Universidade de Aveiro
2017 Departamento de Química
CLÁUDIA ISABEL RODRIGUES MARTINS
Micropartículas de laminarina com incorporação de lisados de plaquetas para adesão e expansão celular Platelet lysates loaded laminarin microparticles for cell attachment and expansion
Universidade de Aveiro
2017 Departamento de Química
CLÁUDIA ISABEL RODRIGUES MARTINS
Micropartículas de laminarina com incorporação de lisados de plaquetas para adesão e expansão celular Platelet lysates loaded laminarin microparticles for cell attachment and expansion Dissertação apresentada à Universidade de Aveiro para cumprimento dos
requisitos necessários à obtenção do grau de Mestre em Biotecnologia
Industrial e Ambiental, realizada sob a orientação científica da Doutora Catarina
Custódio, Investigadora Pós-Doutorada de Departamento de Química da
Universidade de Aveiro, e do Professor Doutor João Mano, Professor
Catedrático do Departamento de Química da Universidade de Aveiro.
o júri
Presidente
vogal – Arguente principal
vogal - orientador
Doutora Mara Guadalupe Freire Martins Equiparado a investigador Coordenador do Departamento de Química da Universidade
de Aveiro
Professora Doutora Cláudia Alexandra Martins Lobato da Silva Professor Auxiliar do Instituto Superior Técnico de Lisboa
Doutora Catarina de Almeida Custódio Bolseiro de pós-doutoramento de Departamento de Química d Universidade de Aveiro
agradecimentos
A última parte da tese e com isto a última parte deste percurso.
Em primeiro lugar gostaria de agradecer aos meus pais, irmão e avós por
estarem sempre presentes e por todo o apoio. Por aturarem o mau feitio,
por me fazerem sorrir sempre que o trabalho não corria da melhor forma
e por me fazerem acreditar que era capaz.
De seguida, não posso deixar de agradecer aos meus amigos, a todos
aqueles que a vida académica me deu e a todos aqueles que estão na minha
vida desde antes. Sem o apoio deste nada seria possível. Aqui não posso
esquecer os meus colegas de laboratório. Durante este ano partilhámos
muitas histórias, muitas aventuras. Sem dúvida que sem eles foram muito
importantes durante este ano de tese. “Aqueles que passam por nós não
vão sós, não nos deixam sós. Deixam um pouco de si, levam um pouco de
nós”. Alguém me disse esta frase este ano e sem dúvida agora que chega
o fim faz todo o sentido. Todas as pessoas do grupo COMPASS com quem
me cruzei neste ano de alguma forma marcaram este ano. E duas pessoas
que marcaram de forma única foram os meus orientadores tiveram sem
dúvida um papel central durante este ano. Ainda me lembro da primeira
reunião em que eu mal sabia o que era um hidrogel… Agora que chegou
ao fim, vejo que foi um ano de muita aprendizagem, uma área nova, um
laboratório novo e uma bagagem sem dúvida bem mais vaste de
conhecimentos.
“Escolhe um trabalho de que gostes, e não terás que trabalhar nem um dia
da tua vida”.
palavras-chave
Engenaria de tecidos, microgeis, bottom-up, microfluídica, microcarriers
resumo
A engenharia de tecidos tem estudado a combinação de vários materiais com células com o intuito de solucionar alguns problemas que as atuais terapias não conseguem dar resposta. Umas das respostas passa pelo desenvolvimento de pequenas estruturas, microgéis, que mimetizam a matriz extracelular e podem interagir de várias formas para criar uma estrutura tridimensional complexa. Este trabalho resume as principais técnicas de microfabricação que têm sido usadas no desenvolvimento de microgéis assim como as principais aplicações destas na engenharia de tecidos. Uma dessas técnicas, a microfluídica foi neste trabalho usada para produzir micropartículas de laminarina. Este polímero foi quimicamente modificado, adicionando grupos metacrilato que permitiram a reticulação das gotículas formadas pela microfluídica quando expostas a luz ultravioleta. Esta tecnologia permite encapsular lisados de plaquetas humanos durante a produção das micropartículas. As micropartículas apresentam elevado grau de monodispersão e uma forma esférica com diâmetro médio de 326.6 μm nas micropartículas com elevado grau de modificação e 303.3 µm nas micropartículas com baixo grau de modificação. Estudos de libertação demonstraram que diferentes graus de modificação da laminarina permite obter diferentes perfis de libertação das proteínas encapsuladas. Ao fim de 14 dias as micropartículas com alto grau de modificação tinham libertado 56.89 ± 1.03% da proteína encapsulada enquanto as micropartículas com baixo nível de modificação libertaram 35.31 ± 2.73%. As partículas funcionais foram então testadas como suporte para cultura de células. Os resultados mostraram que as partículas funcionalizadas com RGD e lisados encapsulados permitem uma eficiente adesão e expansão celular. As células cultivadas na superfície dos microgéis promovem ainda agregação dos mesmos em estruturas tridimensionais. Para aplicações clínicas, hidrogéis injetáveis/moldáveis são muitas vezes necessários para preencher defeitos de geometrias irregulares. Estes microgéis de elevada versatilidade podem vir a traduzir-se no desenvolvimento de sistemas de elevado desempenho para a produção de sistemas injetáveis para aplicações em engenharia de tecidos.
keywords
Tissue engineering, microgels, bottom-up, microfluidic, microcarries
abstract
Tissue engineering has studied the combination of diverse biomaterials with cells in order to overcome some limitations of traditional therapies. One of solutions is related with the development of microstructures, microgels, that mimic the extracellular matrix and can assemble to create a complex three-dimensional structure. This work reviews the microfabrication techniques that been used to fabricate these microstructures as well as the main application of these in tissue engineering. One of this technique, microfluidic flow-focusing has been used to produce laminarin microparticles. This polymer was chemical modified, by adding methacrylated groups that allowed the photopolymerization of laminarin droplets when exposed to UV light. This technology allows to encapsulate human platelet lysates during fabrication of microgels by microfluidic. The microparticles shows high monodisperse and spherical shape with an average diameter around 326.6 μm in the microparticles with high degree of modification and 303.3 µm in the microparticles with low degree of modifications. The release studies demonstrate that different levels of modification of the laminarin microparticles allow to obtain different encapsulated proteins release profiles. At the end of 14 days the high methacrylated laminarin released 56.89 ± 1.03% of total amount of protein while low methacrylated laminarin released 35.31 ± 2.73%. The results showed that RGD functionalized particles with encapsulated platelet lysates allow an efficient cell adhesion and expansion. The cells cultured on the surface of microgels promote the assembly in three dimensional structures. For clinical applications, injectable/moldable hydrogels are several times necessaries to fill defects of irregular geometries. This highly versatility microgels can be translated in the development of the high-performance systems to production of injectable systems for applications in tissue engineering
i
Contents
Chapter 1 – Motivation…………………………………………………………………… .1
Chapter 2 - Recent trends on microfabrication of hydrogels ............................................... 4
1. Introduction .............................................................................................................. 4
2. Microfabrication of hydrogel…………………………………………………………4
2.1. Micromolding ............................................................................................... 6
2.2. Bioprinting ................................................................................................... 7
2.3. Microfluidics ................................................................................................ 9
2.4. Spray based technologies ............................................................................ 11
2.5. Superhydrophobic-based platforms for microgel
production……………………………………………….…………………………13
3. Applications of microfabricated hydrogels .............................................................. 14
3.1. Cell laden microgels as building blocks for 3D constructs ........................... 14
3.2. Cell microcarriers ....................................................................................... 16
3.3. High-throughput screening .......................................................................... 17
3.4. Drug Delivery ............................................................................................. 18
3.5. Assembly of microgels ............................................................................... 19
3.5.1. Surface Tension .......................................................................................... 20
3.5.2. Acoustic assembly ...................................................................................... 20
3.5.3. Magnetic assembly ..................................................................................... 21
3.5.4. Biomolecular recognition ............................................................................ 21
3.5.5. Cellular controlled assembly ....................................................................... 23
4. Conclusions ............................................................................................................ 23
5. References: ............................................................................................................. 23
ii
Chapter 3 – Materials and Methods .................................................................................. 32
1. Laminarin ............................................................................................................... 32
2. Platelet Lysates ....................................................................................................... 32
3. Methods .................................................................................................................. 32
3.1. Synthesis and characterization of methacrylated laminarin .......................... 32
3.2. Fabrication of MeLam microparticles ......................................................... 33
3.3. RGD functionalization of MeLam microparticles ........................................ 34
3.4. Scanning electron microscopy (SEM) ......................................................... 35
3.5. Studies on the release of PL ........................................................................ 35
3.6. Cell culture on MeLam microparticles ........................................................ 35
3.7. Cell morphology analysis ............................................................................ 36
3.7.1. DNA quantification..................................................................................... 37
3.7.2 MTS Viability Assay .................................................................................. 37
3.8. Statistical analysis ....................................................................................... 37
4. References .............................................................................................................. 37
Chapter 4 - Multifunctional laminarin microparticles for cell adhesion and expansion ...... 39
1. Introduction ............................................................................................................ 39
2. Materials and Methods ............................................................................................ 40
2.1. Synthesis and characterization of methacrylated laminarin .......................... 40
2.2. Fabrication of MeLam microparticles ......................................................... 41
2.3. RGD functionalization of MeLam microparticles ........................................ 42
2.4. Scanning electron microscopy (SEM) ......................................................... 43
2.5. Studies on the release of PL ........................................................................ 43
2.6. Cell culture on MeLam microparticles ........................................................ 43
2.7. Cell morphology analysis ............................................................................ 44
iii
2.8. DNA quantification..................................................................................... 45
2.9. MTS Viability Assay .................................................................................. 45
2.10. Statistical analysis ....................................................................................... 45
3. Results and Discussion............................................................................................ 46
3.1. Synthesis and Characterization of Methacrylated Laminarin ....................... 46
3.2. Fabrication of Methacrylated Laminarin microparticles .............................. 47
3.3. Cumulative release of platelet lysates from MeLam microparticles ............. 49
3.4. RGD functionalization of MeLam microparticles ........................................... 51
3.5. L929 fibroblast capture and expansion on MeLam microparticles .................. 52
4. Conclusions ............................................................................................................ 54
5. References: ............................................................................................................. 54
Chapter 5 – General Conclusions ..................................................................................... 59
iv
v
List of figures
Figure 2.1: Schematic representation of different techniques to produce microgels and their
applications. .......................................................................................................................6
Figure 2.2: Micromolding-based fabrication of hydrogel microstructures. (A) Schematic
representation of the surface tension-induced droplet formation.52 (B) Schematic
representation of the fabrication process of Janus microstrips and optical microscope images
of self-bent Janus microstrips in response to changes in the pH. The scale bar in each figure
indicates 300 μm.53 ............................................................................................................7
Figure 2.3: Bioprinting techniques. (A) Inkjet bioprinting: the drops are ejected by thermal
or acoustic forces. (B) Laser-assisted bioprinting constituted by a pulse laser and a ribbon.
(C) Extrusion bioprinting: the application of a continuous force can print bioink droplets and
fibers.69 ..............................................................................................................................9
Figure 2.4: Schematic representation of microfluidic technologies to fabricate hydrogels
microstructures. (A) Schematic representation of microfluidic technology used to produce
protein microgel based in water-in-oil emulsion.80 (B) The capillarity microfluidic device
with a three-barrel injection capillary and three parallel inserted spindlier capillaries for the
generation of microfibers and the biomimetic vessels.82 ................................................... 11
Figure 2.5: Schematic representation of the apparatus used to produce microparticles. (A)
Representation of the dropwise UV curing apparatus developed for the processing PEG-
fibrinogen cell-laden microparticles.90 .............................................................................. 13
Figure 2.6: Schematic representation of droplet microarray platform. Crosslinking of
alginate droplets by performing parallel addition of CaCl2 solutions into the individual
droplets via the sandwiching method. By changing the position of the slide 1 (bottom vs top)
containing CaCl2 droplets, it is possible to form either an array of fixed hydrogel particles
(Step 2a) or detach hydrogel particles to form freefloating hydrogel particles (Step 2b).50 14
vi
Figure 2.7: Schematic representation of the open porous microgels. After cell seeding,
spheroid cells were maintained in the open porous Nutrients could be diffused very well. 30
mg/ml of microgel – human adipose stem cells (hASCs) dispersion were injected to evaluate
the formation of adipose tissue after 14 days.100 ............................................................... 15
Figure 2.8: Chitosan microparticles are functionalized with antibodies and used for specific
cell isolation/separation from an cell heterogenous population and for further cell
expansion.45 ..................................................................................................................... 17
Figure 2.9: Schematic representation of the fabrication process showing the deposition of
cell-laden pre-polymers.118 ............................................................................................... 18
Figure 2.10: (A) Schematic of magnetic directed assembly of microgels. Microgels are
assembled to fabricate three-layer spheroids through the application of external magnetic
fields.139 (B) Assembly of magnetic freestanding hydrogel particles and representative
images of (i) brightfield, (ii) fluorescent, and (iii) overlay of coculture hydrogels. HeLa cells
expressing GFP cells (green) were immobilized in circle-shaped freestanding hydrogels and
MLTy-mCherry cells (expressing fluorescent red) were immobilized in squared-shaped
freestanding hydrogels. Scale bars: 1 mm.50 ..................................................................... 21
Figure 2.11: a. Scheme illustrating microgel formation using a microfluidic water-in-oil
emulsion system. A pre-gel and crosslinker solution are segmented into monodisperse
droplets followed by in-droplet mixing and crosslinking via Michael addition. b. microgels
are purified into an aqueous solution and annealed using FXIIIa into a microporous scaffold,
either in the presence of cells or as a pure scaffold. c. Fluorescent images showing purified
microgel building blocks (left) and a subsequent cell-laden MAP scaffold (right).22 ......... 22
Figure 3.1: Schematic representation of microfluidic system used to produce MeLam
microparticles. (A) Schematic representation of PDMS microchip with T-junction shape. (B)
Droplets formation into the microchip. (C) UV crosslinked MeLam microparticles .......... 34
vii
Figure 3.2: Schematic representation of bioconjugation streptavidin and biotinylated RGD
with MeLam microparticles. After modification, microparticles were cultured with L929
cells. This scheme also represents cell attachment on the microparticles and subsequent
assembly of the structures. ............................................................................................... 36
Figure 4.1: Schematic representation of microfluidic system used to produce MeLam
microparticles. (A) Schematic representation of PDMS microchip with T-junction shape. (B)
Droplets formation into the microchip. (C) UV crosslinked MeLam microparticles .......... 42
Figure 4.2: Schematic representation of bioconjugation streptavidin and biotinylated RGD
with MeLam microparticles. After modification, microparticles were cultured with L929
cells. This scheme also represents cell attachment on the microparticles and subsequent
assembly of the structures. ............................................................................................... 44
Figure 4.3: (A) Schematic illustration of the methacrylation reaction of laminarin with
glycidyl methacrylate. (B) 1H NMR spectrum of laminarin before modification. (C) Low
methacrylated laminarin (D) High methacrylated laminarin. ............................................ 47
Figure 4.4: Images of the high MeLam microparticles in oil (A) in PBS (B) obtained by
optical microscopy and respective histogram of the distribution (C) of microparticles (n =
74) after washed with PBS. Images of the low MeLam microparticles in oil (D) in PBS (E)
obtained by optical microscopy and respective histogram of the distribution (F) of
microparticles size (n = 74) after washed with PBS. SEM image of monodisperse high (G)
and low (H) MeLam microparticles .................................................................................. 48
Figure 4.5: (A) Cumulative protein profile release from low and high methacrylate
laminarin microparticles by incubation in PBS solution up to 14 days quantified by micro-
BCA assay. ELISA assay performance to quantify specific growth factors release from
microparticles. (B) Concentration of VEGF and TGF-β1 presented in PL sample. VEFG
release profiled (C) and TGF-β1 release profile (D) up to 14 days from low and high
methacrylate laminarin. Error bars represent standard deviation (n = 3). ........................... 51
viii
Figure 4.6: Fluorescence imagens showing the functionalization of MeLam microparticles.
