regarding the hiPSCs expansion with plastic microcarriers (PSM), cells adhered to the

vitronectin-coated surface with a 73±6% yield (Figure 7A). Cell adhesion can be observed in bright-field

microscopy images (Figure 7B). This percentage of adhesion was within expectations, as vitronectin

presents itself as an ECM glycoprotein, which promotes cell adhesion via αvβ5 integrins [96]. Moreover,

the adhesion yield was very similar to the one reported by Rowland et al under 2D-configurations [96].

Cells were cultured in this platform until day 5. Bright-field microscopy images also show that the

attached cells were able to grow on the PSM-VTN surface. Additionally, direct quantification shows a

4.95±0.5-fold increase in cell number (Figure 7A), which demonstrated a significant growth in cell

population. According to the literature, this was expected as Badenes et al. reported a 6.6±1.0-fold

increase for the same platform under static conditions. Despite the differences, the value obtained in

this experiment is not significantly different from what was reported, which validates the suitability of this

method to expand hiPSCs [86].

Regardless of the previous results, the focus of this work is the expansion of hiPSCs using

dissolvable microcarriers. Therefore, hiPSCs were cultured onto these microcarriers using different

coatings and culture media combinations. Cells were daily monitored through bright-field microscopy,

which demonstrated cell adhesion to the beads, and further cell growth, regardless of the combination

(Figure 7B). Direct cell quantification shows that the highest adhesion yield was observed in Matrigel-

coated DM (93±8%) followed by DM-SII (71±3%), when both were cultured on mTeSR™1.

Nevertheless, the cell fold increase after 5 days of culture for these two combinations was very similar

(4.42±0.6 and 4.39±0.44, respectively). There are no references in the literature of DM being used as

an expansion scaffold for hiPSCs. However, these results were expected as the use of these substrates

and culture media has been proven to support hiPSCs growth. For instances, Matrigel was firstly used

as a substitute for feeder-cells in 2D-cultures, since it contains ECM components like laminin and

collagen [61]. Its use as a microcarrier coating is also presented in the literature, with Bardy et al

achieving cell densities close to 1.3x106cells/mL and a 7.7±0.2-fold increase, under static conditions

[83]. The results obtained for DM-Mat mT1 demonstrated that the cell density achieved ranged from

34

6.83 – 8.15x105 cells/mL, with an average of 6.69x105 cells/mL over 5 days of expansion. The results

hereby obtained demonstrate that DM-Mat supported the expansion of hiPSCs under static conditions.

Nevertheless, to fairly compare these results with the method developed by Bardy et al., the same

expansion period should be tested.

Figure 7 – Expansion of hiPSCs under static conditions using both polystyrene (PSM) and dissolvable microcarriers (DM). (A) From left to right: Cell adhesion yield and cell fold increase for all the tested combinations of microcarriers, coatings and culture media. (B) From left to right: Bright-field microscopy images from day 1 and day 5 of the previous combinations. Maximum intensity projection of confocal microscopy images of the pluripotency markers for the expanded cells. The nuclei were counterstained with DAPI. Scale bar: 132µm. Abbreviatures: vitronectin-coated polystyrene microcarriers and E8®medium (PSM-VTN E8); Matrigel-coated dissolvable microcarriers and mTeSR™1 medium (DM-Mat mT1); Synthemax-II

35

coated dissolvable microcarriers and mTeSR™1 medium (DM-SII mT1); Synthemax-II coated dissolvable microcarriers and TeSR™2 (DM-SII T2).

Regarding the DM-SII and mTeSR™1 combination, the adhesion yield was similar to what was

observed in the case of PSM-VTN. This can be explained by the chemical nature of SII. This synthetic

peptide-copolymer contains the RGD-sequence of the human vitronectin ECM-protein, which promotes

cell adhesion [71]. Additionally, the fold increase was very similar to the previous cases without any

significant difference between each condition. In the literature, Silva et al reported similar results using

the SII-coated polystyrene microcarriers, which demonstrates the versatility of such substrate as a

microcarrier coating [88].

The former combinations do not preclude the absence of a GMP-compliant system, making

them an unviable option for the expansion of clinical-grade hiPSCs. Therefore, TeSR™2 was used as

a xeno-free option for culturing these cells on DM-SII. Through bright-field microscopy images on day

1, it is possible to observe small aggregates being formed without any attachment to the DM-SII beads

(Figure. 7B). This is translated into a slightly lower adhesion yield among all combinations. However,

the final cell fold increase was not affected by this event, since these small aggregates started to adhere

to the DM-SII over the expansion period. Interestingly, the 4.7±0.4-fold increase of such combination

does not vary significantly from the other options, which makes it a viable option if GMP-compliant

systems were ever to be considered.

Immunocytochemistry was used to assess the pluripotency phenotypes. The results show that

expanded hiPSCs can maintain their pluripotency after 5 days of static expansion, regardless of

medium, coating and microcarrier combinations (Figure. 7B). Nevertheless, the expression of hPSC

markers needs further validation with RT-PCR, flow cytometry and differentiation assays to confirm the

pluripotency of the expanded cells.

Despite the promising results, the focus of this work is the scalability of the expansion process

using dissolvable microcarriers. The DM-SII in combination with mTeSR™1 proved to be the chemically

defined culture system with the best cell adhesion yield. Therefore, this combination was used a starting

platform to expand hiPSCs under dynamic conditions.

