A Novel Strategy for the Expansion of Peripheral Blood Stem Cells in Autologous Patient Serum, including a Proposed Design for Automation Ex Vivo

PAUL FADUOLA

A THESIS SUBMITTED IN PARTIAL FULFILMENT OF

THE REQUIREMENTS FOR THE DEGREE OF

DO C T OR O F PHI L OSOPH Y

IN

STEM CELL BIOLOGY

PANAMA COLLEGE OF CELL SCIENCE

2014

DEDICATION

To my beautiful family, i love you all.

A Novel Strategy for the Expansion of Peripheral Blood Stem Cells in Autologous Patient Serum, including a Proposed Design for Automation Ex Vivo

Paul Faduola

Stem Cell Biology

Panama College of Cell Science

Abstract

Stem Cells are now the favorite cells used for cell therapy in many disease conditions

including spinal cord injury, stroke, Alzheimer’s disease, Parkinson’s disease, and

several other problems. They are also a standard of care in the treatment of some

selected patients with cancer after high-dose chemotherapy (HDC). This is because of

their potential to repair and regenerate damaged cells, tissues and organ. The bottle

neck to the clinical application of Peripheral Blood Stem Cells (PBSCs) has been the

low level of the concentration recovered after apheresis that is mostly insufficient to

produce therapeutic efficacy. Ex vivo expansion of PBSC is a logical step to overcome

such problem, but current efforts in this direction are still inefficient.

The purpose of this thesis is to demonstrate the efficacy of scaling up of PBSC in

manipulated autologous serum coupled with the synergistic action of hematopoietic

growth factors (HGFs) in the presence of stromal cell derived factor.

We derived PDGF from autologous serum using cellaid protocol, then supplement the

serum with interleukin 3 (IL-3), interleukin 6 (IL-6), stem cell factor (SCF), granulocytes

colony stimulating factor (G-CSF), megakaryocytes growth and differentiation factor

(MGDF) and stromal cell derived factor (SCDF)

Our result indicates that bench scale expansion of PBSC is possible in autologous

serum. To translate this success into the clinic, the cells need to be expanded in a large

scale. Here we describe a proposed design for automation ex vivo that will allow our

expansion protocol to be translated into machine/device manipulation to allow for

routine clinical application that favors good manufacturing practice

Keywords:Peripheral,Blood,StemCell,Autologous,Serum,Expansion,Ex

vivo,Hematopoietic,Pluripotent,passage,Cytokines,Bioreactor,Transplant.

TABLE OF CONTENT

Abstract ............................................................................................................................3

Table of Contents ............................................................................................................5

Introduction.......................................................................................................................6

Aim of study......................................................................................................................9

Materials and Methods...................................................................................................10

Results............................................................................................................................16

Discussion......................................................................................................................17

Bioreactor System..........................................................................................................17

Moving in-vitro expanded autologous PBSC into the clinic............................................18

Novel elementary strategy for routine clinical application...............................................20

Conclusion......................................................................................................................23

Acknowledgements ........................................................................................................24

Reference.......................................................................................................................25

Tables ............................................................................................................................30

Figures ...........................................................................................................................36

Abbreviation....................................................................................................................47

Introduction Significant progress has been made lately to position stem cell therapy in clinics. Some

stem cell therapies have already been approved by regulatory bodies like FDA while

many others are either in early or late stage of clinical trials. This is not surprising

considering the potential of stem cells to repair and regenerate damaged tissues and

organ (1). Amongst the different sources of stem cells, peripheral blood stem cells

(PBSCs) are now the most commonly used for transplantation in patients above

20years (Fig 1). The advantage of PBSC as the only stem that constantly travel from

the bone marrow to areas that are damaged in the body through the peripheral blood

circulation is creating opportunities for it applications toward systemic diseases. It is

expected that the expansion and differentiation of PBSC into Mesenchymal stem cells,

muscle stem cells or neural stem cells, will provide treatment for incurable conditions

like skeletal dysplasia, Duchene muscular dystrophy and neurodegenerative diseases

like amyotrophic lateral sclerosis (ALS) and Alzheimer’s disease (AD).