Images of MeLam microparticles with biotin-PEG-SH (A) and MeLam microparticles
without biotin (control) (B) after incubation with fluorescently labeled SaV..................... 52
Figure 4.7: Fluorescence images of the high MeLam microparticles with encapsulated PL
(A-D) culture with L929 cells up to 14 days. These images demonstrate the ability to cell
attach, cytoskeleton was stained with phalloidin (red) and nuclei was stained with DAPI
(blue). (E) Cell viability by MTS assay was determined at 3, 7 and 11 days. (F) DNA
quantification of all formulations tested up to 11 days of culture. Results are present as mean
± standard error of the mean (n = 3). ................................................................................ 54
Figure S4.1: Images of MeLam microparticles with biotin-PEG-SH bioconjugate with pure
SaV (A) and without bioconjugation with pure SaV (control) (B) after incubation with
fluorescence biotin ........................................................................................................... 57
Figure S4.2: Images assessed by optical microscopy with encapsulated PL (A and B) and
without PL (C and D) for 24 hours and 7 days. ................................................................. 58
Figure S4.3: Fluorescence images of the high MeLam microparticles without encapsulated
PL (A-C) culture with L929 cells up to 14 days. These images demonstrate the ability to cell
attach, cytoskeleton was stained with phalloidin (red) and nuclei was stained with DAPI
(blue). .............................................................................................................................. 58
ix
Abbreviations
2D – Bidimensional
3D – Three dimensional
DMAP – 4-(N,N-dimethylamino)pyridine
DMSO – Dimethyl Sulfoxide
DS – Degree of Substituion
ECM – Extracellular Matrix
FBS – Fetal Bovine Serum
FEP – Fluorinated Ethylene Propylene
GFs – Growth Factors
HTS – High-throughput screening
MCS – Maleic Anhydride-modified Chitosan
MeLam – Methacrylated Laminarin
MSCs – Mesenchymal Stem Cells
PBS – Phosphate Buffered Saline
PDMS – Polydimethylsiloxane
PEG – Poly(ethylene glycol)
PDGF – Platelet Derived Growth Factor
PL – Platelet Lysates
PLGA – Poly(DL-lactic-co-glycolic acid)
PRP – Platelet Rich Plasma
RT – Room Temperature
SaV – Streptavidin
TE – Tissue Engineering
TGF-β1 – Transforming Growth Factor
UV – Ultraviolet
VEGF – Vascular Epithelial Growth Factor
CHAPTER 1
Motivation
1
Chapter 1 - Motivation
Chapter 1 - Motivation
Tissue Engineering (TE) is a multidisciplinary field of research that applies the principles
of engineering, biology and materials science to either enhance or replace biological
tissues.1,2 Conventional TE strategies typically employ a “top-down” approach, in which
cells are seeded onto a biodegradable polymeric scaffold with specific biochemical and
physical features (e.g. porosity, pore architecture, chemical composition) to act as a
temporary support matrix in which cells are cultured.3–5 This approach has been shown
promising results towards the development of the scaffolds for the regeneration of
tissues.6,7 Nevertheless, the fabrication of complex and fully functional tissues is still a
challenge due to the limited diffusion of nutrients and oxygen and lack of vascularization
systems.8 For reconstructing hierarchically organized and complex 3D tissues that more
accurately mimic organs, the bottom-up tissue engineering approach has emerged as an
alternative strategy. This strategy employs the use of small units as building blocks with
a specific microarchitecture, that assembly to form complex 3D tissue constructs. Several
methods have been proposed for the fabrication of these tissue building blocks, including
generation and assembly of cell sheets, direct printing of cells and fabrication of cell-
laden microscale hydrogels.
Hydrogels are hydrophilic polymer networks with high water content, which allows high
biocompatibility and similar structure to the ECM.4 The recent convergence of nano and
microtechnologies, has resulted in the emergence of microscale hydrogels (microgels),
synthesized through different microfabrication techniques. For instance, micromolding,
bioprinting, microfluidics, electrospraying and superhydrophobic platforms for microgels
production are some of these techniques, which are describe in detail in chapter 2 of this
thesis. Moreover, these chapter also goes through the materials that have been explored
in the microfabrication of hydrogels as well their main applications.
Laminarin has recently been proposed as an ideal polymer to produce cell laden microgels
while retaining high cell viability.9 Laminarin is a β-1,3-glucan with β-1,6-branchings
extracted from brown algae. The low molecular weight of this polymer associated with
the intrinsic low viscosity and high solubility in aqueous or organic solvents makes this
polymer particularly appealing for the microfabrication of hydrogels.9,10 A main goal in
this project is the fabrication of cell microcarriers that in addition to serve as a support
for cell culture and expansion may be also used as an injectable system for tissue
regeneration purposes. In this project, microfluidics was used to produce laminarin
2
Chapter 1 - Motivation
microparticles with well controlled dimension and high monodispersed. Two parallel
studies were conducted, fabrication of laminarin derived microparticles that were
explored as cell microcarriers for cell expansion. In this strategy, human platelet lysates
(PLs) were used as a source of growth factors. PL are a whole-blood derivative with high
concentration of growth factors that are being established as a safe and efficient cell
culture supplement. Besides the influence of PLs in the cultured cells, the release profile
from the laminarin microparticles was also studied. The results suggest that microfluidics
is an effective technique for the production of laminarin microparticles. The size of the
particles is easily controlled by adjusting parameters such as flow rates and polymer
concentrations. The laminarin microparticles allowed a slow release of PL for about 14
days. Cell culture studies demonstrated the positive effect of PL release on the culture
cells. Only particles containing PL were able to support cell expansion.
References:
1. Boos, A. M. et al. The potential role of telocytes in Tissue Engineering and Regenerative
Medicine. Semin. Cell Dev. Biol. 55, 70–78 (2016).
2. Bhowmick, S. et al. Biomimetic electrospun scaffolds from main extracellular matrix components
for skin tissue engineering application – The role of chondroitin sulfate and sulfated hyaluronan.
Mater. Sci. Eng. C 79, 15–22 (2017).
3. Bajaj, P., Schweller, R. M., Khademhosseini, A., West, J. L. & Bashir, R. 3D Biofabrication
Strategies for Tissue Engineering and Regenerative Medicine. Annu Rev Biomed Eng 16, 247–
276 (2014).
4. Slaughter, B. B. V et al. Hydrogels in Regenerative Medicine. Adv. Mater. 21, 3307–3329 (2009).
5. Pang, Y. et al. Novel integrative methodology for engineering large liver tissue equivalents based
on three-dimensional scaffold fabrication and cellular aggregate assembly. Biofabrication 8, 1–16
(2016).
6. Shen, X. et al. Sequential and sustained release of SDF-1 and BMP-2 from silk fibroin-
nanohydroxyapatite scaffold for the enhancement of bone regeneration. Biomaterials 106, 205–
216 (2016).
7. Huang, B. J., Hu, J. C. & Athanasiou, K. A. Cell-based tissue engineering strategies used in the
clinical repair of articular cartilage. Biomaterials 98, 1–22 (2016).
8. Causa, F., Netti, P. A. & Ambrosio, L. A multi-functional scaffold for tissue regeneration: The
need to engineer a tissue analogue. Biomaterials 28, 5093–5099 (2007).
9. Custódio, C. A., Reis, R. L. & Mano, J. F. Photo-Cross-Linked Laminarin-Based Hydrogels for
Biomedical Applications. Biomacromolecules 17, 1602–1609 (2016).
10. Wang, D. et al. The first bacterial β-1,6-endoglucanase from Saccharophagus degradans 2-40T
for the hydrolysis of pustulan and laminarin. Appl. Microbiol. Biotechnol. 101, 197–204 (2017).
CHAPTER 2 Recent trends on microfabrication of
hydrogels
4
Chapter 2 – Recent trends on microfabrication of hydrogels
Chapter 2 - Recent trends on microfabrication of hydrogels
Martins CR1, Custódio CA1, Mano JF1
1-Department of Chemistry, CICECO, Aveiro Institute of Materials, University of Aveiro Campus
Universitário de Santiago, 3810-193 Aveiro - Portugal
Abstract
Hydrogels are polymeric 3D networks with high water content that have become very
popular in multiple biomedical applications due to their unique properties such as,
biocompatibility and versatile fabrication. Microfabricated hydrogels (microgels) are
presently under intense investigation for application as platforms for controlled release of
drugs and bioactive molecules, cell encapsulation and cell expansion. More recently,
microgels have become popular in the design of 3D hierarchically organized constructs
for tissue engineering applications. Microgels serve as building blocks, where living cells
can be cultured and arranged in complex 3D structures that resemble in vivo complexity
of tissues and organs.
This review highlights the efforts developed in last years regarding microfabrication of
hydrogels, their applications, strategies for microgels assembly and future perspectives in
the field.
Key words: Microfabrication, Hydrogels, Bottom-up, Tissue Engineering, Microgels
1. Introduction
Hydrogels are three dimensional (3D) hydrophilic polymeric networks that partially
resemble the physical characteristics of the native extracellular matrix (ECM).1–6 Their
resemblance to living tissues opens up many opportunities for applications in biomedical
areas. The main areas of hydrogel applications are contact lenses, wound dressings,
controlled release of bioactive molecules and 3D cell culture.6–9 In particular, 3D
biomimetic structures have widespread applications in tissue engineering (TE) and have
been used for cell culture in substitution of flat 2D supports.
Methods for the preparation of 3D structures for applications in TE comprise two large
categories: top-down and bottom-up approaches.10,11 The traditional top-down approach
5
Chapter 2 – Recent trends on microfabrication of hydrogels
involves seeding cells into full sized porous scaffolds to form tissue constructs. These 3D
matrices can be then cultured in static conditions or inserted in bioreactors for dynamic
cell culture. Commonly used techniques to fabricate top-down scaffolds include freeze-
drying,12 solvent-casting,13 electrospinning14 and supercritical fluids.16 Scaffolds
prepared by top-down strategies have been widely explored for engineering tissues such
as skin,17 bone,18,19 or cartilage.20,21 Still, top-down approaches often have difficulty in
recreating the intricate microstructural and hierarchical features of native tissues.
Recently, bottom-up strategies have been proposed, by culturing living cells in small units
and arranging them into 3D large constructs.22 This method is based in the use of small
units as building blocks with a specific microarchitecture and the assembly of these units
to engineer large and well organized constructs.23 The bottom-up approach allows for the
creation of structures with controlled porosity and organization, multiple cell types and
high cellular densities.24 Different structures have been produced using bottom-up
approaches such as, stacked cell sheets,25 cell aggregation to form spheroids26 and
complex and organized assembly of microgels.27,28
Microgels are small soft hydrogel units formed by a crosslinked polymeric structure using
microfabrication methods. Physical and chemical crosslinking methods of microgels
preparation will be discussed in this review. Some examples of hydrogel microfabrication
techniques are micromolding,29 bioprinting,30 microfluidics,31 electrospray32 and spray
drying33 and droplet microarrays34 that will be overviewed. The assembly of microgel
units has an excellent potential for the reconstruction of 3D complex structures for TE
applications.
2. Microfabrication of hydrogels
Traditionally, hydrogels have been classified based on the method of crosslinking as
either chemically (covalently) or physically crosslinked networks. Chemical crosslinking,
includes radical polymerization,35,36 enzyme-catalyzed crosslinking37 and click
chemistry.38 A common method to promote radical polymerization is the use of ultraviolet
(UV), visible or infrared light along with a photoinitiator that allows the formation of a
radical group. The free radical reacts with specific functional groups on the polymer
backbone allowing the polymerization.39–41 The use of click chemistry to produce
hydrogels has been increasing during the last years due to the high reactivity and
selectivity and mild reaction conditions.35 In particular, recent development of radical
6
Chapter 2 – Recent trends on microfabrication of hydrogels
mediated thiol-ene chemistry, Diels-Alder reaction, tetrazole-alkene photo-click
chemistry, and oxime renders it possible to form hydrogels.40,42,43
Physical crosslinking includes ionic interaction,44,45 hydrogen bonds interactions,46
polymer self-aggregation via non-covalent interactions and electrostatic interactions.47,48
Different stimulus can promote these reactions, such as temperature and pH variations.
The physical crosslinked hydrogels can be classified in thermosensitive,49
stereocomplexed30 and ionically50 depending on the crosslinking response.
The previous listed methods have also been used associated with microfabrication
techniques to form a variety of microgels (figure 2.1). Such microfabrication strategies
will be described in the following topics.
Figure 2.1: Schematic representation of different techniques to produce microgels and their applications.
2.1.Micromolding
Micromolding is a versatile technique, with associated low cost and simplicity that can
be used to mold a variety of different materials in multiple geometries and sizes.29 This
technique is based on printing, molding and embossing that allow for the production of
microgels.51 A prepolymer solution is first casted into a mold featuring well designed
arrays of wells, typically fabricated from elastomeric materials such as
polydimethylsiloxane (PDMS). After crosslinking, the microgels are taken out from the
mold. For example, Jung and co-works developed a micromolding-based approach to
synthesize poly(ethylene glycol)-based microspheres with controlled macroporous
structures.52 For that an aqueous prepolymer solutions was used to fill the wells of
7
Chapter 2 – Recent trends on microfabrication of hydrogels
micropatterned PDMS molds (figure 2.2 A) Next, hydrophobic wetting fluid consisting
of hexadecane and a photoinitiator is pipetted onto the molds to allow surface tension-
induced droplet formation of the prepolymer solution. More recently, Oh and co-workers
have explored micromolding to produce a pH-responsive self-bending of bilayered Janus
hydrogel microstrips (figure 2.2 B).53 The prepolymer solution was deposited in PDMS
mold and polymerized under UV radiation. This technique allowed to define the
dimensions and shape of Janus microstrips on demand and precisely tune the chemical
properties.