4.2 Expansion of hiPSCs under dynamic conditions with DM-SII and mTeSR™1 culture media and cell characterization

The results obtained for the expansion of hiPSCs under static conditions using dissolvable

microcarriers demonstrates that this platform is suitable to expand hiPSCs at a laboratory scale. The

next step was to implement a dynamic microcarrier-based culture system in spinner flasks, envisaging

the scalability of the expansion process using the dissolvable matrices.

To achieve the established goals, TCLab cells were previously expanded as a 2D-monolayer

culture. Afterwards, they were transferred to a suspension culture device (spinner-flask) with Synthemax

II-coated dissolvable microcarriers (DM-SII) and mTeSR™1 culture medium. A density of 55,000

cells/cm2 was used for the inoculation of the spinner-vessel. Cells were cultured for a period of 7 days

36

and two samples of 500 μL were daily retrieved for direct quantification of the cell number. After

expansion, samples of cells attached to the beads were retrieved for further pluripotency analysis. The

results are presented in Figure 8.

Figure 8 – Expansion of TCLab hiPSCs under dynamic conditions using Synthemax-II dissolvable microcarriers with mTeSR™1. (A) Total number of cells over 7 days of expansion. Results are presented as the mean average of n=4 experiments. The error bars represent the standard error of mean (SEM) (B) Graphic representation of the adhesion yield and fold increase attained on the first day and throughout the culture, respectively. This is the outcome of the mean of n=4 experiments, with the error bar standing for the Standard Error of Mean (SEM). (C) Bright-field microscopy images of the cells attached to the beads on day 1 and on day 7, respectively. (D) Maximum confocal intensity projection of the immunocytochemistry analysis for expression of intracellular OCT4 and extracellular TRA-1-60 pluripotency markers. (E) Viability test of cells attached to the microcarriers cultured on day 1 and day 7 of the dynamic expansion. Green is the calcein metabolized by the living cells, whereas the dead cells (red) were stained by the ethidium homodimer.

In Figure 8A, it is possible to observe the variation in the total number of cells over the 7 days

of dynamic expansion. This graphic was obtained through direct cell quantifications. Day1 is presented

as the timepoint with the lowest cell number, with a mean of (6.8±0.7)x106 cells, which means

(2.26±0.2)x105 cells/mL. From day 0 to day 1, there is no agitation to promote cell adhesion onto DM-

SII. The attained adhesion yield ranged from 49 – 75% with a mean of 56.6±6.2% (Figure 8B), which is

comparable to what was obtained under static conditions. The adhesion of hiPSC to the DM-SII was

expected due to the chemical nature of Synthemax-II, which simulates the cell-ECM interactions [68,

70, 71].

37

The total number of cells increased as they grew attached to the available surface area. This

correlated with the bright field microscopy images at the final day of culture when compared to the

images obtained in the first day of the culture (Figure 8C). At day 5, the total number of cells ranged

from 1.99 to 3.8x107, with a total mean of (2.86±0.4)x107 cells in the spinner-vessel. This day is

presented as the timepoint with the highest number of cells, after which it started to decline (Figure 8A).

In comparison, Bardy et al. achieved a higher cell density (3.1±0.2x106 cell/mL), when using Matrigel-

coated microcarriers under dynamic conditions. However, this platform precludes the expansion of

hiPSCs under GMP-compliant settings [83].

Regarding the cell fold increase, it varied in a proportional manner, decreasing after day 5

(Figure 8B). This may be due to the existence of cell-to-cell interactions, which leads to the formation of

large cell-bead aggregates (cluster), hampering oxygen and nutrient diffusion (Figure 8C). In figure 8E,

it is presented the result of a viability assay performed on day 7. The presence of a few dead cells within

the cluster is confirmed by the ethidium homodimer staining. This result may be explained by the

limitations of oxygen and nutrient diffusion, which affect cell viability [97]. These observations together

with the direct cell quantifications, suggest that the harvesting procedure should be performed on the

5th day of culture. Nevertheless, at day 7 the cells attached to the DM-SII beads expressed OCT4 and

TRA-1-60 pluripotency markers, which indicated the maintenance of pluripotency characteristics in the

cells cultured in the spinner-flask (Figure 8D).

At day 7, cells were harvested for further pluripotency characterization. These assays comprised

immunocytochemistry, qRT-PCR and flow cytometry analysis of the expression of pluripotency markers.

The results are presented in Figure 9. After harvesting the expanded cells, these were replated onto

Matrigel-coated plates. Human iPSCs maintained their capacity to form colonies, since they stained

positively for OCT4, SOX2 and TRA1-60 pluripotency markers (Figure 9A). Flow cytometry was used to

confirm the previous results. As it is possible to observe from figure 9B, more than 91% of the harvested

cells were positive for the expression of the pluripotency markers SOX2, NANOG, TRA-1-60 and SSEA-

4 after 7 days of dynamic culture. At the beginning and at the end of the culture, mRNA was isolated to

evaluate the expression of pluripotency genes by qRT-PCR (Figure 9C). It was observed the expression

of Oct4 and Nanog pluripotency genes, with further downregulation of differentiation genes, such as

Pax6, Sox17 and T.