When PBSCs were discovered in 1950s, initial thought was that they are non-leukemic

DNA-synthesizing cells capable of self-renewal. These cells were later revealed to be

stem cells that originated from the bone marrow (BM) through a test called shielding

experiment. This experiment demonstrates that after total body irradiation, stem cells

from shielded areas that are rich in hematopoietic stem cells (HSC) re-entered the blood

stream and even replenish the BM. The self-renewal potentials of PBSC became the

point of attraction to scientists. Other experiments like post radiation parabiosis and

cross circulation experiments in large animals were performed to verify this regenerative

potential. They soon came to the conclusion that PBSCs were indeed capable of

replenishing the BM after chemotherapy or radiotherapy. This discovery means that

white blood cells (WBCs) could be recovered from whole blood with the possibilities that

PBSCs will be present in them. Because of the ease of collection, peripheral Blood

Stem Cell Transplantation (PBSCT) was expected to be widely acceptable and

applicable. This expectation did not materialize early due to low concentration of the

stem cells in the blood stream. A breakthrough came in timely with the invention of a

continuous-flow apheresis technology called NCI-IBM blood cell separator in the

1960s.The machine made it possible to process more of the patient’s blood to allow

more of these stem cells to be separated. This was thought to be a crucial step in

getting PBSC into the clinic, but it was not to be because the stem cell recovery from

peripheral blood was still less than the recovery from BM. Though attempts at

transplanting these cells were done, initial outcomes from transplants failed with no

recovery of granulocytes or platelets two months post transplantation. Concerns were

immediately raised about the self renewal properties and proliferative ability of these

cells. This engraftment problem was soon linked to the dose of stem cells being infused.

Another attempt at improving the amount of PBSC available for transplant was achieved

using liquid nitrogen to store these cells until a sufficient dose is harvested. Immediately

after this possibility came to light, successful transplants were achieved with fast and

complete replenishment of the hematopoietic system (2- 4).

Even though the collection of PBSC was less invasive, collecting therapeutic dose

required several apheresis to be performed making the procedure laborious. To address

this challenge, ways to improve the concentration of the stem cells temporally before

apheresis were considered. One of the ways adopted was the use of hematopoietic

growth factors to increase the concentration in the peripheral blood. This was made

possible by discovery that granulocyte colony stimulating factor (G-CSF) and

granulocyte-macrophage colony-stimulating factor (GM-CSF) initially used to manage

the side effects resulting from the reduction of blood cells due to the myeloablative

treatment can also mobilize significant amount of CD34+ hematopoietic stem cell from

the BM to the peripheral blood (5). Current efforts have aimed at ex vivo expansion to

shorten the time for effective repopulation in vivo. This includes the use of cytokines (6-

13), stromal feeder layers or extracellular matrix culture (14-16), regulatory pathway

manipulation (17-21) and chemical compound supplementation (19). None of these

approaches has effectively allowed for clinical scale ex vivo expansion of PBSC.

The time has come for PBSC ex vivo expansion to be translated into bioreactor system

for automated processing in community health centers. This step is necessary to mimic

in vivo environment effectively so that cancer patients and patients needing stem cell

therapy can have sufficient cells for transfusion (22). In cancer patients for example, not

all of them have benefitted from the conventional form of PBSCT. This is because

patients respond differently to mobilizing agent in term of PBSC expansion in the blood

stream. While some patients require a maximum of two collections to achieve

therapeutic effect, other respond poorly even after several collections. This poor

response have been linked to advanced disease status with marrow involvement at the

time of mobilization, extensive prior treatment with chemotherapy and/or radiotherapy,

prolonged disease history, low-grade histology and use of fludarabine-containing

regimens prior to mobilization (22-24). In this poor responder group, ex vivo expansion

is necessary to achieve therapeutic effect (25).

In term of stem cell therapy, PBSC harvested from the peripheral blood have shown

promise in the treatment of many systemic diseases. Stem cells have been expanded

and differentiated into other cell types and used to replace tissue that is damaged by

disease or injury. This form of therapy has been used to replace cells damaged by

spinal cord injury (26-28), stroke (29-30), heart damage (31-35), Parkinson’s disease

(36-37) and several other problems. Unfortunately, only little has been achieved in term

of technologies that will enable efficient and consistent ex vivo expansion of stem cells

to scale up its application in clinics.

AIM OF THE STUDY

The purpose of this thesis is to demonstrate the efficacy of scaling up PBSC in

manipulated autologous serum coupled with the synergistic action of hematopoietic

growth factors (HGFs) in the presence of stromal cell derived factor. We derived PDGF

from autologous serum using cellaid protocol, then supplement the serum with

interleukin 3 (IL-3), interleukin 6 (IL-6), stem cell factor (SCF), granulocytes colony

stimulating factor (G-CSF), megakaryocytes growth and differentiation factor (MGDF)

and stromal cell derived factor (SCDF). We included stromal cell derived factor as part

of the supplementation based on the evidence that extracellular matrix proteins

encourage self-renewal and adhesion of HSCs to progenitor cells. Our choice of

manipulated autologous serum is an effort to move PBSC from bench to clinic. Another

purpose of this study is to describe how this expansion protocol can be translated into

machine/device manipulation to allow for routine clinical application that favors good

manufacturing practice.