The versatility of micromolding allows the fabrication of microgels with different
morphologies and capable to encapsulate cells, proteins, to create responsive systems and
vascularized structures.4,54 This ability to produce microgels molded in any shape enables
great control over the assembly process into 3D structures.4,53
Figure 2.2: Micromolding-based fabrication of hydrogel microstructures. (A) Schematic representation of
the surface tension-induced droplet formation.52 (B) Schematic representation of the fabrication process of
Janus microstrips and optical microscope images of self-bent Janus microstrips in response to changes in
the pH. The scale bar in each figure indicates 300 μm.53
2.2.Bioprinting
3D bioprinting allows for the rapid prototyping of complex 3D constructs and it has been
used as a powerful tool to engineer tissues and organs. This technology allows for the
specific placement of cells and tissues, enabling the production of complex and highly
organized 3D constructs.57–60 In TE bioprinting techniques can be divided into inkjet,
laser-assisted, and extrusion bioprinting.
A B
8
Chapter 2 – Recent trends on microfabrication of hydrogels
Inkjet printing technique allows to handle and print biological materials at high
resolution, precision and speed.61 In this technique electrostatic, thermal62 or acoustic63
forces can be applied to eject droplets from the nozzle (figure 2.3 - A). This system offers
some advantages such as low cost when compared with other similar techniques, high
resolution and high speed and compatibility with many biological materials. Beyond this,
inkjet printing has the potential to introduce concentration gradients of cells, materials or
growth factors throughout the 3D structure by altering drop densities or sizes.64,65 Still,
inkjet printing has some disadvantages, such as the risk of exposing cells and materials
to thermal and mechanical stress, low droplet directionality, non-uniform droplet size,
frequent clogging of nozzle and unreliable cell encapsulation.61 Iwanaga and co-workers
have used inkjet printing to produce alginate microparticles.65 They tested distinct
alginate solutions to analyse the influence on particle size, which exhibit a linear
correlation with the concentration of alginate. Zhu and co-workers developed a gold
nanorod-incorporated gelatin methacryloyl-based bioink for printing 3D functional
cardiac tissue constructs.66 The authors demonstrated that the developed bioink with low
viscosity, allows for the easy integration of cells at high densities and improved cell
adhesion and organization.
Laser-assisted is another bioprinting technique used to engineer 3D constructs. The
essential part of this technique is a donor layer that responds to laser stimulation. This
system is composed by three main components constituted by a pulse laser source beam,
a ribbon that prints the structures and a substrate that collects the printed materials (figure
2.3 – B).61,67 Due to its unprecedented cell printing resolution and precision and ability to
print viscous materials, is an attractive tool for the in printing of tissues and organs
substitutes.67 Keriquel and co-workers reported the use of laser-assisted bioprinting to test
different cell printing geometries. The authors demonstrated for the first time that printed
cells remain viable and proliferate, both in vitro and in vivo, independent of the geometry
used.68 Nevertheless, lasers with high resolution and intensity are expensive compared to
other nozzle-based printing methods, and this could represent a limitation for laser
assisted printing techniques.64,69
An alternative bioprinting technique is extrusion, a modification of inkjet printing.69
Generally, extrusion bioprinting functions by the robotically controlled extrusion of a
material, which is deposited onto a substrate.61 This system can be used to deposit
materials with high viscosity. It is applied to the system a continuous force, that allow to
print uninterrupted cylindrical lines rather than a single bioink droplet (figure 2.3 - C).
9
Chapter 2 – Recent trends on microfabrication of hydrogels
Some concerns about cell viability have been reported using extrusion bioprinting, and
this may be a limitation of this technique. A main advantage of this technique is the
compatibility with a high range of materials such as hydrogels, copolymers and cell
spheroids.61,64 An additional advantage is its good compatibility with photo, chemical and
thermal crosslinking.69 In a recent study, Zhu and co-workers developed a gold nanorod-
incorporated gelatin methacryloyl based bioink for printing 3D functional cardiac tissues.
They used extrusion bioprinting for the printing of cell-laden fibers at a high resolution
and high cell viability.66
Figure 2.3: Bioprinting techniques. (A) Inkjet bioprinting: the drops are ejected by thermal or acoustic
forces. (B) Laser-assisted bioprinting constituted by a pulse laser and a ribbon. (C) Extrusion bioprinting:
the application of a continuous force can print bioink droplets and fibers.69
2.3.Microfluidics
Microfluidics have recently become a highly attractive tool to overcome limitations of
the traditional bulk emulsion methods to generate monodisperse microgels in a reliable
manner. Microfluidic technologies have also been used to produce microplatforms to
study new drugs and to produce microparticles that can be used to building 3D constructs.
The principle of microfluidics is the creation of a stream of polymer solution in a
microchannel (dispersed phase) and the induction of a periodic break-up by flow focusing
with a second immiscible fluid (continuous phase). The two fluids cross each other on a
chip which can have different shapes, such as T-junction, flow Focusing and co-flow.
When these two fluids meet, droplets form in a “drop-by-drop” fashion due to a balance
of interfacial tension and the shear of the continuous phase acting on the dispersed phase
.31,68 The disperse phase may contain a crosslinking agent, cells or biomolecules which
will be encapsulated in the droplet/microparticle.72 An important advantage of this
technology is the ability to produce droplets with highly controlled morphology with the
added benefit of providing droplet manipulation (merging, sorting, deformation, single-
10
Chapter 2 – Recent trends on microfabrication of hydrogels
cell loading, etc.) due to the facility in tuning different flow rates solutions.73 Another
advantage of microfluidics is the possibility to rapidly produce monodispersed
microparticles with encapsulated living cells,74,75 drugs76 or other bioactive compounds
in a unique step. One of the most widely used systems in droplet generation using
microfluidics, is based in water-in-oil emulsifications.77–79 Mao and co-workers reported
a microfluidic-based method for encapsulating single cells within a thin hydrogel layer
using a one-step method.72 The results of this study show that cell viability was
maintained over a three-day period and that the microgels are mechanically tractable. In
another study, Shimanovich and co-workers have described an approach for generating
microgels composed of amyloid fibril networks using a microfluidic droplet maker
device.80 They have demonstrated that these microgels enable the local release of
encapsulated molecules resulting in enhanced antimicrobial action (figure 2.4 - A). An
interesting conclusion from this work was that they were able to generate dynamic
materials that enable the internal content of nanofibrils to be changed at the post-synthetic
stage in response to exposure to monomeric protein molecules in solution. Importantly,
the protein nanofibril microgels demonstrated to be nontoxic to a human cell line and
have the potential for effective drug encapsulation.
Krutkramelis and co-workers have described the photopolymerization of aqueous
hydrogel forming solutions within microscale emulsion droplets.81 They studied the
inhibitory effect of oxygen in photopolymerization. After understanding this limitation,
it was possible to fabricate a continuous PEG-DA microgel on a chip, a nitrogen-jacketed
microfluidic device for in situ oxygen purging has been developed. The works previously
reported confirm precise control offered by microfluidic technology under the structures
produced. This demonstrate the great importance of the technology can offer to create
tissue constructs.
The microgels may be formed in situ by the action of crosslinking reactions occurring
along the time (the crosslinking agent already exists in the aqueous solution), through
high irradiation or by a temperature change. The droplets can also gellify at the exit of
the tube using spatial precipitation co-axial baths.
Cheng and co-workers used microfluidics to fabricate bioactive microfibers for creating
different tissue constructs.82 By employing a multiple laminar flow they were able to
fabricate a series of cell-laden hydrogel microgels with tunable morphological and
structural features from designed multiple injection flows (figure 2.4 - B). They proposed
11
Chapter 2 – Recent trends on microfabrication of hydrogels
this technology to generate complex fiber-shaped cellular building-blocks continuously
in a one-step microfluidic spinning process.
Figure 2.4: Schematic representation of microfluidic technologies to fabricate hydrogels microstructures.
(A) Schematic representation of microfluidic technology used to produce protein microgel based in water-
in-oil emulsion.80 (B) The capillarity microfluidic device with a three-barrel injection capillary and three
parallel inserted spindlier capillaries for the generation of microfibers and the biomimetic vessels.82
2.4.Spray based technologies
Electrospray is a broadly used technique to generate submicrometric particles with high
yields. In electrospraying, the solution is forced through a nozzle spray machine or
syringe needle and a Taylor cone is created due to the applied electric field forming
droplets that are further crosslinked.56,83,84 Electrospraying can be used to produce small,
nearly monodisperse particles when a colloidal suspension of solid nanoparticles or a
solution of a material is sprayed. Although this technique allows to produce monodisperse
particles, microfluidic technique allows to improve the monodispersity of the
microparticles. Different parameters can be manipulated to control morphological
features of the microgels in terms of size and distribution, shape, surface roughness and
porosity.85 The droplets size can be controlled by the liquid flow rate and by adjusting the
voltage applied to the nozzle.86 Electrospraying allows the encapsulation of living cells,
growth factors or drugs in a single step procedure and this could be an advantage when
12
Chapter 2 – Recent trends on microfabrication of hydrogels
compared with other time consuming techniques.87,88 Kim and co-workers described the
fabrication of RGD-alginate microgels formed by electrospraying for the encapsulation
of endothelial cells and growth factors.87 They have proved that encapsulated endothelial
cells maintain viability and are able to proliferate within the microgels. In addition, the
RGD-alginate microgels demonstrated the long-term release of the encapsulated growth
factors in vitro. In a similar study, Yao and co-workers developed a fibrin nanofiber
hydrogel loaded with drug encapsulated poly(DL-lactic-co-glycolic acid) (PLGA)
microspheres prepared via electrospray and electrospinning.89 Electrospray allowed the
production of PLGA microspheres with high drug efficiency and convenient drug release
control. After, by electrostretching process microspheres was encapsulated into
microfibers hydrogel. This system showed a good release behaviour comparing to the
PLGA microspheres and hUMSCs showed a better adhesion demonstrating the great
potential in the neural regeneration to the system. Another method based on droplet
formation and subsequent conversion into particles is co-axial air-flow. Here, the solution
is forced through a nozzle and injected air led the solution to break up into a spray at the
outlet of nozzle.27 Oliveira and co-workers developed a system to fabricate PEG-
fibrinogen cell laden particles.90 The polymer solution was exposed to UV light prior to
its entry into the jet-in-air nozzle that promote the increases the viscosity of the solution
(figure 2.5).
Spray-drying involves the use of a spray drier, mainly consisting of an atomizer and
drying chamber. The solvent is atomized in droplets induced by a stream of hot air that
promotes solvent evaporation in the spray-drying chamber, resulting in the formation of
microparticles, which are then separated from the air through a cyclone or a filter bag.91,92
Spray drying technique offers many advantages, including efficiency, reproducibility,
simplicity, scale-up and the ability to produce homogenous powders.93 These advantages,
led to the use of this system in the production of microgels, coatings and powders.33 Zhao
and co-workers reported the synthesis of stimuli-responsive hemicellulose microgels via
a single-step crosslinking chemistry using spray drying.33 The crosslinking reaction
occurred during spray drying, and the functional hemicellulose microgels were made
responsive to different external stimuli such as pH, electroactivity, magnetic field, and
dual-stimuli (pH and electric field). The simultaneous use of the both methods offers the
potential for fabrication of microgels with rapid stimuli response and good
biocompatibility. Some disadvantages of spray-drying include the possible degradation
of the heat sensitive fine particles, inefficient yields and heterogeneous size
13
Chapter 2 – Recent trends on microfabrication of hydrogels
distribution.92,94 Take into account this, spray dry method can be used to produce
hydrogels microparticles, although it is not better option, being more used in
pharmaceutical industry to produce powders.
Figure 2.5: Schematic representation of the apparatus used to produce microparticles. (A) Representation
of the dropwise UV curing apparatus developed for the processing PEG-fibrinogen cell-laden
microparticles.90
2.5.Superhydrophobic-based platforms for microgel production
In most of the techniques previously reported, the hydrogels were produced in liquid
environment. The superhydrophobic-based platforms are an alternative microparticle
processing method. The droplets are formed using a superhydrophobic surface that allows
to change the contact angle between the aqueous solution and the surface. When the liquid
is suspended on the surface, a sphere is formed involving a basic liquid-air interface.
Superhydrophobic-based platforms are potentially attractive to produce multiple
microgels with encapsulated proteins or other biomolecules and living cells.95 Song and
co-workers were pioneers in the processing of biomaterials at the microscale using
superhydrophobic platforms.95 Such technology was later on adapted to produce
multilayered particles96 or liquified particles.97 However, the proposed strategy,
encompasses multiple pipetting and subsequent crosslinking, resulting in the production
of particles not smaller than 1 mm in diameter, hampering in consequence their tentative
use for cell encapsulation. In order to overcome these limitations, Costa and co-workers
have proposed a new method to produce droplet microarrays that allows the production
of microgels with sizes under 500m.98 The deposition of droplets over fully covered
14
Chapter 2 – Recent trends on microfabrication of hydrogels
superhydrophobic surfaces enables the production of sphere-like objects. Recently, Neto
and co-workers developed a versatile platform (figure 2.6) that allows to create a thousand
microdroplets of specific geometry and volume, based on the use of superhydrophobic
surfaces patterned with a wettable superhydrophilic domains.50 This approach enables the
dispensing of aqueous solutions into thousands of droplets without the need for manual
pipetting or robotic devices. This is very promising due to its ability to easily create and
manipulate thousands of microgels of controlled size and geometry.
Figure 2.6: Schematic representation of droplet microarray platform. Crosslinking of alginate droplets by
performing parallel addition of CaCl2 solutions into the individual droplets via the sandwiching method.
By changing the position of the slide 1 (bottom vs top) containing CaCl2 droplets, it is possible to form
either an array of fixed hydrogel particles (Step 2a) or detach hydrogel particles to form freefloating
hydrogel particles (Step 2b).50
3. Applications of microfabricated hydrogels
Microfabricated hydrogels have found multiple applications as cell encapsulation, cell
expansion used as building units to engineer complex and hierarchal 3D constructs, drug
delivery or even as microreactors. In the next topics, recent developments in microgel
applications are presented and discussed.
3.1.Cell laden microgels as building blocks for 3D constructs
Most living tissues are composed of repeating microunits, which are combinations of
multiple cell types with well-defined 3D microarchitectures and tissue-specific functional
properties. A major challenge in TE is to engineer biomimetic tissues that contain
appropriate cell microenvironment and the multicellular architectural features found in
vivo. Taking this into account, microgels became attractive entities for TE applications,
15
Chapter 2 – Recent trends on microfabrication of hydrogels
namely for the fabrication of complex and hierarchal 3D structures. Du and co-workers
were pioneers in the fabrication of biomimetic 3D tissue constructs using cell laden PEG
microgels opening the paradigm for directing the assembly of mesoscale materials.25
Yuebi and co-workers demonstrated the self-assembly of alginate microgels modified
with combinations of binding pair molecules.99 These molecules bind to their
complements rapidly and under physiological conditions. The modified alginate
microgels were shown to support viable cell encapsulation. The modified microgels can
be induced to self-assemble either in suspension or following a brief centrifugation step
forming complex 3D constructs that can combine multiple cell types. In a more recent
work, Xia and co-workers developed a strategy to fabricate open porous microgels with
high hydrophobicity and great injectability (figure 2.7).100 The microgels were based on
double bonded poly-(L-glutamic acid)-g-2-Hydroxylethyl methacrylate (PLGA-g-
HEMA) and maleic anhydride-modified chitosan (MCS). The high hydrophobicity of the
system, made the stem cell shape spheroid to favour adipogenic differentiation. The
porous structure promoted the nutrients diffusion that promotes the high stem cell
viability. Lienemann and co-workers developed a strategy based on microfluidic to create
monodisperse pre-microgel droplets.101 This work presented a strategy to bypass Poisson
encapsulation statistics in synthetic microniches by selection of only cell-laden microgels.