The pluripotency of the expanded hiPSCs was also assessed by evaluating their ability to

differentiate into progeny of the three embryonic germ layers. The harvested cells were replated onto

Matrigel-coated plates and finally inoculated in ultra-low attachment plates (ULA) as suspended cell

aggregates. Cells were able to from Embryoid Bodies (EBs). After 4 weeks of culture, the EBs were

replated onto laminin-coated plates. The expression of the three germ lineages was assessed through

immunocytochemistry. In figure 9D, it is possible to observe the expression of specific markers for

endoderm, ectoderm and mesoderm, such as SOX17, TUJ1 and α-SMA, respectively.

The expanded hiPSCs were also directly differentiated towards neural progenitors, based on

the work developed by Fernandes et al. [98]. For that, cells were replated onto Matrigel-coated plates.

When 90 – 100% confluence was achieved, the dual-SMAD inhibition was used to induce neural

commitment. At day 12, neural progenitors were replated onto laminin-coated plates and cultured in

38

N2B27 medium, without the chemical inhibitors SB and LDN. The bFGF was used to enhance the

viability and formation of neuroepithelial rosettes. The structures obtained resemble in vitro the

configuration of the neural tube, from which neurons are derived. In figure 9E, it is possible to observe

a positive result for the immunocytochemistry of SOX2 and apical ZO1 markers, which demonstrate the

polarization of the neuroepithelial cells [98].

Figure 9 – Characterization assay of the TCLAB cell line after expansion under dynamic conditions combining the use of DM-SII and mTeSR™1. (A) Confocal microscopy images of immunocytochemistry for the pluripotency markers: SOX2, TRA-1-60 (Scale bar: 132 µm) and OCT4 (Scale bar: 66 µm) The nuclei were counterstained with DAPI. (B) Flow cytometry analysis of the hiPSCs harvested after 7 days of expansion in the spinner flask. Cells were stained for Oct4 and SOX2 intracellular markers and TRA-1-60 and SSEA-4 cell surface markers. The error bars represent the SEM of n=4 experiments. (C) Quantitative RT-PCR

39

analysis of the pluripotency and differentiation genes of hiPSCs after seven days of culture. mRNA was isolated at the beginning and at end of the culture. (D) Immunostaining showing the formation of cells expressing TUJ1 (ectoderm), SOX17 (endoderm) and α-SMA (mesoderm) after the EB formation and spontaneous differentiation assay with hiPSC cultured in spinner-flask. Scale bar: 100µm for TUJ1 and 50 µm for SOX17 and α-SMA. (E) Confocal microscopy images for immunostaining for SOX2 and ZO-1. The nuclei were counterstained with DAPI. Scale bar: 33 µm.

To prove the standardization of this culture platform, the expansion of another cell line was

performed under the same conditions. Therefore, Gibco hiPSCs were inoculated at the same initial

density as in the previous case. The results are presented in Figure 10.

Figure 10 –Expansion of GIBCO hiPSC line under dynamic conditions using Synthemax-II dissolvable microcarriers with mTeSR™1. (A) Total number of cells over 7 days of expansion. This graphical representation is the mean average of n=4 experiments. The error bars represent the standard error of mean (SEM) (B) Graphic representation of the adhesion yield and cell fold increase attained on the first day and throughout the culture, respectively. This is the outcome of the mean of n=4 experiments, with the error bar standing for the Standard Error of Mean (SEM). (C) Bright-field microscopy images of the cells attached to the beads on day 1 and on day 7, respectively. Scale bar: 100 µm. (D) Maximum intensity projection, obtained with confocal microscopy, for immunocytochemistry of OCT4 (Scale bar: 94 µm) and TRA-1-60 (Scale bar: 66 µm) pluripotency markers.

The cell adhesion yield and cell fold increase values were assessed in the same manner as

previously mentioned for the expansion of The TClab cell line. On day 1, 47±3% of the GIBCO cells

adhered to the DM-SII. As cells grew attached to the beads, (6.4±1.2)x105 cells/mL were achieved on

day 5, which also proved to be the timepoint with the highest number of cells. A mean of (1.9±0.4)x107

total cells were achieved on this day, with an isolated experiment achieving almost 3x107 cells

(Figure10A). The fold increase also varied in the same manner, decreasing after day 5 (Figure 10B).

When comparing these results to the expansion of TCLab cell line, the Mann-Whitney statistical test did

not present any significant difference between the former parameters, namely cell adhesion and fold

40

increase (data not shown). Moreover, the pluripotency phenotype was maintained throughout the

culture, as the expanded cells positively stained for the OCT4 and TRA-1-60 markers, when attached

to the DM-SII beads (Figure 10D).

After the harvesting procedure, cells were characterized to evaluate their phenotype. For that

purpose, expanded hiPSCs were harvested and then replated onto Matrigel-coated plates for further

immunocytochemistry (Figure 11A) and flow cytometry analysis (Figure 11B). Differentiation assays by

using the EB’s assay were also performed to assess the potency of the harvested cells (Figure 11C).

The results of such characterization panel are presented in figure 11.