MATERIALS AND METHODS

Materials

Consumables

FercomApS - Virumvej 90 A - 2830 Virum - Denmark

Falcon Tubes 15 ml & 50 ml Falcon

Forceps

Glass Pasteur Pipettes

Glass slides

Pipette Tips 10µl, 100µl, 500µl,

Plastic Pasteur Pipettes

Scalpels Feather

Syringes, 1 ml, 2 ml, 5 ml Braun

Sterile filters 0.22µm millipore

Surgical Tweezers

Tissue Culture & Multiwell Plates BD Falcon

35-mm Petri dish Nunc

C.C. Obi Nig. Ltd, 161, Herbert Macaulay Street, Yaba, Lagos, Lagos Surgical cap

Surgical mask

Gloves

Equipment

FercomApS - Virumvej 90 A - 2830 Virum - Denmark

Autoclave

DM IRB Leica inverted microscope

Fridge & Freezer Combination

Hot Cabinet Heraeus

Incubator Thermo Scientific

Laboratory Centrifuge Heraeus

Melting-press machine

K-SYSTEMS .KivexBiotecLtd .KlintehøjVaenge1. DK-3460 Birkerød . Denmark

G 185 tri-gas incubator

IVF L426Dual Lamina Flow Cabinet

C.C. Obi Nig. Ltd, 161, Herbert Macaulay Street, Yaba, Lagos, Lagos Waterbath

Hemocytometer

Kits

Amersham Biosciences AB Björkgatan 30, SE-751 84 Uppsala, Sweden

Ficoll-paque

StemCell Technologies. United Kingdom

EasySep® Human CD34 Positive Selection Cocktail

EasySep® Magnetic Nanoparticles

MethoCult SF

JMS Co., Ltd ,Hiroshima, Japan

Cellaid® human serum collection kit

Cell culture media, supplements and reagents

Zayo-Sigma Chemicals Ltd. Jos, Nigeria

Recombinant human interleukin 3

Recombinant human interleukin 6

Recombinant human stem cell factor

Recombinant human megakaryocytes growth and differentiation factor

Recombinant human granulocytes colony stimulating factor

Recombinant Stromal cell derived factor

HTDS International, 3 rue du Saule Trapu – BP 246 91882 Massy Cedex France

Dulbecco’s Phosphate buffered Saline

C.C. Obi Nig. Ltd, 161, Herbert Macaulay Street, Yaba, Lagos, Lagos Wright stain

Giemsa stain

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Methods

Mononuclear Cell preparation

Ethical permission was received before the donor was recruited.10mls of venous blood

was collected from the after mobilization with G-SCF. The concentration of the white

blood cell was determined manually using a hemocytometer and adjusted to 1-2 x

106cell/ml with PBS before 4mL of the cells were layered on 3mL of the Ficoll-Paque

and centrifuged at 400g for 25 minutes at 24°C. The Peripheral Blood Mononuclear Cell

(PBMNC) interface band were harvested, wash twice with phosphate-buffered saline

(PBS). This was followed by CD34+ cells selection using human CD34 selection kit.

Manual EasySep® CD34+ Cell Positive Selection using Purple EasySep® Magnet

They concentration of the PBMNC was adjusted to 2 x108 cells/ml with PBS and placed

in 5mL falcon polystyrene round tubes. 100µl of the EasySep® positive selection

Cocktail was added to 1mL of the cells. This was mixed well and incubated at room

temperature for 15 minutes.100µl of EasySep® magnetic nanoparticles was then added

to 1mL of cells, mixed well and incubated at room temperature for 10 minutes. The cell

suspension was then adjusted to 2.5mL with PBS, mixed thrice in the tube by pipetting

up and down and placed in a magnet (Fig 2) with the cap of the tube off. After 5

minutes, the magnet and the tube were inverted in one motion for 3 seconds to discard

the supernatant and then returned to the upright position leaving the magnetically

labeled cells inside the tube. The tube was then removed from the magnet and the cells

were diluted with 2.5mL of PBS with gentle pipetting up and down. The rinsing of the

magnetically CD34+ labeled cells was continued as detailed in the protocol. The CD34+

positively selected cells were then re-suspended in 2mL of PBS.