They demonstrated of the versatility of this work, developing a strategy that allows
efficient encapsulation of individual cells that can be used for different cell types.
Cell-laden microgels can be applied in various applications as shown the works
previously reported. Encapsulation of single cells can be useful to study biological
responses a specific cellular type.
Figure 2.7: Schematic representation of the open porous microgels. After cell seeding, spheroid cells were
maintained in the open porous Nutrients could be diffused very well. 30 mg/ml of microgel – human adipose
stem cells (hASCs) dispersion were injected to evaluate the formation of adipose tissue after 14 days.100
16
Chapter 2 – Recent trends on microfabrication of hydrogels
3.2.Cell microcarriers
Microcarriers have been widely used in biotechnology industry for the large-scale
production of proteins or viruses.102,103 Cell microcarriers are also a promising culture
system for producing great quantities of anchorage-dependent cells for biomedical and
biochemical applications.104–106 In this case, cells are usually cultured over the surface of
microparticles and are expanded in dynamic conditions using bioreactors. Expanded cells
are recovered from the microcarriers by tripsinization. The coating of microparticles
using smart polymers could be also envisaged to use temperature or other mild stimuli
for cell recovery.107 From cell microcarriers used up to date, Cytodex 3 is probably the
mostly used. This commercial microcarrier consists of a thin layer of denatured collagen
chemically coupled to a matrix of crosslinked dextran. For instance, Nie and co-workers
have used Cytodex to expand and differentiate human embryonic stem cells (ESCs) into
the three germ layers.108 Gelatin based microcarriers (CultiSpher-S) have also been
broadly used for cell expansion. Bender and co-workers have shown the potential of these
gel microcarriers in enhanced cortical neuron adhesion, viability and neurite extension.109
In a more recent work, Soure and co-workers developed a xenogeneic-free microcarrier-
based system for the effective expansion of umbilical cord matrix derived from
mesenchymal stem cells (MSCs) under dynamic conditions.110 In this work, they used a
bioreactor-based manufacturing of MSCs cultured in medium supplemented with human
platelet lysates.
These principals were extrapolated to TE strategies where these systems might be used
both as a strategy for cell expansion and simultaneously injectable systems to be used in
vivo using minimally invasive procedures.111 For example, Custódio and co-workers
reported the use of chitosan microparticles to capture and expand a specific cell type that
can also be regarded as an injectable biomaterial (figure 2.8).45 The microparticles were
modified with specific antibodies enabling the particles to select specific cell types from
mixed cell populations and further cell expansion. The authors suggest that this system
may be used in vivo for tissue regeneration using minimally invasive procedures. Kim
and co-workers also developed an injectable multifunctional microparticle system.87
However, while the work of the Custódio and co-workers promotes cell adhesion on the
surface of microparticle, in Kim´s work cells and growth factors were encapsulated in
microparticles. Cells encapsulated within the microgel exhibited a time-dependent
proliferation with enhanced cell viability, and the size-controlled microgels resulted in
17
Chapter 2 – Recent trends on microfabrication of hydrogels
sustained release of growth factors for enhanced new vessel formation. This method
shows a decrease in the host immune response, but also may be a promising strategy to
improve the therapeutic efficacy for the delivery of therapeutic cells and angiogenic
proteins. Such kind of assembled particles mediated by multiple cell attachment may be
compartmentalized in liquified environments to produce microtissues that can be used in
therapies or as disease models.112,113
Figure 2.8: Chitosan microparticles are functionalized with antibodies and used for specific cell
isolation/separation from an cell heterogenous population and for further cell expansion.45
3.3.High-throughput screening
Engineering tissues combines the use of different biomaterials with proteins and/or cells.
Reactions and interactions between the cells and biomaterials need be studied to evaluated
the cell biology and cytotoxicity of the material. This led to the emerging of high-
throughput screening (HTS) approaches. This powerful technology allows to evaluate a
large combination of structural, biophysical and biochemical parameters and screening
the individual and combined effects of multivariate biological cues.50,114,115
The HTS is developed under the principles: sufficiently sensitive to measure a relevant
cellular response, easy to automate and reproducible.114,116 Basically, this system consists
of a library of distinct material or molecular entities, displayed on a single substrate at
specific addressed locations, so that comparative cell response to each test entity can be
assessed.117 The main advantages of this technology are the significant decrease in cost
and time for screening a biological target. Based on these principles, Guermani and co-
workers developed a microprinting approach to print cellular structures capable to mimic
native tissues architecture.118 The goal of their work, was the synthesis of a high-
18
Chapter 2 – Recent trends on microfabrication of hydrogels
throughput platform to understand the effects of structural, cellular and
microenvironmental heterogeneity on human mesenchymal stem cells differentiation.
(figure 2.9). In a similar approach, superhydrophobic surface patterned with wettable
spots have been used to dispense and fix small volumes of aqueous based solutions that
can give rise to patterned microgels.119 Such technology permits the distribution of a large
amount of controlled combinations of materials and cells in the hydrogels that can be
analysed directly in the chip using image analysis. Besides the biological behaviour of
the cells other properties of the individual microgels can be assessed. Oliveira and co-
workers developed an on-chip microarray platform based on this type of surfaces to test
the mechanical and viscoelastic properties of miniaturized hydrogels and their effect in
cell function.120
Figure 2.9: Schematic representation of the fabrication process showing the deposition of cell-laden pre-
polymers.118
19
Chapter 2 – Recent trends on microfabrication of hydrogels
3.4.Drug Delivery
The polymer network can easily be tuned by controlling the density of crosslinks in the
gel matrix and the affinity of the hydrogels for the aqueous environment in which they
are swollen. The properties of the hydrogel at the macromolecular scale will influence
diffusion and interfacial properties. Due to such versatility, microgel beads have been
widely recognized as a promising material for controlled drug delivery.121,122 The
presence of macromolecular chains in the hydrogels that react with external stimuli
permits to develop microgels able to control the release profile of encapsulated drugs
depending on parameters such as pH, temperature or the presence of specific
molecules.107,123,124 Lee and co-workers demonstrated the possibility of ascorbic acid
delivery in response to different pH stimulus using P(MAA-co-EGMA) hydrogel
microparticles.125 The swelling of the microparticles also showed different responses to
pH changes. This system allows to control the ascorbic acid delivery to control pH of the
environment. Sivakumaran and co-workers explored the application of soft
nanocomposite injectable hydrogels containing entrapped microgels for drug delivery.126
Copolymer microgels based on N-isopropylacrylamide and acrylic acid were synthesized
that exhibited both ionic and hydrophobic affinity for binding to bupivacaine. Microgels
were subsequently immobilized within an in situ-gelling hydrogel network to achieve
longer and more constant drug release kinetics than can be achieved with microgels alone.
In a more recent work, Lai and co-workers developed an approach based of electrospray
technique to produce core-shell hydrogel microspheres.127 They used
carboxymethylcellulose (CMC) and alginate polymers to produce microparticles with
multi cores with distinct properties. This system allows a better control in drug delivery
and a great potential for multi-drug therapy, due to the possibility to separate drugs in
different cores. Different materials can be used to produce hydrogels that enables the
encapsulation of different drugs, proteins or other biomolecules. Controlling the porosity,
the shape and the layers of hydrogels are possible to control the drug delivery.
3.5.Assembly of microgels
In TE, there has been significant interest in using assembly of small building blocks to
form biological tissues in a bottom-up engineering strategy. To date, bottom-up assembly
of microgels has been achieved by a variety of techniques. Such methods can be divided
in two main groups: self-assembly or directed assembly. Self-assembly is the process by
which an organized structure spontaneously forms from individual components into
20
Chapter 2 – Recent trends on microfabrication of hydrogels
patterns or structures.11,128 Self-assembly properties have been exploited for the
preparation of 3D nanostructured bioactive scaffolds, by using specific complementary
molecular interactions. Microgels have been conjugated with ionic-complementary self-
assembling peptides, oligosaccharides, binding pair molecules and nucleic acids allowing
the self-organization of the microgels.99,129,130
Directed assembly is the use of specific driving forces to assemble microgels in a directed
manner. Directed assembly approaches include programmable molecular recognition and
binding scheme, hydrophilic-hydrophobic interactions, surface template, microfluidic
and magnetic assembly are promising technologies to assemble microgels.131
3.5.1. Surface Tension
Whitesides and co-workers were pioneers in the microfabrication of 3D constructs that
assemble by surface tension. Using this strategy, hydrophilic microgels can be assembled
in millimetre-scale objects into well-defined 3D structures through minimization of the
interfacial free energy of the liquid–liquid interface.132,133 Parameters such as external
energy input, surface tension, and microgel dimensions can be controlled and influence
the assembly process. Du and co-workers also reported about shape-controlled microgels
that direct assemble within multiphase reactor systems into predetermined geometric
configurations.25,134 By increasing the hydrophilicity of the microgels and reducing the
surface tension of the surrounding solution they were able to improve the assembly
process. In addition, by combining this directed assembly process with a second
crosslinking they were able produce 3D structures with increased stability. The increase
of hydrophobicity at the surface of hydrogels can lead to floatable microbjects that can
move over the surface of aqueous media with minimum friction.135
3.5.2. Acoustic assembly
Acoustics has been also used in the biomedical field to manipulate droplets, cells and
biomolecules. The application of the surface waves has showed some advantages like as
non-invasive, simple and inexpensive approach towards the precise and rapid patterning
of microparticles and cells.136 For example, Feng Xu and co-workers reported the use of
acoustic fields to direct the assembly of PEG-methacrylate microgels (figure 2.11).131 By
applying an acoustic field to droplet encapsulating microgels of different shapes and sizes
the particles are agitated and self-assemble. They also investigated the effect of acoustic
frequency and amplitude on the assembly process, and the effect of acoustic excitation
21
Chapter 2 – Recent trends on microfabrication of hydrogels
on the viability of cells encapsulated in the microgels. The results indicated that
crosslinking and acoustic excitation did not have significant effect on cell viability.
Despite the very promising results in acoustic assembly, a main limitation of this method
is the uni-directional patterning, that limited the creation of line patterns.
3.5.3. Magnetic assembly
Magnetic fields have different applications in TE such as cellular manipulation, cell
sorting and isolation, 3D cell culture and clinical in vivo imaging.137 Recently, magnetic
hydrogels have emerged as a novel biocomposite for their active response properties and
extended applications.138 The potential of the magnetic hydrogels for the fabrication of
modular 3D scaffolds have been also explored to promote directed assembly between
structures. Xu and co-workers reported about the microfabrication of gelatin methacrylate
(GelMA) and PEG microgels incorporating magnetic nanoparticles that were easily
assembled into microscopic hydrogels (figure 2.10 - A).139 These microgels were used as
building blocks to create 3D complex multilayer constructs using external magnetic
fields. More recently, Neto and co-workers reported about the fabrication of freestanding
hydrogel particles with defined geometries and sizes containing magnetic particles and
cells (figure 2.10 - B).50 The full control of the magnetic fields is still a challenge to truly
be able to build organized and predefined 3D structures using such magnetic-responsive
building blocks.
Figure 2.10: (A) Schematic of magnetic directed assembly of microgels. Microgels are assembled to
fabricate three-layer spheroids through the application of external magnetic fields.139 (B) Assembly of
magnetic freestanding hydrogel particles and representative images of (i) brightfield, (ii) fluorescent, and
(iii) overlay of coculture hydrogels. HeLa cells expressing GFP cells (green) were immobilized in circle-
shaped freestanding hydrogels and MLTy-mCherry cells (expressing fluorescent red) were immobilized in
squared-shaped freestanding hydrogels. Scale bars: 1 mm.50
22
Chapter 2 – Recent trends on microfabrication of hydrogels
3.5.4. Biomolecular recognition
Biomolecular recognition has been also explored to assemble microgels into 3D
structures. For example, Griffin and co-workers synthesized microgel building blocks
composed of multi-armed poly(ethylene) glycol-vinyl sulphone backbones decorated
with a cell adhesive peptide and two transglutaminase peptide substrates (K and Q).
(figure 2.11).22 The microgel building blocks were then assembled through Michael-type
addition between the K and Q peptides mediated by activated Factor XIII (FXIIIa), a
naturally occurring enzyme responsible for stabilizing blood clots. This enzyme-mediated
annealing process allowed incorporation of living cells into a dynamically forming MAP
scaffold that contained interconnected microporous networks.
In a different strategy, using DNA nucleotide bases as programmable, sequence-specific
‘glues’, shape-controlled hydrogel units have been self-assembled into complex 3D
structures.140 Li and co-workers modified the surface of cell-laden PEG microgels with
complementary DNA to form DNA-templated assembly of PEG microtissues. The
recognition capabilities of DNA are the key to achieve rapid assembly of multiple
building blocks. In a similar approach Qi and co-workers have developed a strategy to
use complementary DNA molecules as glue to direct the self-assembly of hydrogel
cubes.141 Using hydrogel cubes that display giant DNA on multiple designated faces, they
were able to construct linear chain structures and net-like structures.99 The use more than
one pair of complementary DNA chains (on other kind of biochemical recognition) would
enable the independent assembly of different points and to produce more controlled and
complex structures.
Figure 2.11: a. Scheme illustrating microgel formation using a microfluidic water-in-oil emulsion system.
A pre-gel and crosslinker solution are segmented into monodisperse droplets followed by in-droplet mixing
and crosslinking via Michael addition. b. microgels are purified into an aqueous solution and annealed using
23
Chapter 2 – Recent trends on microfabrication of hydrogels
FXIIIa into a microporous scaffold, either in the presence of cells or as a pure scaffold. c. Fluorescent
images showing purified microgel building blocks (left) and a subsequent cell-laden MAP scaffold (right).22
3.5.5. Cellular controlled assembly
A number of works have been focused on the development of materials that self-assembly
through the presence of cells. Cruz and co-workers have shown that chitosan microgels
support cell adhesion and proliferation.142 When cells spread actively on a number of
microgels they trigger the assembly of the microstructures, forming a 3D scaffold.
Oliveira and co-workers have reported similar outcomes using protein based microgels to
form cell-induced aggregation 3D constructs.143 More recently, Custódio and co-workers
developed a refined strategy using functional microgels capable of binding to receptors
on the cell surface.27 They demonstrated that microgels when combined with cells, are
able to establish interconnected networks and lead to the formation of stable and robust
3D structures. They has also reported that chitosan microgels bioconjugated with specific
antibodies provide suitable surfaces to capture a target cell type, subsequent expansion of
the captured cells and assembly in a complex 3D structure.45
4. Conclusions
Microgels are microfabricated hydrophilic polymeric networks in the range of several
micrometers down to nanometers. In the past decades, multiple methods and technologies
have been developed to microfabricate hydrogels. Microfabrication has been used to
produce microgels for cell encapsulation with high cell viability due to the increased
diffusion of oxygen and nutrients. Microfabrication techniques have also been used for
encapsulation of drugs and other biomolecules without compromising their bioactivity.