Figure 11 – Characterization assay of the GIBCO cell line after expansion under dynamic conditions resorting to DM-SII and mTeSR™1. (A) Immunostaining for the OCT4 and SOX2 intracellular markers and TRA-1-60 and SSEA-4 extracellular markers for cells replated after dynamic expansion. The nuclei were counterstained with DAPI. Scale bar: 100 µm. (B) Flow cytometry analysis of the hiPSCs harvested after 7 days of expansion in the spinner flask. Cells were stained for Oct4 and SOX2 intracellular marker and TRA-1-60 and SSEA-4 extracellular marker. The error represents the SEM of n=4 experiments. (C) Immunostaining showing the formation of cells expressing TUJ1 (ectoderm), SOX17 (endoderm) and α-SMA (mesoderm) after the EB formation and spontaneous differentiation assay with hiPSC cultured in spinner-flask. The nuclei were counterstained with DAPI. Scale bar: 100µm.

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The harvesting procedure did not affect the ability of cells to form undifferentiated colonies,

neither their differentiation capabilities. As it is possible to observe from figure 11A, the replated cells

maintained their pluripotent phenotype, since they positively stained for OCT4, SOX2, SSEA-4 and TRA-

1-60 pluripotency markers. Flow cytometry analysis confirmed the expression of these markers for more

than 92% of the expanded cell population (Figure 11B). The differentiation capabilities of expanded

hiPSCs was assessed through the formation of EBs. The positive staining for TUJ1, SOX17 and α-SMA

markers indicated the presence of hiPSCs-derived progeny from the three germ layers (Figure 11C).

Overall, these results proved that the use of DM-SII for the expansion of hiPSCs is cell line-

independent. This is in agreement with previous results described in the literature, as Synthemax-II has

been proven to support the proliferation of hPSCs under static conditions [68-71]. Despite the

differences between hESCs, Jin et al. demonstrated that hiPSCs could be expanded onto Synthemax-

II coated surfaces as efficiently as hESCs. The authors took advantage of the same combination of

substrate and medium (SII and mTeSR™1) to expand cells in 2D-culture. In this work, cells could

maintain their undifferentiated state up to 10 passages, with the cell-SII interactions mediated via αvβ5

integrins [99]. However, this study entailed a static platform which is not easily scalable and devoid from

shear stress of the dynamic cultures, which have been proven to improve homogeneity of the culture

environment and regulate stem cell fate.

Regarding expansion methods for hPSCs, microcarriers have been used as a 3D-platform that

can be further incorporated into suspension cultures. Oh et al. developed a protocol for the expansion

of hESCs, with Matrigel-coated microcarriers. In this study, the two cell lines tested achieved cell

densities close to 3.5x106 cells/ml, which demonstrated the robustness and efficiency of such system

[82]. Analogously, Bardy et al. also reported the use of Matrigel as a microcarrier coating for the

expansion hiPSCs, which yielded a cell density similar to the previous study [83]. In both cases, the

harvested cells exhibited a phenotype consistent with a pluripotent stem cell, as they expressed

pluripotency markers and were able to generate progeny derived from the three germ layers. On the

other hand, other animal-derived substrates have been reported to support hPSCs growth onto

microcarriers. Chen et al. observed that shear-resistant hES cell lines would exhibit a comparable

growth when cultured onto microcarriers coated with both Matrigel and mouse-derived laminin.

Nevertheless, shear-sensitive cells would exhibit a reduced cell growth, viability and pluripotency when

propagated on laminin-coated microcarriers. The authors postulated that the gelatinous thick nature of

the Matrigel substrate would offer a shear protective element [84].

Despite the results, such platforms were not GMP-compliant, which hampers the clinical

translation of the expanded hPSCs. Therefore, other alternatives were developed to counteract such

disadvantage. For instances, Badenes et al reported the use of SII-coated polystyrene microcarriers for

the development of an expansion protocol for hiPSCs. In this work, the authors highlighted the possibility

of integrating this platform into a fully controlled bioreactor configuration [100]. Within this context, Silva

et al used similar microcarriers and mTeSR™1 media for the dynamic expansion of hESCs. The authors

were able to achieve 5x105 cells/ml over five days. At the end of the culture, the harvested cells retained

their undifferentiated phenotype [88]. In comparison, the cell density attained by DM-SII was higher than

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the one reported by Silva et al. Therefore, the use of DM-SII promises to be an efficient alternative for

the expansion of hESCs under defined conditions.

4.2.1 Metabolic profile of TCLab expansion using DM-SII and mTeSR1 media.

The direct measurement of glucose, lactate and glutamine concentrations was performed to

assess the metabolism of the TCLAB cell line, during the 7 days of dynamic expansion. At each daily

culture medium change, a sample of fresh and exhausted medium was retrieved to establish the typical

concentration profile for each nutrient and metabolite (Figure 12).

Figure 12 – Metabolic profile of TCLAB cell line during expansion under dynamic conditions with DM-SII and mTeSR™1. The culture media was daily changed for newly mTeSR™1 culture media. The results are the mean of n=4 experiments, with the error bars standing for the standard error of mean (SEM) (A) Concentration of glucose (mM) over seven days of expansion. (B) Specific rate of glucose consumption over seven days of expansion (µM.cell-1.day-1). (C) Concentration of lactate (mM) over 7 days of expansion. (D) Specific production rate of lactate per day over seven days of expansion (µM.cell-1.day-1). (E) Apparent yield of lactate produced from glucose over seven days of expansion.