Culture Medium Preparation

The culture medium was prepared under a lamina air flow hood (Fig 8) with the same

donor’s serum and separated based on the cellaid protocol. 200mls of venous whole

blood was collected in a bag containing glass beads and incubated at room temperature

for 30minutes with constant gentle agitation to allow for platelet attachment. The serum

was separated from the whole blood by centrifuging at 2500rpm for 10 minutes and heat

inactivated at 560c for 30 minutes. This serum was then used to dilute IL-3 (10ng/mL),

IL-6 (10ng/mL), SCF (100ng/mL), G-CSF (100ng/mL), MGDF (100ng/mL) and SCDF

(100ng/mL).

Bench Scale Ex Vivo Expansion Protocol

The CD34+ positively selected cells were expanded ex vivo in a freshly prepared culture

media described above. It is generally believed that when cells are seeded in lower

concentration, they tend to expand more. We initiated our seeding at a concentration of

5000/mL of the culture media in 16 well culture plates in triplicates. The culture plates

were placed inside a G-185 tri gas incubator (Fig 9) at a temperature of 37oC, 5% CO2

and 5% O2 for 14days.

Assessment of Cell Culture

The culture assessment was done on day 7, 10 and 14 to determine the % increase,

CFU-GM numbers and morphology change of the seeded cells. Cells from at least 2

wells were washed once in PBS and used for the analysis on selected days.

Percentage Increase Check

The % increase was determined after filling the hemocytometer chamber with a pipette

containing the washed cells. A cover slip was properly mounted on the hemocytometer

to allow the fluid containing the cells to enter the chamber by capillary action when the

pipette came in contact with the edge of the cover slip. Care was taken to ensure air

bubbles were not formed and the fluid did not overflow into the trenches. The cells were

counted as described in the hemocytometer manual. The % increase was obtained by

comparing the concentration of cells seeded before and after culture.

CFU-GM Assay

This assay was used for the measurement of clonogenicity in the PBSC culture. A

35mm petri dish containing MethoCult SF was used. 1x 103 expanded cells (day 7)

,1x104 expanded cells (day 10) and 1x106 expanded cells (day 14) were seeded on

cytokine supplemented with methylcellulose and cultured in a G 185 tri-gas incubator at

37°C ,5% CO2, 5% O2 for 14 days. The culture was done alongside 700 freshly isolated

CD34+ cells/plate in methylcellulose for 14days as control. The numbers of CFU-GM

were counted in an inverted light microscope (Fig 10).

Morphology Assessment

Morphological evaluation was done on 200 differential cell counts to determine

morphological changes. This was achieved by staining slides prepared from the cells

with Wright-Giemsa stain. The stained slides were assessed for myeloid precursor cells

based on the characteristics summarized (table 1).

RESULT

Percentage increase on seeded cell in culture

The percentage CD34+ recovery in experiment 1 was 32.3%, experiment 2 was 29.5%

and experiment 3 was 35.0. There was a mean percentage increase of 66.7%, 128.7%

and 206% on day 7, 12 and 14 respectively (Table 2).

Rate of CFU-GM Expansion

They mean percentage increase in CFU-GM peak on day 7 by 69.5% above the initial

CFU-GM. We observed a 47.5% and 32.6% decline on day 12 and 14 respectively

(Table 3).

Morphological changes in cultured myeloid cells

Apart from myeloblasts that decreased on day 7 and 12, there was a mean increase in

promyelocytes, myelocytes/metamyelocytes on day 7 and 12 (Table 4).

DISCUSSION

Bioreactor System

Tissue culture flask and well plates have been the mainstay of stem cell culture and

expansion. Their wide acceptance globally was mainly due to the fact that they are

cheap and easy to use. Despite these advantages, automated bio-processing platforms

are better positioned to encourage culture and expansion of stem cells for clinical

applications. This is because they can guarantee efficient, robust and large scale

production.