Biological systems are highly hierarchical in structure. To mimic such organization
microfabrication tools that can generate and manipulate multi-scale building units are
needed. Is in this context microgels are particularly attractive as building blocks to
engineer complex and organized tissues.
Still, producing functional constructs is challenged by the limitations in the assembly
processes and the effective fabrication of tissue constructs with relevant length scales.
5. References:
1. Geckil, H., Xu, F., Zhang, X., Moon, S. & Demirci, U. Engineering hydrogels as extracellular
24
Chapter 2 – Recent trends on microfabrication of hydrogels
matrix mimics. Nanomedicine 5, 469–84 (2010).
2. Shapiro, J. M. & Oyen, M. L. Hydrogel composite materials for tissue engineering scaffolds. Jom
65, 505–516 (2013).
3. Zhu, J. & Marchant, R. E. Design properties of hydrogel tissue-engineering scaffolds. Expert Rev.
Med. Devices 8, 607–626 (2011).
4. Yeh, J. et al. Micromolding of shape-controlled, harvestable cell-laden hydrogels. Biomaterials
27, 5391–5398 (2006).
5. Ahmed, E. M. Hydrogel: Preparation, characterization, and applications: A review. J. Adv. Res. 6,
105–121 (2015).
6. Slaughter, B. B. V et al. Hydrogels in Regenerative Medicine. Adv. Mater. 21, 3307–3329 (2009).
7. Wu, J. et al. Fabrication and characterization of monodisperse PLGA-alginate core-shell
microspheres with monodisperse size and homogeneous shells for controlled drug release. Acta
Biomater. 9, 7410–7419 (2013).
8. Riederer, M. S., Requist, B. D., Payne, K. A., Way, J. D. & Krebs, M. D. Injectable and
microporous scaffold of densely-packed, growth factor-encapsulating chitosan microgels.
Carbohydr. Polym. 152, 792–801 (2016).
9. Caló, E. & Khutoryanskiy, V. V. Biomedical applications of hydrogels: A review of patents and
commercial products. Eur. Polym. J. 65, 252–267 (2015).
10. Oliveira, S. M., Reis, R. L. & Mano, J. F. Towards the design of 3D multiscale instructive tissue
engineering constructs: Current approaches and trends. Biotechnol. Adv. 33, 842–855 (2015).
11. Lu, T., Yuhui, L. & Chen, T. Techniques for fabrication and construction of three-dimensional
scaffolds for tissue engineering. Int. J. Nanomedicine 8, 337–350 (2013).
12. Correia, C. R. et al. Chitosan scaffolds containing hyaluronic acid for cartilage tissue engineering.
Tissue Eng. Part C. Methods 17, 717–730 (2011).
13. Kong, J., Hwang, I.-W. & Lee, K. Top-Down Approach for Nanophase Reconstruction in Bulk
Heterojunction Solar Cells. Adv. Mater. 26, 6275–6283 (2014).
14. Jayakumar, R., Prabaharan, M., Nair, S. V. & Tamura, H. Novel chitin and chitosan nanofibers in
biomedical applications. Biotechnol. Adv. 28, 142–150 (2010).
15. Wijesena, R. N. et al. A method for top down preparation of chitosan nanoparticles and
nanofibers. Carbohydr. Polym. 117, 731–738 (2015).
16. Santo, V. E. et al. Enhancement of osteogenic differentiation of human adipose derived stem cells
by the controlled release of platelet lysates from hybrid scaffolds produced by supercritical fluid
foaming. J. Control. Release 162, 19–27 (2012).
17. Bhowmick, S. et al. Biomimetic electrospun scaffolds from main extracellular matrix components
for skin tissue engineering application – The role of chondroitin sulfate and sulfated hyaluronan.
Mater. Sci. Eng. C 79, 15–22 (2017).
18. Sun, X. et al. Modeling vascularized bone regeneration within a porous biodegradable CaP
scaffold loaded with growth factors. Biomaterials 34, 4971–4981 (2013).
19. Shen, X. et al. Sequential and sustained release of SDF-1 and BMP-2 from silk fibroin-
nanohydroxyapatite scaffold for the enhancement of bone regeneration. Biomaterials 106, 205–
25
Chapter 2 – Recent trends on microfabrication of hydrogels
216 (2016).
20. Parmar, P. A. et al. Collagen-mimetic peptide-modifiable hydrogels for articular cartilage
regeneration. Biomaterials 54, 213–225 (2015).
21. Huang, B. J., Hu, J. C. & Athanasiou, K. A. Cell-based tissue engineering strategies used in the
clinical repair of articular cartilage. Biomaterials 98, 1–22 (2016).
22. Griffin, D. R., Weaver, W. M., Scumpia, P. O., Di Carlo, D. & Segura, T. Accelerated wound
healing by injectable microporous gel scaffolds assembled from annealed building blocks. Nat
Mater 14, 737–744 (2015).
23. Causa, F., Netti, P. A. & Ambrosio, L. A multi-functional scaffold for tissue regeneration: The
need to engineer a tissue analogue. Biomaterials 28, 5093–5099 (2007).
24. Khan, O. F., Voice, D. N., Leung, B. M. & Sefton, M. V. A novel high-speed production process
to create modular components for the bottom-up assembly of large-scale tissue-engineered
constructs. Adv. Healthc. Mater. 4, 113–120 (2015).
25. Du, Y., Lo, E., Ali, S. & Khademhosseini, A. Directed assembly of cell-laden microgels for
fabrication of 3D tissue constructs. Proc. Natl. Acad. Sci. 105, 9522–9527 (2008).
26. Cheng, N. C., Wang, S. & Young, T. H. The influence of spheroid formation of human adipose-
derived stem cells on chitosan films on stemness and differentiation capabilities. Biomaterials 33,
1748–1758 (2012).
27. Custódio, C. A. et al. Functionalized microparticles producing scaffolds in combination with
cells. Adv. Funct. Mater. 24, 1391–1400 (2014).
28. Chan, H. F. et al. Rapid formation of multicellular spheroids in double-emulsion droplets with
controllable microenvironment. Sci. Rep. 3, 3462 (2013).
29. Tekin, H. et al. Responsive micromolds for sequential patterning of hydrogel microstructures. J.
Am. Chem. Soc. 133, 12944–12947 (2011).
30. Poldervaart, M. T. et al. Prolonged presence of VEGF promotes vascularization in 3D bioprinted
scaffolds with de fi ned architecture. J. Control. Release 184, 58–66 (2014).
31. Seiffert, S. & Weitz, D. A. Microfluidic fabrication of smart microgels from macromolecular
precursors. Polymer (Guildf). 51, 5883–5889 (2010).
32. Correia, D. M. et al. Electrosprayed poly(vinylidene fluoride) microparticles for tissue
engineering applications. RSC Adv. 4, 33013–33021 (2014).
33. Zhao, W. et al. In situ cross-linking of stimuli-responsive hemicellulose microgels during spray
drying. ACS Appl. Mater. Interfaces 7, 4202–4215 (2015).
34. Berthuy, O. I. et al. Multiplex cell microarrays for high-throughput screening. Lab Chip 16,
4248–4262 (2016).
35. Jiang, Y., Chen, J., Deng, C., Suuronen, E. J. & Zhong, Z. Click hydrogels, microgels and
nanogels: Emerging platforms for drug delivery and tissue engineering. Biomaterials 35, 4969–
4985 (2014).
36. Fraser, A. K., Ki, C. S. & Lin, C. C. PEG-based microgels formed by visible-light-mediated thiol-
ene photo-click reactions. Macromol. Chem. Phys. 215, 507–515 (2014).
37. Henke, S. et al. Enzymatic Crosslinking of Polymer Conjugates is Superior over Ionic or UV
26
Chapter 2 – Recent trends on microfabrication of hydrogels
Crosslinking for the On-Chip Production of Cell-Laden Microgels. Macromol. Biosci. 1524–1532
(2016). doi:10.1002/mabi.201600174
38. El-Sagheer, A. H. & Brown, T. Click chemistry with DNA. Chem. Soc. Rev. 39, 1388–1405
(2010).
39. Wang, J. & Wei, J. Hydrogel brushes grafted from stainless steel via surface-initiated atom
transfer radical polymerization for marine antifouling. Appl. Surf. Sci. 382, 202–216 (2016).
40. Custódio, C. A., Reis, R. L. & Mano, J. F. Photo-Cross-Linked Laminarin-Based Hydrogels for
Biomedical Applications. Biomacromolecules 17, 1602–1609 (2016).
41. Nguyen, K. T. & West, J. L. Photopolymerizable hydrogels for tissue engineering applications.
Biomaterials 23, 4307–4314 (2002).
42. Malkoch, M. et al. Synthesis of well-defined hydrogel networks using Click chemistry. Chem.
Commun. 2774–2776 (2006).
43. Hein, C. D., Liu, X.-M. & Wang, D. Click chemistry, a powerful tool for pharmaceutical
sciences. Pharm. Res. 25, 2216–2230 (2008).
44. Zhou, Y. et al. Chitosan microspheres with an extracellular matrix-mimicking nanofibrous
structure as cell-carrier building blocks for bottom-up cartilage tissue engineering. R. Soc. Chem.
8, 309–317 (2016).
45. Custódio, C. A., Cerqueira, M. T., Marques, A. P., Reis, R. L. & Mano, J. F. Cell selective
chitosan microparticles as injectable cell carriers for tissue regeneration. Biomaterials 43, 23–31
(2015).
46. Wang, M. & Kim, J. C. Microgels of poly(hydroxyethyl acrylate-co-coumaryl acrylate-co-
octadecyl acrylate): Photo-responsive release. Colloid Polym. Sci. 291, 2319–2327 (2013).
47. Ye, X. et al. Self-healing pH-sensitive cytosine- and guanosine-modified hyaluronic acid
hydrogels via hydrogen bonding. Polymer (Guildf). 108, 348–360 (2017).
48. Berger, J. et al. Structure and interactions in covalently and ionically crosslinked chitosan
hydrogels for biomedical applications. European Journal of Pharmaceutics and
Biopharmaceutics 57, 19–34 (2004).
49. Laurenti, M. et al. Synthesis of thermosensitive microgels with a tunable magnetic core. Am.
Chem. Soc. 27, 10484–10491 (2011).
50. Neto, A. I. et al. Fabrication of Hydrogel Particles of Defined Shapes Using Superhydrophobic-
Hydrophilic Micropatterns. Adv. Mater. 28, 7613–7619 (2016).
51. Tran, K. T. M. & Nguyen, T. D. Lithography-based methods to manufacture biomaterials at small
scales. J. Sci. Adv. Mater. Devices 2, 1–14 (2017).
52. Jung, S. & Yi, H. Facile Micromolding-Based Fabrication of Biopolymeric - Synthetic Hydrogel
Microspheres with Controlled Structures for Improved Protein Conjugation. Chem. Mater. 27,
3988–3998 (2015).
53. Oh, M. S. et al. Control of Reversible Self-Bending Behavior in Responsive Janus Microstrips.
ACS Appl. Mater. Interfaces 8, 8782–8788 (2016).
54. Heath, D. E. et al. Regenerating the cell resistance of micromolded PEG hydrogels. Lab Chip 15,
2073–2089 (2015).
27
Chapter 2 – Recent trends on microfabrication of hydrogels
55. Oh, J. K., Drumright, R., Siegwart, D. J. & Matyjaszewski, K. The development of
microgels/nanogels for drug delivery applications. Prog. Polym. Sci. 33, 448–477 (2008).
56. Farjami, T. & Madadlou, A. Fabrication methods of biopolymeric microgels and microgel-based
hydrogels. Food Hydrocoll. 62, 262–272 (2017).
57. Demirci, U. & Montesano, G. Single cell epitaxy by acoustic picolitre droplets. Lab Chip 7,
1139–1145 (2007).
58. Tasoglu, S. & Demirci, U. Bioprinting for stem cell research. Trends in Biotechnology 31, 10–19
(2013).
59. Wu, Z. et al. Bioprinting three-dimensional cell-laden tissue constructs with controllable
degradation. Sci. Rep. 6, 1–10 (2016).
60. Yanagawa, F., Sugiura, S. & Kanamori, T. Hydrogel microfabrication technology toward three
dimensional tissue engineering. Regen. Ther. 3, 45–57 (2016).
61. Murphy, S. V & Atala, A. 3D bioprinting of tissues and organs. Nat. Biotechnol. 32, 773–785
(2014).
62. Udey, R. N., Jones, a D. & Farquar, G. R. Aerosol and Microparticle Generation Using a
Commercial Inkjet Printer. Aerosol Sci. Technol. 47, 361–372 (2013).
63. Gao, Q., He, Y., Fu, J. Z., Qiu, J. J. & Jin, Y. an. Fabrication of shape controllable alginate
microparticles based on drop-on-demand jetting. J. Sol-Gel Sci. Technol. 77, 610–619 (2016).
64. Zhu, W. et al. 3D printing of functional biomaterials for tissue engineering. Current Opinion in
Biotechnology 40, 103–112 (2016).
65. Iwanaga, S., Saito, N., Sanae, H. & Nakamura, M. Facile fabrication of uniform size-controlled
microparticles and potentiality for tandem drug delivery system of micro/nanoparticles. Colloids
Surfaces B Biointerfaces 109, 301–306 (2013).
66. Zhu, K. et al. Gold Nanocomposite Bioink for Printing 3D Cardiac Constructs. Adv. Funct.
Mater. 27, 1–12 (2017).
67. Li, J., Chen, M., Fan, X. & Zhou, H. Recent advances in bioprinting techniques: approaches,
applications and future prospects. J. Transl. Med. 14, 1–15 (2016).
68. Keriquel, V. et al. In situ printing of mesenchymal stromal cells , by laser-assisted bioprinting ,
for in vivo bone regeneration applications. Sci. Rep. 7, 1–10 (2017).
69. Mandrycky, C., Wang, Z., Kim, K. & Kim, D. H. 3D bioprinting for engineering complex tissues.
Biotechnol. Adv. 34, 422–434 (2016).
70. Huang, R., Wang, Y., Qi, W., Su, R. & He, Z. Chemical catalysis triggered self-assembly for the
bottom-up fabrication of peptide nanofibers and hydrogels. Mater. Lett. 128, 216–219 (2014).
71. Wang, Q., Liu, S., Wang, H., Zhu, J. & Yang, Y. Alginate droplets pre-crosslinked in
microchannels to prepare monodispersed spherical microgels. Colloids Surfaces A Physicochem.
Eng. Asp. 482, 371–377 (2015).
72. Mao, A. S. et al. Deterministic encapsulation of single cells in thin tunable microgels for niche
modelling and therapeutic delivery. Nat. Mater. 1, 1–10 (2016).
73. Hu, Y., Azadi, G. & Ardekani, A. M. Microfluidic fabrication of shape-tunable alginate
microgels: Effect of size and impact velocity. Carbohydr. Polym. 120, 38–45 (2015).
28
Chapter 2 – Recent trends on microfabrication of hydrogels
74. Konry, T., Dominguez-Villar, M., Baecher-Allan, C., Hafler, D. A. & Yarmush, M. L. Droplet-
based microfluidic platforms for single T cell secretion analysis of IL-10 cytokine. Biosens.