Glucose concentration decreased thoroughly due to an increased consumption by the growing

cell population (Figure 12A). Human PSCs require large amount of glucose to fulfill their metabolic

needs, namely cell growth. Consequently, lactate concentration increases over time, as a waste product

(Figure 12C). From day 5 until day 7, lactate concentration raises above 15mM. Chen et al. observed

that hPSCs growth was hampered by lactate concentrations above 20mM [101]. On the other hand,

Horiguchi et al. demonstrated that lactate concentration higher than 15mM would exert an inhibitory

effect upon cell growth [102], which might explain the decrease in cell density after day 5. Overall, these

observations explain, at least partially, why the hPSCs culture media must be changed on a daily basis.

The apparent yield of lactate from glucose (Y´qLac/qGlu) was also calculated (Figure 12E). This

parameter gives an estimation of the glucose fraction converted into lactate. The theoretical maximum

yield is equal to 2, as one molecule of glucose can only give rise to two molecules of lactate via

glycolysis. Overall, the attained Y´qLac/qGlu ranged from 1.5 to 2 (Figure 12E). Kropp et al. also obtained

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similar results for hiPSCs cultured as cell aggregates in a repeated batch strategy [92]. This is consistent

with the majority of glucose being converted into lactate, rather that entering the tricarboxylic acid cycle

(TCA).

In the presence of oxygen, somatic cells direct glucose-derived pyruvate for oxidative

phosphorylation, where electron transfer to oxygen is catalyzed to produce ATP and CO2. In contrast,

hPSCs are highly proliferative cells with immature mitochondria that need to synthetize proper

intermediates for cell growth, namely nucleic acids, proteins and substrates for membrane biosynthesis.

Instead of metabolizing all glucose into CO2, pyruvate is deviated from entering the mitochondria which

slows the rate of TCA cycle. Therefore, hPSCs rely on glycolysis to produce ATP even if there is oxygen

available to conduct the alternative metabolic path. This is known as the Warburg effect, where cells

exploit a less profitable ATP metabolic pathway in order to channel this energy for the biomass formation

[103-105]. As this analysis was only performed for the expansion of TCLab cell line, it would be

interesting to see if the same effect would be observed on Gibco cell line expansion.

4.3 Expansion and characterization of TCLab cell line under xeno-free conditions.

From the previous results, it was possible to conclude that hiPSCs were able to grow onto the

DM-SII, maintaining their phenotype. It was also proven that this expansion is cell line independent, as

two hiPSC lines were tested. Despite the positive results, the previous combination of DM-SII and

mTeSR™1 did not comply with GMP conditions. The mTeSR™1 culture medium contains bovine serum

albumin. Therefore, the use of TeSR™2 in combination with DM-SII was evaluated for the expansion of

clinical-grade hiPSCs, as this culture media is free of animal proteins [57]. The results of this expansion

experiment are presented in Figure 13.

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Figure 13 Expansion of TCLab hiPSC line under dynamic conditions using Synthemax-II dissolvable microcarriers with TeSR™2. (A) Total number of cells over 7 days of expansion. This graphical representation is the mean average of n=4 experiments. The error bars represent the standard error of mean (SEM) (B) Graphic representation of the adhesion yield and fold increase attained on the first day and throughout the culture, respectively. This is the outcome of the mean of n=4 experiments, with the error bar standing for the Standard Error of Mean (SEM). (C) Bright-field microscopy images of the cells attached to the beads on day 1 and on day 7, respectively. (D) Maximum intensity projection obtained with confocal microscopy of the immunocytochemistry results for intracellular OCT4 and extracellular SSEA-4 pluripotency markers. (E) Representative images from cell-viability assays (Calcein, live cells in green; Ethidium homodimer, dead cells in red). Scale bar: 100 µm.

In Figure 13A, it is possible to observe the total number of cells over 7 days of expansion using

DM-SII along with TeSR™2. As in the previous cases, an initial cell density of 55,000 cells/cm2 was

used to inoculate the spinner-flask. At day 1, the attained 61±4.2%-adhesion yield proved to be higher

than what was observed for the expansion with mTeSR™1. Despite the results, Mann-Whitney statistical

test demonstrated a p-value of 0.3429, therefore, no significant difference was observed between the

two conditions (data not shown). The chemical nature of Synthemax-II explains the cell adhesion to the

DM surfaces, as it simulates the cell-ECM [68, 70, 71].

As cells grew attached to the DM-SII surface, the 4th day was proven to be the timepoint where

the highest number of cells was achieved. On this day, the value ranged from 1.36 – 2.26x107, with a

mean of (1.87±0.2)x107cells in the spinner-flask. Interestingly, the total number of cells started to

decrease thoroughly only after day 6, with a cell density of (5.56±0.8)x105cells/mL being achieved at

the end of the culture (day 7). The previous combination of DM-SII and mTeSR™1 yielded a higher cell

density over the same period, with the Mann-Whitney statistical test providing a p-value lower than 0.05,

which demonstrates significant differences between the two expansion conditions. Despite the results,

45

the use of one platform over the other depends on the biomedical applications that is intended for the

expanded hiPSCs. If clinical use as cell therapies is to be considered, then the use of TeSR™2 over

mTeSR™1 is preferable as the former avoids the use of xenogeneic components.