Bioreactor designs are being explore to allow stem cell expansion in clinical scale. The

efficiency of such a design will depend on its ability to control how cytokines and other

growth factors are fed into the system and how the products of their metabolism are

eliminated. An efficient bioreactor system is expected to mimic in vivo scenario. To

achieve this, the system must have a very good control of aeration, temperature, light

and PH compatible to physiological life. Expansion capacity and quality can be

improved upon by understanding the appropriate mode of introducing the nutrients,

proper concentration and composition of the growth factors to be used for the

expansion. The application of such knowledge in bioreactor system will be a step in the

right direction. Presently, nutrients are introduced into the system in batch, fed batch or

continuous and perfusion. Perfusion strategy is more widely applied because it ensures

continuous renewal of nutrient and removal of metabolites from the system which is a

solid requirement for self-renewal and differentiation of stem cells. The fed-batch

method however has the advantage of supplying the nutrient in a more appropriate

manner than the perfusion strategy thus ensuring that their release and utilization are

better controlled. In this way, less metabolite that is inhibitory to expansion will be found

in the culture system.

Several bioreactor designs like stirred culture vessels (Fig 3), cell culture bags (Fig 4),

bubble column or air-lift vessels (Fig 5), rotary cell culture systems (Fig 6) and

microfluidic devices (Fig 7) have been developed. The bioreactor design that will

improve the expansion of PBSC should have an efficient control of the

microenvironment. Apart from the micro environment, more efforts should be made to

know the growth factors that improve expansion synergistically with the different

designs. Each design has its advantage and disadvantage (table 5), choosing the

appropriate culture design is therefore very critical.

Moving Ex Vivo Expanded Autologous PBSC into the Clinic

Apart from the capacity of stem cells to repopulate, they are also expected to be safe for

patient use. To fulfill this requirement, the expansion must be free of feeder cell, animal

proteins or microbial agents that might contaminate these cells. The expansion of stem

cells have since been done using different cytokine combination and concentration,

autologous serum, serum free media, fetal bovine serum, different mode of culture and

varied initial culture density. The heterogeneous nature of these trials have made

conclusion very difficult considering that one variable could have significant effect on the

outcome. Traditionally, stem cells have been cultured in FBS because they are rich in

attachment, growth factors, nutritional and physiochemical compounds that support cell

growth. However, translating this process to clinics is being hampered by the genuine

fear of contamination with undesirable pathogens such as viruses, mycoplasma, prions,

and other zoonotic agents. The use of serum free media has been suggested as a

possible way to overcome the danger of FBS. They problem with serum free media is

that they may only support expansion for single passage. Even when they are able to

support expansion for multiple passages, the rate of growth is slower. Human serum

has been offered as suitable alternative to the safety ridden FBS and the poor efficiency

serum free media. Types of human serum that have been studied include; autologous,

allogeneic and cord blood. Results from allogeneic human serum have been mixed and

the drive for cord blood serum has been largely hinged on the primitive nature of the

source. Positive outcomes have been reported with autologous human serum. The

argument against the use of autologous serum so far is harvesting enough quantity and

the fact that serum from older patients may not support optimal expansion. These two

concerns can be simply addressed if people are encouraged to cryopreserve their

serum early enough. The major problem limiting the application of human serum is by

far the poor understanding of components in the serum that are inhibitory to the

expansion process. Serum contains growth factors and inhibitors, cytotoxic substances,

differentiation agents, attachments etc. Some of these components support a particular

cell type while they inhibit the growth of other cells. More knowledge of the cytotoxic

substances and other components that interfere with the growth factors and hormones

in the serum is crucial for future progress

Efforts should also be made to optimize the growth factors in autologous serum use for

expansion. Already, the process of serum collection has been manipulated to allow the

derivation of growth factors. An example of this manipulation is the derivation of PDGF

using cellaid. PDGF is a growth factor necessary for the stimulation and expansion of

early progenitors and committed progenitors cells. It is possible that more growth factors

could be sufficiently derived from autologous human serum without the need for

supplementation as more innovative manipulation strategies are explored.

Interestingly, more clinicians and biotechnology companies have begun to look in the

direction of autologous stem cell therapy. This renew interest has opened a new way in

which trial is being done to assess safety and efficacy of stem cell therapy outside the

traditional clinical trial rules. Examples of new ways in which an expansion protocol can

be assessed include patient sponsored studies where the patients pay the bill instead of

the government, open label study and institutional review board approval. This is the

right time to harness innovative approaches that will revolutionize stem cell therapy

globally.

Novel Elementary Strategy for Routine Clinical Application

One reason why stem cell therapy has been somewhat restricted to transplant referral

centers and some few stem cell treatment centers, stems from the fact that stem cell

transplantation where originally done with bone marrow cells. Despite the current reality

that PBSC can be easily harvested than bone marrow cells, our clinical, scientific and

regulatory authorities have sadly turned Luddite. Luddite is a term used to describe

group of English textile artisans who, beginning in the 19th century protested against,

and smashed, new labor saving machinery in the early industrial revolution, and now a

term for one opposed to new technology or new way of doing things.