Bioelectron. 26, 2707–2710 (2011).
75. Shi, Y. et al. High throughput generation and trapping of individual agarose microgel using
microfluidic approach. Microfluid. Nanofluidics 15, 467–474 (2013).
76. Dashtimoghadam, E., Mirzadeh, H., Taromi, F. A. & Nyström, B. Microfluidic self-assembly of
polymeric nanoparticles with tunable compactness for controlled drug delivery. Polymer (Guildf).
54, 4972–4979 (2013).
77. Bawazer, L. A. et al. Combinatorial microfluidic droplet engineering for biomimetic material
synthesis. Sci. Adv. 2, 1–12 (2016).
78. Shui, L., Eijkel, J. C. T. & Berg, A. van den. Multiphase flow in microfluidic systems – Control
and applications of droplets and interfaces. Adv. Colloid Interface Sci. 133, 35–49 (2007).
79. Yang, C. H. et al. Microfluidic-assisted synthesis of hemispherical and discoidal chitosan
microparticles at an oil/water interface. Electrophoresis 33, 3173–3180 (2012).
80. Shimanovich, U. et al. Protein microgels from amyloid fibril networks. ACS Nano 9, 43–51
(2015).
81. Krutkramelis, K., Xia, B. & Oakey, J. Monodisperse polyethylene glycol diacrylate hydrogel
microsphere formation by oxygen-controlled photopolymerization in a microfluidic device. Lab
Chip 16, 1457–1465 (2016).
82. Cheng, Y. et al. Controlled Fabrication of Bioactive Microfibers for Creating Tissue Constructs
Using Microfluidic Techniques. ACS Appl. Mater. Interfaces 8, 1080–1086 (2016).
83. Bock, N., Woodruff, M. A., Hutmacher, D. W. & Dargaville, T. R. Electrospraying, a
reproducible method for production of polymeric microspheres for biomedical applications.
Polymers (Basel). 3, 131–149 (2011).
84. Guarino, V., Khartini, W., Abdul, W. & Ambrosio, L. Biodegradable microparticles and
nanoparticles by electrospraying techniques. J. Appl. Biomater. Funct. Mater. 10, 191–196
(2012).
85. Altobelli, R., Guarino, V. & Ambrosio, L. Micro- and nanocarriers by electrofludodynamic
technologies for cell and molecular therapies. Process Biochem. 51, 2143–2145 (2016).
86. Jaworek, A. et al. Electrospinning and electrospraying techniques for nanocomposite non-woven
fabric production. Fibres Text. East. Eur. 17, 77–81 (2009).
87. Kim, P. H. et al. Injectable multifunctional microgel encapsulating outgrowth endothelial cells
and growth factors for enhanced neovascularization. J. Control. Release 187, 1–13 (2014).
88. Di, J., Kim, J., Hu, Q., Jiang, X. & Gu, Z. Spatiotemporal drug delivery using laser-generated-
focused ultrasound system. J. Control. Release 220, 592–599 (2015).
89. Yao, S., Yang, Y., Wang, X. & Wang, L. Fabrication and characterization of aligned fibrin
nanofiber hydrogel loaded with PLGA microspheres. Macromol. Res. 25, 528–533 (2017).
90. Oliveira, M. B., Kossover, O., Mano, J. F. & Seliktar, D. Injectable PEGylated fibrinogen cell-
laden microparticles made with a continuous solvent- and oil-free preparation method. Acta
Biomater. 13, 78–87 (2015).
29
Chapter 2 – Recent trends on microfabrication of hydrogels
91. Oh, J. K., Lee, D. I. & Park, J. M. Biopolymer-based microgels/nanogels for drug delivery
applications. Prog. Polym. Sci. 34, 1261–1282 (2009).
92. Silva, A. S., Tavares, M. T. & Aguiar-Ricardo, A. Sustainable strategies for nano-in-micro
particle engineering for pulmonary delivery. J. Nanoparticle Res. 16, 1–17 (2014).
93. Sollohub, K. & Cal, K. Spray drying technique: II. Current applications in pharmaceutical
technology. Journal of Pharmaceutical Sciences 99, 587–597 (2010).
94. Beck-Broichsitter, M., Strehlow, B. & Kissel, T. Direct fractionation of spray-dried polymeric
microparticles by inertial impaction. Powder Technol. 286, 311–317 (2015).
95. Song, W., Lima, A. C. & Mano, J. F. Bioinspired methodology to fabricate hydrogel spheres for
multi-applications using superhydrophobic substrates. Soft Matter 6, 5868–5871 (2010).
96. Lima, A. C., Custódio, C. A., Alvarez-Lorenzo, C. & Mano, J. F. Biomimetic methodology to
produce polymeric multilayered particles for biotechnological and biomedical applications. Small
9, 2487–2492 (2013).
97. Costa, A. M. S. & Mano, J. F. Solvent-free strategy yields size and shape-uniform capsules. J.
Am. Chem. Soc. 139, 1057–1060 (2017).
98. Costa, A. M. S., Alatorre-Meda, M., Oliveira, N. M. & Mano, J. F. Biocompatible polymeric
microparticles produced by a simple biomimetic approach. Langmuir 30, 4535–4539 (2014).
99. Hu, Y. et al. Controlled self-assembly of alginate microgels by rapidly binding molecule pairs.
Lab Chip 17, 2481–2490 (2017).
100. Xia, P. et al. Injectable Stem Cell Laden Open Porous Microgels That Favor Adipogenesis: In
Vitro and in Vivo Evaluation. ACS Appl. Mater. Interfaces 9, 34751–34761 (2017).
101. Lienemann, P. S. et al. Single cell-laden protease-sensitive microniches for long-term culture in
3D. Lab Chip 17, 727–737 (2017).
102. Alfred, R. et al. Efficient suspension bioreactor expansion of murine embryonic stem cells on
microcarriers in serum-free medium. Biotechnol. Prog. 27, 811–823 (2011).
103. Van Wezel, A. L. Growth of cell-strains and primary cells on micro-carriers in homogeneous
culture. Nature 216, 64–65 (1967).
104. Zhang, Z. Injectable Biomaterials for Stem Cell Delivery and Tissue Regeneration. Expert Opin.
Biol. Ther. 17, 49–62 (2017).
105. Oliveira, M. B. & Mano, J. F. Polymer-based microparticles in tissue engineering and
regenerative medicine. Biotechnol. Prog. 27, 897–912 (2011).
106. Li, B. et al. Past, present, and future of microcarrier-based tissue engineering. Journal of
Orthopaedic Translation 3, 51–57 (2015).
107. Mano, F. J. Stimuli-Responsive Polymeric Systems for Biomedical Applications. Adv. Eng.
Mater. 10, 515–527 (2008).
108. Nie, Y., Bergendahl, V., Hei, D. J., Jones, J. M. & Palecek, S. P. Scalable Culture and
Cryopreservation of Human Embryonic Stem Cells on Microcarriers. Biotechnol. Prog. 25, 20–31
(2009).
109. Bender, M. D., Bennett, J. M., Waddell, R. L., Doctor, J. S. & Marra, K. G. Multi-channeled
biodegradable polymer/CultiSpher composite nerve guides. Biomaterials 25, 1269–1278 (2004).
30
Chapter 2 – Recent trends on microfabrication of hydrogels
110. de Soure, A. M. et al. Integrated culture platform based on a human platelet lysate supplement for
the isolation and scalable manufacturing of umbilical cord matrix-derived mesenchymal
stem/stromal cells. J. Tissue Eng. Regen. Med. 11, 1630–1640 (2017).
111. Mano, J. F. et al. Natural origin biodegradable systems in tissue engineering and regenerative
medicine: present status and some moving trends. J. R. Soc. Interface 4, 999–1030 (2007).
112. Correia, C. R. et al. Semipermeable capsules wrapping a multifunctional and self-regulated co-
culture microenvironment for osteogenic differentiation. Sci. Rep. 6, 1–12 (2016).
113. Correia, C. R. et al. In vivo osteogenic differentiation of stem cells inside compartmentalized
capsules loaded with co-cultured endothelial cells. Acta Biomater. 53, 483–494 (2017).
114. Oliveira, M. B. & Mano, J. F. High-throughput screening for integrative biomaterials design:
Exploring advances and new trends. Trends Biotechnol. 32, 627–636 (2014).
115. Oliveira, M. B. et al. Superhydrophobic chips for cell spheroids high-throughput generation and
drug screening. ACS Appl. Mater. Interfaces 6, 9488–9495 (2014).
116. Mohanraj, B. et al. A high throughput mechanical screening device for cartilage tissue
engineering. J. Biomech. 47, 2130–2136 (2014).
117. Rasi Ghaemi, S., Harding, F. J., Delalat, B., Gronthos, S. & Voelcker, N. H. Exploring the
mesenchymal stem cell niche using high throughput screening. Biomaterials 34, 7601–7615
(2013).
118. Guermani, E. et al. Engineering complex tissue-like microgel arrays for evaluating stem cell
differentiation. Sci. Rep. 6, 1–8 (2016).
119. Salgado, C. L., Oliveira, M. B. & Mano, J. F. Combinatorial cell–3D biomaterials
cytocompatibility screening for tissue engineering using bioinspired superhydrophobic substrates.
Integr. Biol. 4, 318 (2012).
120. Oliveira, M. B., Luz, G. M. & Mano, J. F. A combinatorial study of nanocomposite hydrogels:
on-chip mechanical/viscoelastic and pre-osteoblast interaction characterization. J. Mater. Chem.
B 2, 5627–5638 (2014).
121. Lyon, L. A. & Serpe, M. J. Hydrogel Micro and Nanoparticles. Wiley (2012).
doi:10.1002/9783527646425
122. Smeets, N. M. B. & Hoare, T. Designing responsive microgels for drug delivery applications. J.
Polym. Sci. Part A Polym. Chem. 51, 3027–3043 (2013).
123. Rodríguez-Velázquez, E., Alatorre-Meda, M. & Mano, J. F. Polysaccharide-Based
Nanobiomaterials as Controlled Release Systems for Tissue Engineering Applications. Curr.
Pharm. Des. 21, 4837–4850 (2015).
124. Sood, N., Bhardwaj, A., Mehta, S. & Mehta, A. Stimuli-responsive hydrogels in drug delivery
and tissue engineering. Drug Deliv. 23, 748–770 (2016).
125. Lee, E. et al. Development of smart delivery system for ascorbic acid using pH-responsive P (
MAA-co-EGMA ) hydrogel microparticles. Drug Deliv. 17, 573–580 (2010).
126. Sivakumaran, D., Maitland, D. & Hoare, T. Injectable microgel-hydrogel composites for
prolonged small-molecule drug delivery. Biomacromolecules 12, 4112–4120 (2011).
127. Lai, W.-F. et al. Electrospray-mediated preparation of compositionally homogeneous core–shell
31
Chapter 2 – Recent trends on microfabrication of hydrogels
hydrogel microspheres for sustained drug release. RSC Adv. 7, 44482–44491 (2017).
128. Rahman, A., Majewski, P. W., Doerk, G., Black, C. T. & Yager, K. G. Non-native three-
dimensional block copolymer morphologies. Nat. Commun. 7, 1–8 (2016).
129. Guven, S. et al. Multiscale assembly for tissue engineering and regenerative medicine. Trends
Biotechnol. 33, 269–279 (2015).
130. Franchi, S. et al. Self-assembling peptide hydrogels immobilized on silicon surfaces. Mater. Sci.
Eng. C 69, 200–207 (2016).
131. Xu, F. et al. The assembly of cell-encapsulating microscale hydrogels using acoustic waves.
Biomaterials 32, 7847–7855 (2011).
132. Breen, T. L., Tien, J., Oliver, S. R. J., Hadzic, T. & Whitesides, G. M. Design and Self-Assembly
of Open, Regular, 3D Mesostructures. Science (80-. ). 284, 948–951 (1999).
133. Choi, I. S., Bowden, N. & Whitesides, G. M. Macroscopic, hierarchical, two-dimensional self-
assembly. Angew. Chemie - Int. Ed. 38, 3078–3081 (1999).
134. Du, Y. et al. Surface-directed assembly of cell-laden microgels. Biotechnol. Bioeng. 105, 655–
662 (2010).
135. Oliveira, N. M. et al. Hydrophobic Hydrogels: Toward Construction of Floating
(Bio)microdevices. Chem. Mater. 28, 3641–3648 (2016).
136. Naseer, S. M. et al. Surface acoustic waves induced micropatterning of cells in gelatin
methacryloyl ( GelMA ) hydrogels Surface acoustic waves induced micropatterning of cells in
gelatin methacryloyl ( GelMA ) hydrogels. Biofabrication 9, 1–11 (2017).
137. Li, Y. et al. Magnetic hydrogels and their potential biomedical applications. Adv. Funct. Mater.
23, 660–672 (2013).
138. Gil, S. & Mano, J. F. Magnetic composite biomaterials for tissue engineering. Biomater. Sci. 2,
812–818 (2014).
139. Xu, F. et al. Three-dimensional magnetic assembly of microscale hydrogels. Adv. Mater. 23,
4254–4260 (2011).
140. Li, C. Y., Wodd, D. K., Hsu, C. M. & Bhatia, S. N. DNA-templated assembly of droplet-derived
PEG microtissues. Lab Chip 11, 2967–2975 (2011).
141. Qi, H. et al. DNA-directed self-assembly of shape-controlled hydrogels. Nat. Commun. 4, 1–10
(2013).
142. Cruz, D. M. G. et al. Chitosan microparticles as injectable scaffolds for tissue engineering. J.
Tissue Eng. Regen. Med. 2, 378–380 (2008).
143. Oliveira, M. B. et al. Development of an injectable system based on elastin-like recombinamer
particles for tissue engineering applications. Soft Matter 7, 6426–6434 (2011).
CHAPTER 3
Materials and methods
32
Chapter 3 – Materials and Methods
Chapter 3 – Materials and Methods
1. Laminarin
Laminarin is a storage glucan found in many macro-algae and most phytoplankton, and
is one of the most abundant carbon sources in the marine ecosystem. It is a glucan wherein
units of glucose are bonded through β-(1,3) repeating units, with sporadic β-(1,6)
branches, easily soluble in aqueous and organic solvents forming clear and stable
solutions.12 Laminarin has been shown to stimulate immunity, and to have antitumor
effects and antibacterial activity.1,3 An attractive feature of this particular polymer is its
inherent low viscosity and high water solubility over a temperature range of 4ºC to 40ºC
that facilitates processing. This is especially useful for microfabrication protocols such
microfluidics processing.3
2. Platelet Lysates
Platelet derived products include Platelet Lysates (PLs) and Platelet Rich Plasma (PRP)
and have been studied and used since the 1970s.4 It is now well established that platelets
are an important source of autologous GFs that can modulate stem cell proliferation and
differentiation. Human PLs can be generated through a simple freeze-thaw procedure of
platelet units. In this work, PLs were purchased from STEMCELL Technologies.
Multiple donor units are pooled during manufacturing to minimize lot-to-lot variability
and multiple lots were used during the experiments.