Characterization assays demonstrated the maintenance of a pluripotent phenotype for the

hiPSC while being expanded on DM-SII and TeSR™2. Both nuclear (OCT4) and surface (SSEA-4)

markers were expressed by cells attached to the beads (Figure 13D). After the harvesting procedure

and replating, cells formed undifferentiated colonies that positively stained for OCT4, SOX2 and SSEA-

4 pluripotency markers (Figure 14A). Flow cytometry analysis confirmed the maintenance of a

pluripotent phenotype, as the results showed more than 92% of expanded cells expressing SSEA-4

(92±2%) and TRA-1-60 (95±2%) extracellular markers. Additionally, the expression of intracellular

markers was also measured, with 89±4% of cells expressing SOX2 marker whereas 75±10% of cells

expressed OCT4 marker (Figure 14B).

At the beginning and end of the culture, mRNA was isolated for further RT-PCR analysis. In

Figure 14C, it is possible to observe the downregulation of differentiation genes, such as PAX6 and T,

at the end of the culture. The maintenance of a pluripotency core network was also confirmed by the

upregulation of OCT4 and NANOG genes. Nevertheless, SOX17 gene was found to be slightly

upregulated in expanded hiPSCs, which is consistent with hPSCs-derived endodermal progeny. In the

literature, the differentiation towards definitive endoderm is coupled with the decrease in NANOG

expression [106-108]. Therefore, the results hereby presented are not consistent with a differentiation

state of expanded hiPSCs.

46

Figure 14 – Characterization assay for the TCLAB cell line propagated onto DM-SII and TeSR™2. (A) Confocal microscopy images of the immunocytochemistry for SOX2, OCT4 intracellular markers and SSEA-4 extracellular marker. The nuclei were counterstained with DAPI. Scale bar: 94 µm (B) Flow cytometry analysis of the hiPSCs harvested after 7 days of expansion in the spinner flask. Cells were stained for Oct4 and SOX2 intracellular marker and TRA-1-60 and SSEA-4 extracellular marker. The error represents the SEM of n=4 experiments. (C) Quantitative RT-PCR analysis of the pluripotency and differentiation genes of hiPSCs after seven days of culture. mRNA was isolated at the beginning and end of the culture. The error bars represent the SEM of n=4 experiments for the pluripotency genes, where the differentiation genes were the outcome of n=3 experiments. (D) Immunostaining showing the formation of cells expressing TUJ1 (ectoderm), SOX17 (endoderm) and α-SMA

47

(mesoderm) after the EB formation and spontaneous differentiation assay with hiPSC cultured in spinner-flask. The nuclei were counterstained with DAPI. Scale bar: 100µm (E) Confocal microscopy images for the immunostaining of cells expressing SOX2 and apical ZO-1 neural progenitor markers. The nuclei were counterstained with DAPI. Scale bar: 33 µm. (F) Immunostaining of cells expressing cTnT cardiac marker of hiPSCs differentiated into cardiac marker, after seven days of dynamic expansion. The nuclei were counterstained with DAPI. Scale bar: 50 µm.

The pluripotent phenotype of the expanded hiPSC was also assessed through spontaneous

differentiation in EB’s. After the formation of EB’s, these were replated onto laminin-coated plates for

further immunocytochemistry analysis. In Figure 14D, it is possible to observe the expression of SOX17,

TUJ1 and α-SMA, which confirmed the ability of harvested hiPSCs to differentiate into cells originated

from the three germ layers. Neural progenitors were also obtained through direct differentiation. The

Dual-SMAD inhibition protocol led to the formation of polarized cells, which expressed SOX2 and apical

ZO-1 markers (Figure 14E). As it was previously mentioned, these structures correspond to neuro-

epithelial rosettes, which recapitulates the neural tube formation in vitro [98, 109]. Likewise, expanded

cells were also directly differentiated towards cardiomyocytes (Figure 14F). Expanded hiPSCs were

replated onto Matrigel-coated plates for further cardiac differentiation. At day 12, cells started to

spontaneously contract, which is the first indicator of a successful differentiation protocol. Other studies

using to the same protocol reported the first beating cells between day 8 or 10, which may be due to the

different cell lines used [110]. On day 15, cells were fixed for further immunocytochemistry analysis,

which positively stained for the cardiac troponin T marker (Figure 14F). Overall, the characterization

assays could prove the maintenance of the phenotype of the expanded hiPSCs, under xeno-free

conditions.

The preliminary results hereby obtained, demonstrated that hiPSCs can be expanded on DM-

SII under xeno-free conditions. Moreover, expanded cells would maintain their phenotype throughout

the culture, being able to differentiate into progeny of the three germ layers.

According to the literature, there are two xeno-free methods reported for the dynamic expansion

of hiPSCs. Fan et al. used polystyrene microcarriers coated with cation poly-L-lysine and vitronectin, in

combination with TeSR™2 media. The authors attained a 38.7±6.6%-yield in terms of cell adhesion

(n=3), which was even lower when culturing hiPSCs clumps at the same cell-to-bead ratio. When

comparing these results to the use of dissolvable microcarriers, the authors could achieve higher cell

densities (2x106cells/ml). Nevertheless, this was the outcome of five microcarrier passages, where the

authors removed the clusters from the spinner and added new microcarriers under static conditions in

the presence of mTeSR™1 culture medium [87]. On the other hand, a different hiPSC line was used,

which may also influence the outcome of such experiments.