It is now a standard of care to treat some selected patients with hematological disorder

with high-dose chemotherapy (HDC) using PBSC as support. Apart from cancer

treatment, stem cell therapy can also be used to regenerate damaged tissues and

organ including neurodegenerative diseases, spinal cord injury, heart disease etc. They

availability of stem cell therapy to practicing physicians will certainly benefit patients

who do not have access to this kind of treatment.

In this study, we use autologous serum harvested according to cellaid protocol and

supplemented with growth factors to expand PBSC in bench scale. Our result (table 2, 3

and 4) confirm the potential of autologous serum in the expansion of PBSC. It indicates

that bench scale expansion of PBSC is possible. To translate this success into the

clinic, the cells need to be expanded in a large scale. However, current efforts for large

scale clinical expansion have failed to continue at cell densities above 1.1 - 1.4 x 106

cell/ml. Some reasons suggested for this arrest includes; insufficient nutrients for the

cells in static culture and accumulation of metabolic products as a result of the

expansion. This condition might have produced the inhibitory effect in the culture

system which often promotes cell death. A fed batch system that continually increases

the volume of cultures in order to increase the space for the cells to expand optimally

and dilute away the inhibitory factors is a logical step to overcome the effect. This is

because the increasing culture volume of the fed batch strategy will eventually maintain

a lower cell density, thereby slowing the rate and impact of endogenous factor

accumulation. Incorporating automated fed batch into a system that allows continuous

observation through time lapse monitoring and a control of the physical and chemical

environment have not been adequately explored. Novel strategies incorporating this

should have a culture system where nutrient need, oxygen or pH profiles and culture

parameters known to have crucial influence over stem cell fate is rightly adjusted.

Exploring this strategy is extremely necessary for a successful clinical scale expansion

protocol.

Here, we present our novel clinical scale automated expansion system (Fig 11) which

consists of an optical fiber and sensor, pneumatic valves, cocktail media reservoir, filter

fluorescence spectrometer for optical biomass measurement, a CO2 incubator, time

lapse monitoring device, automated immune-staining device, a PH and temperature

controlling device. The culture setup is mounted inside a CO2 incubator and is

connected to an external valve which controls the flow of pressurized air from a

reservoir containing the cocktail media outside the incubator. The pressure component

was designed to force the cocktail media into a micro-valve pneumatic connection which

then supplies the media through a tube into a culture plate positioned inside an

incubator. Once the dose volume of the cocktail media per pump is known, the precise

flow rate can be established by defining the number of pumps per time unit. It has a

software program designed to signal and take measurement of scattered light through

the fiber optic from a fluorescence spectrophotometer. The scattered light of the culture

provides information regarding the accumulation of inhibitory factors and current nutrient

profile and is controlled by a motorized XY stage so that this information is available on

a fixed time. This software also regulates the CO2, O2 and temperature control box to

provide appropriate conditions inside the incubator through separate optic fibers. Based

on information analyzed by the software from the culture media in the incubating

system, a signal is sent to the valve to pump or halt the supply of nutrient from the

cocktail reservoir depending on the concentration of the inhibitory factors detected or

nutrients available. Time lapse monitoring device was fixed inside the incubating system

to provide a real time picture of the cultured cells so that a closed system is maintained

during culture and expansion. A tube is also positioned close to the culture plate and

directed outside the incubator so that occasionally a small volume of the cultured cell is

aspirated from the plate for automated immune-staining outside the incubator. This

function is also controlled by the software.

Stem cell expanded by our strategy can be translated into clinical settings by using

autologous human serum in preparing the cocktail media. A strategy like ours when fully

developed could encourage the use of autologous serum to expand PBSC in

community health center in accordance with good manufacturing practice.

CONCLUSION

In autologous stem cell therapy, patients donate their own blood cells and serum prior to

therapy and the expanded cells are infused back to the patients when it is needed. We

have demonstrated that autologous serum manipulated to derive PDGF can allow

bench scale expansion of PBSC, a lot more still need to be done to understand how

other growth factors and components that support expansion can be manipulated during

serum harvest. If autologous serum is sufficiently manipulated to derive other factors

that encourage expansion without the need for supplementation with recombinant

growth factors, it will further encourage the availability and reduce the cost of

autologous stem cell treatment in community healthcare centers. This is because all

that will be needed is the automated system that process and expand these cells, since

the serum and the cell to be expanded will be harvested from the patient.