3. Methods
3.1.Synthesis and characterization of methacrylated laminarin
Methacrylated laminarin (MeLam) was modified by a common chemical reaction
following the protocol previously described.3 Briefly, MeLam was synthetized by
reacting laminarin (6kDa) (Carbosynth, U.K.) with glycidyl methacrylate (Acros
Organics, Germany). Laminarin (1g) and 4-(N,N-dimethylamino)pyridine (DMAP) (167
mg) (Acros Organic, Germany) were dissolved in 10 mL of dimethyl sulfoxide (DMSO)
(Sigma-Aldrich, Germany) under nitrogen atmosphere. Varying the amount of glycidyl
methacrylate added is possible to manipulate the degree of modification; low degree of
modification was obtained by adding 2.9 × 10-3 mol of glycidyl methacrylate to the
33
Chapter 3 – Materials and Methods
previously prepared solution and high degree of modification by adding 5.1 × 10-3 mol of
glycidyl methacrylate. The mixture was stirred at room temperature (RT) for 48 hours
protected from light, being stopped by adding HCl solution (37%) (Sigma-Aldrich, USA)
to neutralize DMAP. Subsequently, the solution was purified by dialysis using a
benzoylated membrane (2000 MWCO) (Sigma-Aldrich, USA) for at least 7 days in
distilled water. The final product was freeze-dried and stored at RT until further use.
Degree of substitution (DS, fraction of modified hydroxyl groups per repeating unit) was
calculated by 1H NMR (Bruker Avance III (300 MHz)) by integrating the peak
correspondent to the acetyl group of the methacrylate centered at ∼2 ppm (IAc) against
the polymer backbone region ∼3 –5.5 ppm (ILam). The following formula (Eq. 1) was
used to calculate the DS value:
𝐷𝑆 =𝐼𝐴𝐶
𝐼𝐿𝑎𝑚 Equation 1
3.2.Fabrication of MeLam microparticles
For the aqueous phase, MeLam was dissolved in phosphate buffered saline (PBS, pH 7.4)
(Sigma, USA) at a concentration of 15% (w/v) with 0.5% (w/v) 2-hydroxy-4-(2-
hydroxyethoxy)-2-methylpropiophenone (Sigma, USA). Moreover, biotin-PEG-thiol
(Polypure AS, Norway) (0.5 mg/mL) was also added to the previously prepared solution.
Biotinylated microparticles are then formed by a Michael-type addition between thiol
group of biotin and alkene group of MeLam. The previously prepared solution was loaded
into plastic syringes (BD Luer-Lok syringe) and connected to the inlets with Fluorinated
Ethylene Propylene (FEP) tubing. For the continuous phase, mineral oil (Fisher) was
loaded into the same type of syringes. Syringe pumps (Harvard Apparatus, PhD Ultra,
USA) were used to inject fluids at controlled flow rates into the microfluidic chip (figure
3.1 A). For these experiments, a T-junction hydrophobic microfluidic chip with header
(190µm etch depth) (Dolomite, UK) was used. This chip allows for the fabrication of
water-in-oil droplets in the size range Ø100 – 300µm (figure 3.1 B). Upon formation in
the flow-focusing device, droplets were photopolymerized with UV light (Omnicure
S2000, Canada) to form microparticles. The outlet tubing (0.5 mm of diameter) was
coiled to make a spiral microchannel ensuring that the microdroplets are kept for at least
60 seconds under UV light (6.12 W/cm2) for the efficient crosslinking of microparticles
(figure 3.1 C). For the experiments, flow rates are set by the syringe pumps to be 8 μl/min
34
Chapter 3 – Materials and Methods
for the aqueous phase (QAq) and 160 μl/min for the continuous phase (QC). The MeLam
microparticles were collected to a falcon (15 mL) and the mineral oil removed after
centrifugation.
To produce microparticles with encapsulated platelet lysates (PL) the protocol was
slightly changed by including PL in the aqueous phase. Briefly, PL (25% w/v) was mixed
with the MeLam solution previously dissolved in PBS and loaded into the plastic
syringes.
Figure 3.1: Schematic representation of microfluidic system used to produce MeLam microparticles. (A)
Schematic representation of PDMS microchip with T-junction shape. (B) Droplets formation into the
microchip. (C) UV crosslinked MeLam microparticles
3.3.RGD functionalization of MeLam microparticles
To access the functionalization with biotin, modified particles were incubated with
DyLight 488 Streptavidin (BioLegend) (10μg/ml), washed with PBS and observed under
fluorescence microscopy (Zeiss, Axio Imager M2). Polymeric microparticles with
covalently attached biotin are proposed as versatile targeting vehicles for multiple
biomolecules, in this work the particles were further functionalized with the tripeptide
Arg-Gly-Asp (RGD) to promote cell adhesiveness.
Biotinylated microparticles were incubated with purified streptavidin (Promega)
(25μg/ml) in PBS under constant stirring for 15min at room temperature. A washing step
was then performed to remove unbound streptavidin. Finally, the particles were incubated
with biotinylated RGD (25 μg/mL) (AnaSpec, USA) in PBS under constant stirring for
15min at room temperature (figure 3.2). The microparticles were washed with PBS
solution to remove all unbound RGD biotinylated.
35
Chapter 3 – Materials and Methods
3.4.Scanning electron microscopy (SEM)
SEM was performed as a means to evaluate the morphology and porosity of the
microparticles produced by microfluidics. For SEM analysis, particles were dried at 25ºC
for 3 days), sputtered with gold and evaluated by SEM (Hitachi SU-70, Japan).
3.5.Studies on the release of PL
Aliquots of the microparticles (triplicate samples from each batch) were suspended in 5
mL phosphate buffered saline (PBS, pH 7.4); samples were gently shaked at 60 rpm in a
water bath at 37ºC. At defined time intervals, 500 μL of PBS were removed and replaced
with 500 μl fresh PBS. The removed supernatants were stored frozen until required and
were then assayed for total protein content using the Micro BCA assay kit. Briefly, 50 μL
of the collected samples were diluted in 100 μL of PBS solution and mixed with 150 μL
of the working solution and incubated for 2 hours at 37ºC. Afterwards, the quantity of
protein was measured by absorbance at a wavelength 592 nm in a Synergy HTX multi-
mode reader (Biotek Instruments, Inc, USA).
The total protein release was calculated following equation described by Hailong5
equation (2):
𝐶𝑢𝑚𝑢𝑙𝑎𝑡𝑖𝑣𝑒 𝑃𝐿 𝑟𝑒𝑙𝑒𝑎𝑠𝑒 (%) = 𝑉𝑒 ∑ 𝐶𝑖+ 𝑉0𝐶𝑛
𝑛−1𝑖
𝑚𝑑𝑟𝑢𝑔 ×100 Equation 2
ELISA assay (ThermoFisherScientific, USA) was also performed to evaluate the release
of transforming growth factor (TGF-β1) and vascular epithelial growth factor (VEGF)
from the microparticles. The assay was performed according to the manufacturer’s
standard protocols. The optical density values were measured using a Synergy HTX
multi-mode reader (Biotek Instruments, Inc, USA) set at 450 nm.
3.6.Cell culture on MeLam microparticles
For the cell culture tests, microparticles prepared with high degree of substitution were
used. Mouse fibroblast (L929) were used to verify the potential of methacrylated
laminarin microparticles as cell carriers (figure 3.2). L929 were cultured in Dulbecco’s
modified Eagle’s medium DMEM (Thermo Scientific, USA) supplemented with 10% of
fetal bovine serum (FBS) and 1% antibiotic/antimycotic at 37ºC under 5% CO2. The high
MeLam 15% (w/v) microparticles were seeded with cells in non-adherent 48 well
microplates in portion 200 microparticles per 1×104 cells under gently shaking (150 rpm)
36
Chapter 3 – Materials and Methods
and at cell culture conditions (5% CO2, 37ºC). The cultured microparticles were
maintained in culture for 11 days. At pre-determined time points, cell morphology, cell
viability and proliferation were assessed. The assembly process was followed and imaged
at day 11. Constructs were evaluated for cell proliferation.
Figure 3.2: Schematic representation of bioconjugation streptavidin and biotinylated RGD with MeLam
microparticles. After modification, microparticles were cultured with L929 cells. This scheme also
represents cell attachment on the microparticles and subsequent assembly of the structures.
3.7.Cell morphology analysis
Phalloidin and DAPI staining were used to visualize actin cytoskeleton and to label the
DNA, respectively. The assay was conducted as outlined by the supplier’s protocol
(Sigma, Germany). Briefly, cultured microparticles were washed with PBS, fixed in 4%
formaldehyde/PBS (v/v) for 1h at room temperature (RT) and washed extensively in PBS
to remove all traces of the fixative. Cells were then stained with 50 μg/ml fluorescent
phalloidin-conjugate solution in PBS for 45 min at room temperature. DAPI labeling
solution 0.5 μg/ml was incubated for 5 min at room temperature. The microparticles were
washed in PBS to remove remaining staining solutions and imaged using a fluorescent
microscope Zeiss Imager M2 (Carl Zeiss SMT Inc).
37
Chapter 3 – Materials and Methods
3.7.1. DNA quantification
Cell proliferation on laminarin microparticles was determined by DNA quantification
using a fluorimetric dsDNA quantification kit (PicoGreen, Invitrogen). This assay allows
measurement of the fluorescence produced when PicoGreen dye is excited by UV light
while bounded to dsDNA. After each time-point, cells were lysed by osmotic and thermal
shock and the supernatant used for double-stranded DNA (dsDNA) content analysis.
Briefly, samples collected after each time point were washed with PBS and immersed in
1 ml of ultrapure water, frozen for at −80 °C, thawed at room temperature, and sonicated
for 30 min. Fluorescence was measured on a microplate reader (Synergy HTX multi-
mode reader). The DNA amount was calculated from a standard curve
3.7.2 MTS Viability Assay
The MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2Htetrazolium) assay (Promega) was performed to evaluate cell viability.
Briefly, after each time period, culture media was removed and cells were washed with
PBS solution. The MTS solution 1:10 ratio to PBS was added to the cells and incubated
for 4 h (37ºC, 5% CO2). After the incubation period, the optical density (OD) was read
at 490 nm in a microplate reader (Synergy HTX multi-mode reader).
3.8.Statistical analysis
All results were subjected a statistical analysis and results were presented as mean ±
standard deviation. Statistical analysis of results was performed using Student’s t-test,
with a significant level of 95% (p < 0.05).
4. References
1. Anjugam, M. et al. A study on β-glucan binding protein (β-GBP) and its involvement in
phenoloxidase cascade in Indian white shrimp Fenneropenaeus indicus. Mol. Immunol. 92, 1–11
(2017).
2. Wang, D. et al. The first bacterial β-1,6-endoglucanase from Saccharophagus degradans 2-40T
for the hydrolysis of pustulan and laminarin. Appl. Microbiol. Biotechnol. 101, 197–204 (2017).
3. Custódio, C. A., Reis, R. L. & Mano, J. F. Photo-Cross-Linked Laminarin-Based Hydrogels for
Biomedical Applications. Biomacromolecules 17, 1602–1609 (2016).
4. Soomekh, D. J. Current Concepts for the Use of Platelet-Rich Plasma in the Foot and Ankle. Clin.
Podiatr. Med. Surg. 28, 155–170 (2011).
38
Chapter 3 – Materials and Methods
5. Che, H. et al. CO 2 -switchable drug release from magneto-polymeric nanohybrids. Polym. Chem.
6, 2319–2326 (2015).
CHAPTER 4
Multifunctional laminarin microparticles
for cell adhesion and expansion
39
Chapter 4 - Multifunctional laminarin microparticles for cell adhesion and
expansion
Chapter 4 - Multifunctional laminarin microparticles for cell adhesion
and expansion
Martins CR,1 Custódio CA,1 Mano JF1
1-Department of Chemistry, CICECO, Aveiro Institute of Materials, University of Aveiro Campus
Universitário de Santiago, 3810-193 Aveiro - Portugal
Abstract
Microfabrication technologies have been explored to produce microgels that can be
assembled in functional constructs for tissue engineering applications. Here, we propose
microfluidics coupled to a source of UV light to produce monodisperse multifunctional
laminarin microparticles by photopolymerization. In an attempt to enhance cell adhesion
and proliferation, the microparticles were loaded with platelet lysates and further
conjugated with an adhesive peptide. The modified microparticles were cultured with
L929 cells, the results showed a high cellular adhesion on microparticles with
encapsulated platelet lysates. The multifunctional laminarin microparticles provide an
effective support for cell attachment and cell expansion, moreover the microparticles tend
to aggregate in robust 3D structures. This showed the potential for using the
microplatforms to rapidly produce large tissue engineered constructs.
Key words: Microfluidic, microcarrier, microgels, platelet lysates
1. Introduction
Living tissues are hierarchically organized three-dimensional (3D) structures composed
of multiple types of cells and extracellular matrix (ECM).1 Thus, effective strategies to
engineer living constructs that mimic native tissues requires the development of structures
with well-defined spatial distributions of different cells embedded in ECM.
Current strategies for tissue and organ development include “bottom-up” tissue
engineering, that consists in the self-assembly of smaller units to build a 3D construct or
“top-down” approaches, involves scaffold-based cell-seeding.
Bottom-up approaches for the directed or random assembly of microunits, which mimic
the living tissue architecture from repeating functional units, have been gaining increasing
40
Chapter 4 - Multifunctional laminarin microparticles for cell adhesion and
expansion
attention in tissue engineering applications.2–5 In the last years, different microfabrication
strategies have been explored for the production of miniaturized structures for cell culture
and cell encapsulation.5–7 Given the advantages including high continuity, reproducibility
and scalability, microfluidics have been widely used to fabricate microparticles with
controlled sizes and structures.8,9
Laminarin is a natural polymer obtained from brown algae with low molecular weight
and low viscosity.10 These properties make this polymer particularly appealing to be used
in microfabrication techniques. Laminarin hydrogels have recently been proposed as a
platform for cell encapsulation and good cell viability has been demonstrated.11 Addition
of methacrylate groups to the hydroxyl-containing groups of laminarin was performed to
make it light polymerizable into a hydrogel.
In this work, we report a simple and efficient microfluidic approach to produce
monodisperse laminarin microgels with encapsulated PL. The methacrylate groups act
also as anchoring sites for the immobilization of thiol-biotin molecules and streptavidin,
which subsequently form complexes with biotin-RGD. Cell adhesion to the microgels
was enhanced by the conjugation of the microparticles with an adhesive peptide (RGD).
The use of streptavidin–biotin should be applicable to a broad range of proteins,
expanding the options available for microparticles modification. By using a
biocompatible and biodegradable polymer as support for cell culture, we provide
simultaneously a support for cell culture and expansion that can be also used to fill tissue
defects in tissue engineering strategies.