Another reported method consisted on the use of vitronectin-coated polystyrene microcarriers

(PSM-VTN) in combination with E8 culture medium. Badenes et al. optimized the use of this type of

microcarriers through a three-level factorial design. The highest cell density reported by the authors was

of 1.4x106 cells/mL after ten days of culture [86]. Nevertheless, both DM-SII and PSM-VTN platforms

achieved almost 20x106 cells in the spinner flask, after 4 days of culture.

The differences between the results obtained and these two platforms, must be due to the

different cell lines used, as well as the lack of an optimized protocol that better exploits the use of

dissolvable microcarriers. Most importantly, the same three-level factorial design carried by Badenes et

48

al., should be performed for the system here presented, using DM-SII and TeSR™2, to obtain the

optimal expansion conditions, namely in term of agitation and seeding cell densities.

The previous studies show a feasible protocol for the xeno-free expansion of hiPSCs. However,

the authors did not study the process of cell harvesting from the microcarriers. This crucial aspect of

stem cell bioprocessing will be discussed further on.

4.4 Growth kinetics and Cell Harvesting.

The results hereby obtained, demonstrate the efficiency of using DM-SII to expand hiPSCs

under xeno-free conditions. Growth kinetic parameters were also analyzed, such as the specific growth

rate (µ, day-1) and the doubling time (t2, day) and productivity (cells.mL-1.day-1), which entailed five days

of exponential growth phase. The results can be observed in table 5.

Table 6 – Growth kinetic parameters analyzed for the expansion of two hiPSC lines, using DM-SII. The results are the mean average of n=4 experiments, with the error representing the SEM.

The Mann-Whitney statistical test showed no significant statistical differences between the

parameters of the different studied conditions, as the p-values were higher than 0.05 (data not shown).

Therefore, the use of DM-SII proved to be a suitable microcarrier type for efficient incorporation under

xeno-free conditions.

After being cultured in the spinner-flask, the hiPSCs were recovered using a harvesting

procedure adapted from Nienow et al. [93]. In this study, the authors resorted to a short period of intense

agitation, coupled with an enzyme to detach the expanded cells from the microcarriers. In the case of

DM-SII, the harvesting solution was a mixture of EDTA and Pectinase to promote the respective

destabilization and further dissolution of the PAG-matrix. Accutase was used as a detaching agent to

promote cell dissociation. In the case of polystyrene microcarriers (PSM), the harvesting protocol was

similar with the difference that only Accutase was used. Moreover, a filtration mesh was used to

physically separate the cells from the beads. To quantify the recovery yield, cells were counted before

and after the harvesting protocol. The results can be observed in figure 15.

Cell line TCLAB GIBCO

Culture medium mTeSR™1 TeSR™2 mTeSR™1

Specific growth rate (day-1) [3.3±0.4]x10-1 [2.4±0.5]x10-1 [2.7±0.3]x10-1

Doubling time (day) [2.16±0.2]x100 [3.39±0.6]x100 [2.66±0.3]x100

Productivity (cells.mL-1.day-1) [9.5±1]x104 [7.9±1]x104 [6±0.9]x104

49

Figure 15 – Graphical representation of the Harvesting yield for different hiPSCs lines cultured on dissolvable and polystyrene microcarriers. The protocol developed by Nienow et al. [93] was adapted for the harvesting of the expanded cells. The results for the harvesting yield resorting to polystyrene microcarriers are the outcome of a single experiment, whereas for DM is the mean of n=7 experiments. The error bar represents the standard error of mean

Suspension cultures proved to yield relevant numbers for disease modelling, drug screening

and even cell therapies. Consequently, several groups have been developing PSM-based platforms for

the xeno-free expansion of hiPSCs [86, 87, 100]. However, current protocols often give little focus to the

downstream processing. Nienow et al. highlighted the equal importance of cell proliferation, as well as

a harvesting procedure that can be effectively scaled-up. The authors defined harvesting as a two-step

process. The first step consists on the cell-bead detachment, whereas the second is the separation

technique (centrifugation/filtration) that leaves cells in suspension without the presence of microcarriers.

Enzymatic dissociation has been reported to efficiently detach cells from the beads [93]. Regarding the

use of polystyrene microcarriers, the prior cell dissociation envisages the use of a filtration unit

proceeding the bioreactor unit. From the results obtained, this unit operation yielded 36% of the cell

content inside the spinner-flask (Figure 15). Regarding the use of DM-SII, the harvesting procedure was

integrated into the spinner-flaks, which ensure the scalability of the downstream processing. Moreover,

the harvesting yield for all cell lines was above 90%, being considerably higher than what was attained

by the alternative method. It should be noticed the need for further experiments, as the filtration yield

was the result of an isolated experiment.

In the literature, Fan et al. specified that the use of biodegradable matrices would be

advantageous as a microcarrier scaffold, since it would reduce steps of downstream separations of cells

from the beads, and thus decreasing the overall cost [87]. Regarding the expansion of hMSCs, other

studies resorted to thermo-responsive polymers that need further validation on hPSCs model.

Nevertheless, the use of DM-SII proved to be advantageous over polystyrene microcarriers, not only

because similar cell densities were achieved under defined conditions (data not shown), but also,

because the majority of cells were recovered without losing their core properties.