ACKNOWLEDGEMENT

I sincerely thank my director of Admission and Student Affairs, Professor Doctor Piotr

Beck for supervising and advising me during this thesis.

My gratitude also goes to Dr Drake for his valuable comments during my studies.

A big thank you to the team at Nordica Fertility Centre Lagos, were this work was

carried out and to Androcare Laboratories and cryobank for providing the donor.

God bless you.

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24. Ketterer N, et al. Factors associated with successful mobilization of peripheral blood

progenitor cells in 200 patients with lymphoid malignancies. Br J Haematol. 1998; 103:

235:242.

25. Sato N, et al. In Vitro Expansion of Human Peripheral Blood CD34+ Cells.Blood.

1993; 82: 3600-3609.

26.Spinal cord injury. See July 14, 2004 Senate testimony by Dr. Jean Peduzzi-

Nelson.Available at:

http://commerce.senate.gov/hearings/testimony.cfm?id=1268&wit_id=3671.

27.Spinal cord injury .A more extensive testimony. Available at:

http://www.stemcellresearch.org/testimony/peduzzi-nelson.htm.

28.Spinal cord injury .Ms. Laura Dominguez. Available at:

http://commerce.senate.gov/hearings/testimony.cfm?id=1268&wit_id=3673.

29.Stilley CS et al. Changes in cognitive function after neuronal cell transplantation for

basal ganglia stroke, Neurology.2004; 63: 1320-1322.

30.Kondziolka D et al. Transplantation of cultured human neuronal cells for patients with

stroke.Neurology.2000; 55: 565-569.

31.Wollert KC et al. Intracoronary autologous bone-marrow cell transfer after myocardial

infarction: the BOOST randomised controlled clinical trial. Lancet.2004; 364: 141-148.

32.Britten MB et al. Infarct remodeling after intracoronary progenitor cell treatment in

patients with acute myocardial infarction. Circulation.2003; 108: 2212-2218.

33.Perin EC et al. Transendocardial, autologous bone marrow cell transplantation for

severe, chronic ischemic heart failure. Circulation.2003; 107: r75-r83.

34.Stamm C et al. Autologous bone-marrow stem-cell transplantation for myocardial

regeneration.Lancet.2003; 361: 45-46.

35.Tse H-F et al. Angiogenesis in ischaemic myocardium by intramyocardial autologous

bone marrow mononuclear cell implantation; Lancet.2003; 361: 47-49.

36.Love S et al. Glial cell line-derived neurotrophic factor induces neuronal sprouting in

human brain, Nature Medicine.2005; 11: 703-704.

37.Gill SS et al. Direct brain infusion of glial cell line-derived neurotrophic factor in

Parkinson disease. Nature Medicine.2003; 9: 589-595.

38. Pasquini MC, Wang Z. Current use and outcome of hematopoietic stem cell

transplantation: CIBMTR Summary Slides, 2013. Available at: http://www.cibmtr.org.

39. Diagram of Stirred bioreactor operated as a chemostat, with a continuous inflow

(feed) and outflow. Available at http://en.m.wikipedia.org/wiki/chemostat.

40.WAVE BioreactorTM 500/1000 system. Available at :

www.bioprocessonline.com/doc/wave-bioreactor-system-0002.

41.Bubble-Column and Air-Lift Bioreactor. Avalaible at

http://www.metal.ntua.gr/~pkousi/e-learning/bioreactors/page 11.htm.

42.Rotary Cell Culture System-motorized base with power supply and assorted vessels

Available at http://www.genengnews.com/gen-articles/rotating-bioreactors-for-

manufacturing/1899/.

43. Kim, SJ,et al. Microfluidic Automation Using Elastomeric Valves and Droplets:

Reducing Reliance on External Controllers. Small. 2012.

44. Ang M. Ex vivo expansion of hematopoietic stem cells: preclinical studies

and clinical application.Thesis, Univerisity of hongkong.2003.

Tables Table 1.Characteristics of Myeloid Cell Types

Adapted from (44)

Table 2.Percentage increase on seeded cell in culture

Table 3.Rate of CFU-GM Expansion

Table 4.Morphological changes in cultured myeloid cells

Day of

Culture

Initial Cell

Density.