50

51

5. Conclusion

In the field of regenerative medicine, hiPSCs lend themselves as extremely valuable assets.

Not only they are suitable for human disease modelling and drug screening, but also, they could

potentially serve as cell-based therapies. However, the latter application of hiPSCs is hampered by the

lack of a reproducible method for scalable production of clinically-relevant cell numbers, under xeno-

free conditions. Several approaches have been developed over the years. The use of polystyrene

microcarriers proves to be an efficient platform. However, little focus has been given to the downstream

processing, which often leads to cell losses and reduced viability.

Regarding the use of dissolvable microcarriers, the static screening demonstrated that different

combinations of coatings and culture media, would prompt a good cell adhesion yield and fold increase

as efficiently as previously established platforms. Additionally, expanded cells could maintain their

pluripotency and differentiation potential, as it was confirmed by immunocytochemistry assays.

The dynamic expansion with DM-SII and mTeSR™1 yield 56±5% in terms of cell adhesion.

Moreover, as cells grew attached to the available surface are, high cell densities were achieved, with

(2.86±0.4)x107 cells being present after 5 days of culture. The results were found to be reproducible

with another hiPSC line. Regarding the metabolic pathway followed by expanding hiPSCs, further

analysis is needed, namely the direct measurement of glutamine and ammonium for both cell lines.

Nevertheless, envisaging the clinical applications of expanded hiPSCs, mTeSR™1 was replaced by the

xeno-free alternative, the TeSR™2 culture media. The results demonstrated the effective translation of

DM-SII into a xeno-free culture system. When compared to the previous conditions, cell expansion

yielded a lower cell density, which may be explained by the lack of an optimized expansion protocol.

In terms of the downstream processing, the cells harvested from polystyrene microcarriers were

subjected to a filtration step. This unit operation achieved a 36%-harvesting yield, which is considerable

lower than the 95±2% of cells recovered from DM-SII. Moreover, the latter harvesting protocol was

integrated into the spinner-flask, which suppresses downstream processing steps, such as filtration, and

thus reducing the overall costs. From the characterization of the expanded hiPSCs, pluripotency

maintenance was confirmed by immunocytochemistry, flow cytometry and qRT-PCR. The differentiation

capabilities were also demonstrated by the spontaneous differentiation into cells derived from the three

germ layers – ectoderm, mesoderm and endoderm. Additionally, for both dynamic conditions using

mTeSR™1 and TeSR™2, the replated cells were directly differentiated towards neural progenitors and

cardiomyocytes.

Overall, the use of DM-SII under defined and xeno-free conditions achieved rather similar results

to the platform resorting to polystyrene microcarriers. Nevertheless, the use of DM-SII prove to be

advantageous over the established platform, as it presented an efficient and integrated bioprocess for

the harvesting of expanded hiPSCs. Other advantages comprise, the relatively easy manipulation of

52

such microcarriers, as they only need a hydration step prior to utilization, and its transparency, which

facilitates the observation of attached cells.

53

6. Future Work

The preliminary results hereby presented, demonstrated the feasible expansion of hiPSCs

resorting to SII-coated dissolvable microcarriers. When compared to polystyrene microcarriers, the

harvesting procedure yield higher cell numbers in a cost-effective manner, as it eliminated the filtration

step.

Nevertheless, to establish a reproducible and xeno-free expansion, this protocol would benefit

from further improvements. For instances, Badenes et al. performed a three-level factorial design [86],

which should also be performed for the use of DM-SII beads. With this analysis, the expansion protocol

would be optimized in terms of initial seeding densities and the agitation throughout the culture.

Regarding the metabolic profile of the expansion method, further analysis is in need to confirm which

pathway is carried by the growing cell population. Within this context, the concentrations of glutamine

and ammonium should also be directly measured, as these are nutrients and waste products of the

hPSCs cell metabolism, respectively. Moreover, this analysis should be performed for all the dynamic

conditions tested in this work, namely the culture of GIBCO and TCLab using mTeSR™1 and TeSR™2,

respectively.

The harvesting protocol would also benefit from an optimized agitation that promoted an efficient

detachment of expanded cells from the DM-SII beads. In parallel, a protease-free method should be

developed, as it would decrease the overall cost for the downstream processing. Regarding the

harvesting procedure for the polystyrene microcarriers, the results obtained should be further validated,

as these were the outcome of a single experiment. It would also be important to complement the

characterization panel of expanded cells with further analysis, namely the alkaline phosphatase,

karyotyping and the formation of teratomas in immunocompromised mice.

Another important aspect is the scalability of the expansion platform. Kropp et al demonstrated

the use of single-use instrumented stirred-tank bioreactors for the expansion of hPSCs as cell

aggregates [92]. In this study, the perfusion feeding strategy achieved higher cell densities [92]. Within

this context, the use of DM-SII could be incorporated in the same type of bioreactors, coupled with a

comparison between repeated batch and perfusion feeding strategies. Analogously, it should be

envisaged the incorporation of a differentiation stage proceeding the expansion of hiPSCs. This would

reduce the risk of contamination and labor-intensive tasks, as media exchanges can be fully automated

in bioreactors. Ultimately, the integrated bioprocess resorting to DM-SII – expansion, differentiation and

harvesting – should be automatically performed in one closed system and, most importantly, in

compliance with GMP-guidelines.

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