(/ml)

Myeloblasts Promyelocytes Myelocytes/Metamyelocytes

0 5000/ml 36 ±6.2

2.8 ±1.4

1.3 ±0.4

7 5000/ml 8.9±0.3 14 ±3.9

22 ±4.8

12 5000/ml 1.2±0.7 27 ±5.3

41± 8.2

Table 5.Advantages and Disadvantages of Bioreactor Designs

Design Advantage Disadvantage

Stirred Culture Vessels Can operate in batch, fed-

batch, and perfusion and

can be adapted for

differentiation and/or

expansion

The hydrodynamic stress

promoted by stirring

Cell Culture Bags The come as disposable,

single-use, pre-sterilized

bioreactors. This make

them more convenient to

use

They can only be applied to

non-adherent stem cells or

those that grow as

aggregates or on

biocompatible micro-carrier

Bubble-Column and Air-Lift

Bioreactors

They permit high-efficiency

mass transfer with

excellent flow and mixing

properties

There is considerable back-

mixing between gas and

liquid phases, high

pressure drops, and bubble

coalescence

Rotary Systems They provide a well-mixed

environment for cell growth

There is limited control of

aggregate size and

nutrient/gas concentrations

as well as efficient gas

transfer through a silicon

membrane

throughout the vessel.

Necrotic centers can form,

leading to cell death inside

the aggregates.

Concentration gradients

resulting from mass

transfer limitations can

create uncontrolled

microenvironments

Microfluidic Culture

Systems

The microenvironment can

be controlled by adjusting

for example the perfusion

rate, providing a high-

throughput system for

evaluating the effects of

soluble factor concentration

gradients on different cell

processes

There may be high shear

stresses associated with

perfusion and the

continuous removal

of important molecules

secreted by cells

that could ultimately

compromise their

performance

Figures

Fig 1.Unrelated Donor Stem Cell Sources by Recipient Age Adapted from (38)

Fig 2.Photo of EasySep® Magnetic

Fig 3.Stirred bioreactor operated as a chemostat, with a continuous inflow (feed)and

outflow.The rate of medium inflow is controlled to keep the culture volume constant.

Adapted from (39)

Fig 4.GE Healthcare Life Science WAVE BioreactorTM 500/1000 system.

Adapted from (40)

Fig 5.Bubble column and Air-Lift Bioreactor. A)bubble column;b)air lift reactor;c)air lift

with particle separator; packed bed air lift.

Adapted from (41)

Fig 6.Rotary Cell Culture System-motorized base with power supply and assorted

vessels

Adapted from (42)

Fig 7.Microfluidic Bioreactor . Adapted from (43)

Fig 8.K System Lamina Air Flow Hood

Fig 9.G-185 Incubator K-System

Fig 10.Leica DM IRB inverted Microscope

Fig 11.A schematic diagram of our clinical scale expansion system for PBSC

ABBREVIATION BFU-E - Erythroid Burst-forming units

B M- Bone marrow

BSA -Bovine serum albumin

CAFC -Cobblestone area forming cell granulocyte-erythrocyte-macrophage

megakaryocyte colony

CFU-GM -Granulocyte-macrophage colony-forming units

CSF- Colony stimulating factors

DM -Defined media

DPBS- Dulbecco's Phosphate Buffered Saline

EPO -Erythropoietin

FACs -fluorescence-activated cell sorting

FBS -Fetal bovine serum

FCS -Fetal calf serum

FITC- Fluorescein isothiocyanate

G-CSF Granulocyte colony stimulating factor

GM -Granulocyte-macrophage

GM-CSF Granulocyte-macrophage colony-stimulating factor

GMP -Good manufacturing practices

HDC -High dose chemotherapy

HGF -Hematopoietic growth factors

HPC -Hematopoietic progenitor cells

PBSC-Peripheral Blood Stem Cell

DNA-Deoxyribonucleic Acid

HSC-Hematopoietic stem cell

BMT- Bone Marrow Transplant

PBSCT- Peripheral Blood Stem Cell Transplant

ANC-Absolute Neutrophil Count

CB- Cord Blood

IL-1b-Interleukin 1beta

IL-3-interleukin 3

IL-6- interleukin 6

SCF-stem cell factor

PDGF-Platelet Derived Growth Factor

TPO-Thrombopoietin

FGF-1 Fibroblast Growth Factor - 1

MSC-Mesenchymal Stem Cell

PGE2-Prostaglandin E2

HOXB4 �Homebox B4

SALL4-Sla like 4

EPO-Erythropoietin

HS-human serum

tPRP-thrombin-activated platelet releasate in plasma

pHPL- Pooled human platelet lysate

MGDF-megakaryocytes growth and differentiation factor

SCDF- stromal cell derived factor

PBMNC-Peripheral Blood Mononuclear Cell