Additionally, encapsulation of platelet lysates (PL) within the microparticles showed
improved cell attachment and expansion in the laminarin microgels. PL, offer much
potential owing to its autogenous nature and high content of proteins and growth factors
(GFs). PL have been used as a natural source of GFs for tissue regeneration purposes and
as a substitute of animal derived components for in vitro cell culture.12
2. Materials and Methods
2.1.Synthesis and characterization of methacrylated laminarin
Methacrylated laminarin (MeLam) was modified by a common chemical reaction
following the protocol previously described.11 Briefly, MeLam was synthetized by
reacting laminarin (6kDa) (Carbosynth, U.K.) with glycidyl methacrylate (Acros
Organics, Germany). Laminarin (1g) and 4-(N,N-dimethylamino)pyridine (DMAP) (167
41
Chapter 4 - Multifunctional laminarin microparticles for cell adhesion and
expansion
mg) (Acros Organic, Germany) were dissolved in 10 mL of dimethyl sulfoxide (DMSO)
(Sigma-Aldrich, Germany) under nitrogen atmosphere. Varying the amount of glycidyl
methacrylate added is possible to manipulate the degree of modification; low degree of
modification was obtained by adding 2.9 × 10-3 mol of glycidyl methacrylate to the
previously prepared solution and high degree of modification by adding 5.1 × 10-3 mol of
glycidyl methacrylate. The mixture was stirred at room temperature for 48 hours protected
from light, being stopped by adding HCl solution (37%) (Sigma-Aldrich, USA) to
neutralize DMAP. Subsequently, the solution was purified by dialysis using a
benzoylated membrane (2000 MWCO) (Sigma-Aldrich, USA) for at least 7 days in
distilled water. The final product was freeze-dried and stored at room temperature until
further use.
Degree of substitution (DS, fraction of modified hydroxyl groups per repeating unit) was
calculated by 1H NMR (Bruker Avance III (300 MHz)) by integrating the peak
correspondent to the acetyl group of the methacrylate centered at ∼2 ppm (IAc) against
the polymer backbone region ∼3 –5.5 ppm (ILam). The following formula (Eq. 1) was
used to calculate the DS value:
𝐷𝑆 =𝐼𝐴𝐶
𝐼𝐿𝑎𝑚 Equation 1
2.2.Fabrication of MeLam microparticles
For the aqueous phase, MeLam was dissolved in phosphate buffered saline (PBS, pH 7.4)
(Sigma, USA) at a concentration of 15% (w/v) with 0.5% (w/v) 2-hydroxy-4-(2-
hydroxyethoxy)-2-methylpropiophenone (Sigma, USA). Moreover, biotin-PEG-thiol
(Polypure AS, Norway) (0.5 mg/mL) was also added to the previously prepared solution.
Biotinylated microparticles are then formed by a Michael-type addition between thiol
group of biotin and alkene group of MeLam. The previously prepared solution was loaded
into plastic syringes (BD Luer-Lok syringe) and connected to the inlets with Fluorinated
Ethylene Propylene (FEP) tubing. For the continuous phase, mineral oil (Fisher) was
loaded into the same type of syringes. Syringe pumps (Harvard Apparatus, PhD Ultra,
USA) were used to inject fluids at controlled flow rates into the microfluidic chip (figure
4.1 A). For these experiments, a T-junction hydrophobic microfluidic chip with header
(190µm etch depth) (Dolomite, UK) was used. This chip allows for the fabrication of
water-in-oil droplets in the size range Ø 100 – 300µm (figure 4.1 B). Upon formation in
42
Chapter 4 - Multifunctional laminarin microparticles for cell adhesion and
expansion
the flow-focusing device, droplets were photopolymerized with UV light (Omnicure
S2000, Canada) to form microparticles. The outlet tubing (0.5 mm of diameter) was
coiled to make a spiral microchannel ensuring that the microdroplets are kept for at least
60 seconds under UV light (6.12 W/cm2) for the efficient crosslinking of microparticles
(figure 4.1 C). For the experiments, flow rates are set by the syringe pumps to be 8 μl/min
for the aqueous phase and 160 μl/min for the continuous phase. The MeLam
microparticles were collected to a falcon (15 mL) and the mineral oil removed after
centrifugation.
To produce microparticles with encapsulated platelet lysates (PL) the protocol was
slightly changed by including PL in the aqueous phase. Briefly, PL (25% w/v) was mixed
with the MeLam solution previously dissolved in PBS and loaded into the plastic
syringes.
Figure 4.1: Schematic representation of microfluidic system used to produce MeLam microparticles. (A)
Schematic representation of PDMS microchip with T-junction shape. (B) Droplets formation into the
microchip. (C) UV crosslinked MeLam microparticles
2.3.RGD functionalization of MeLam microparticles
To access the functionalization with biotin, modified particles were incubated with
DyLight 488 Streptavidin (BioLegend) (10μg/ml), washed with PBS and observed under
fluorescence microscopy (Zeiss, Axio Imager M2). Polymeric microparticles with
covalently attached biotin are proposed as versatile targeting vehicles for multiple
biomolecules, in this work the particles were further functionalized with the tripeptide
Arg-Gly-Asp (RGD) to promote cell adhesiveness.
Biotinylated microparticles were incubated with purified streptavidin (Promega)
(25μg/ml) in PBS under constant stirring for 15min at room temperature. A washing step
was then performed to remove unbound streptavidin. Finally, the particles were incubated
43
Chapter 4 - Multifunctional laminarin microparticles for cell adhesion and
expansion
with biotinylated RGD (25 μg/mL) (AnaSpec, USA) in PBS under constant stirring for
15min at room temperature. The microparticles were washed with PBS solution to
remove all unbound RGD biotinylated.
2.4.Scanning electron microscopy (SEM)
SEM was performed as a means to evaluate the morphology and porosity of the
microparticles produced by microfluidics. For SEM analysis, particles were dried at 25ºC
for 3 days), sputtered with gold and evaluated by SEM (Hitachi SU-70, Japan).
2.5.Studies on the release of PL
Aliquots of the microparticles (triplicate samples from each batch) were suspended in 5
mL phosphate buffered saline (PBS, pH 7.4), samples were gently shaked at 60 rpm in a
water bath at 37ºC. At defined time intervals, 500 μL of PBS were removed and replaced
with 500 μL fresh PBS. The removed supernatants were stored frozen until required and
were then assayed for total protein content using the Micro BCA assay kit. Briefly, 50 μL
of the collected samples were diluted in 100 μL of PBS solution and mixed with 150 μL
of the working solution and incubated for 2 hours at 37ºC. Afterwards, the quantity of
protein was measured by absorbance at a wavelength 592 nm in a Synergy HTX multi-
mode reader (Biotek Instruments, Inc, USA).
The total protein release was calculated following equation described by Hailong13
equation (2):
𝐶𝑢𝑚𝑢𝑙𝑎𝑡𝑖𝑣𝑒 𝑃𝐿 𝑟𝑒𝑙𝑒𝑎𝑠𝑒 (%) = 𝑉𝑒 ∑ 𝐶𝑖+ 𝑉0𝐶𝑛
𝑛−1𝑖
𝑚𝑑𝑟𝑢𝑔 ×100 Equation 2
ELISA assay (ThermoFisherScientific, USA) was also performed to evaluate the release
of transforming growth factor (TGF-β1) and vascular epithelial growth factor (VEGF)
from the microparticles. The assay was performed according to the manufacturer’s
standard protocols. The optical density values were measured using a Synergy HTX
multi-mode reader (Biotek Instruments, Inc, USA) set at 450 nm.
2.6.Cell culture on MeLam microparticles
For the cell culture tests, microparticles prepared with high degree of substitution were
used. Mouse fibroblast (L929) were used to verify the potential of methacrylated
44
Chapter 4 - Multifunctional laminarin microparticles for cell adhesion and
expansion
laminarin microparticles as cell carriers (figure 4.2). L929 were cultured in Dulbecco’s
modified Eagle’s medium DMEM (Thermo Scientific, USA) supplemented with 10% of
fetal bovine serum (FBS) and 1% antibiotic/antimycotic at 37ºC under 5% CO2. The high
MeLam 15% (w/v) microparticles were seeded with cells in non-adherent 48 well
microplates in portion 200 microparticles per 1×104 cells under gently shaking (150 rpm)
and at cell culture conditions (5% CO2, 37ºC). The cultured microparticles were
maintained in culture for 11 days. At pre-determined time points, cell morphology, cell
viability and proliferation were assessed. The assembly process was followed and imaged
at day 11. Constructs were evaluated for cell proliferation.
Figure 4.2: Schematic representation of bioconjugation streptavidin and biotinylated RGD with MeLam
microparticles. After modification, microparticles were cultured with L929 cells. This scheme also
represents cell attachment on the microparticles and subsequent assembly of the structures.
2.7.Cell morphology analysis
Phalloidin and DAPI staining were used to visualize actin cytoskeleton and to label the
DNA, respectively. The assay was conducted as outlined by the supplier’s protocol
(Sigma, Germany). Briefly, cultured microparticles were washed with PBS, fixed in 4%
45
Chapter 4 - Multifunctional laminarin microparticles for cell adhesion and
expansion
formaldehyde/PBS (v/v) for 1h at room temperature (RT) and washed extensively in PBS
to remove all traces of the fixative. Cells were then stained with 50 μg/ml fluorescent
phalloidin-conjugate solution in PBS for 45 min at room temperature. DAPI labeling
solution 0.5 μg/ml was incubated for 5 min at room temperature. The microparticles were
washed in PBS to remove remaining staining solutions and imaged using a fluorescent
microscope Zeiss Imager M2 (Carl Zeiss SMT Inc).
2.8.DNA quantification
Cell proliferation on laminarin microparticles was determined by DNA quantification
using a fluorimetric dsDNA quantification kit (PicoGreen, Invitrogen). This assay allows
measurement of the fluorescence produced when PicoGreen dye is excited by UV light
while bounded to dsDNA. After each time-point, cells were lysed by osmotic and thermal
shock and the supernatant used for double-stranded DNA (dsDNA) content analysis.
Briefly, samples collected after each time point were washed with PBS and immersed in
1 mL of ultrapure water, frozen for at −80 °C, thawed at room temperature, and sonicated
for 30 min. Fluorescence was measured on a microplate reader (Synergy HTX multi-
mode reader). The DNA amount was calculated from a standard curve.
2.9.MTS Viability Assay
The MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2Htetrazolium) assay (Promega) was performed to evaluate cell viability.
Briefly, after each time period, culture media was removed and cells were washed with
PBS solution. The MTS solution 1:10 ratio to PBS was added to the cells and incubated
for 4 h (37ºC, 5% CO2). After the incubation period, the optical density (OD) was read
at 490 nm in a microplate reader (Synergy HTX multi-mode reader).
2.10. Statistical analysis
All results were subjected a statistical analysis and results were presented as mean ±
standard deviation. Statistical analysis of results was performed using Student’s t-test,
with a significant level of 95% (p < 0.05).
46
Chapter 4 - Multifunctional laminarin microparticles for cell adhesion and
expansion
3. Results and Discussion
3.1.Synthesis and Characterization of Methacrylated Laminarin
Laminarin is a low-molecular-weight polysaccharide and bioactive compound present in
brown algae.10 The abundance of hydroxyl groups in the laminarin structure may be used
to insert a polymerizable moiety or to chemically bind a bioactive agent. To synthesize
laminarin with UV crosslinking ability via grafting acrylate units, the modification of
laminarin using glycidyl methacrylate has recently been proposed.11 Briefly,
methacrylated laminarin was synthesized as a hydrogel precursor by taking advantage of
the functionality of the hydroxyl groups in laminarin as well as the reactivity of the epoxy
group in glycidyl methacrylate. (figure 4.3 A). In this work, the functionalization protocol
was followed as previously described and two different degree of modification were
obtained. The chemical modification and degree of substitution of laminarin was assessed
by 1H NMR. Comparing the original 1H NMR spectrum of laminarin (figure 4.3 B) with
the 1H NMR spectrum (figure 4.3 C and D) after modification it was possible to observe
the appearance of two new peaks. The peaks at δ = 5.7 ppm and δ = 6.1 ppm corresponds
of vinylic protons (C=CH2) and the peak at δ = 1.9 ppm corresponds of the protons of
methyl group (CH3). Integration and normalization of the methyl group peak in the
methacrylate segment in relation to the hydrogen peaks of the laminarin backbone (3 ∼
5.5 ppm) provides consistent means for calculating the degree of substitution of hydroxyl
groups in laminarin by the methacrylate group. The average degree substitution increased
from 30% (Low - MeLam) to 60% (High -MeLam), by adding 7% (400 μl) and 14%
(700 μl) (v/v) glycidyl methacrylate to laminarin during the synthesis.11 This value is
relative to the total amount of hydrogens per monomer of laminarin. The equation 1 give
the value of the substitutions taking account the total of hydrogens.
47
Chapter 4 - Multifunctional laminarin microparticles for cell adhesion and
expansion
Figure 4.3: (A) Schematic illustration of the methacrylation reaction of laminarin with glycidyl
methacrylate. (B) 1H NMR spectrum of laminarin before modification. (C) Low methacrylated laminarin
(D) High methacrylated laminarin.
3.2.Fabrication of Methacrylated Laminarin microparticles
Based on the promising results of laminarin hydrogels as cell culture platforms,11 and on
the intrinsic low viscosity of this polymer (6 KDa) we hypothesized that microfabricated
laminarin hydrogels could provide effective microplatforms for cell culture. MeLam was
processed in a microfluidic flow-focusing device. Aqueous MeLam droplets were formed
using a water-in-oil emulsion. The continuous stream of laminarin is broken into droplets
by the shear forces exerted from the oil phase using a microchip with a T-junction (figure
4.1). Microfluidics technique allowed to efficiently produce high monodisperse
microparticles with spherical shape.14,15 The microparticles size and shape can be tuned
to a desired value by adjusting the flow rates of the aqueous and dispersed phase or by
adjusting the diameter of the microfluidic channel. In the present work, different flow
rates were tested being noticeable that the increase of the ratio aqueous phase/continuous
phase (QAq)/QC decreases the size of microparticles. The flow rates were optimized to
prepared microparticles with average diameter of 100 µm. A continuous phase at 160
µL/min and aqueous phase of 8 µL/min were found to be the ideal flow rates (figure 4.4
A and D). The formed droplets go through a FEP tube under UV light with intensity of
6.12w/cm2 for 60 seconds that allowed the crosslinking of MeLam microspheres. The
formed MeLam microparticles were collected immersed in mineral oil. The excess of
48
Chapter 4 - Multifunctional laminarin microparticles for cell adhesion and
expansion
mineral oil was cleared and the microparticles were washed with PBS solution to remove
the remaining oil. Some previous works have reported the use of surfactants or organic
solvents (e.g. ethanol 70%) for a complete removal of the mineral oil.14 In this particular
work this was not possible, as such solvents may denature the encapsulated proteins. The
fabricated particles exhibit an increase in size after the removal of oil and incubation of
PBS, due to the swelling of the microgels. The average diameter of the microparticles
was 326.6 µm to high degree of methacrylation (figure 4.4 B and C) and 303.3 µm to low
degree of methacrylation (figure 4.4 E and F) and they exhibit a smooth surface. The
homogenous and spherical shape and smooth surface of microparticles is notable in the
SEM images in both degrees of modification microparticles (figure 4.4 G and H).
Figure 4.4: Images of the high MeLam microparticles in oil (A) in PBS (B) obtained by optical microscopy
and respective histogram of the distribution (C) of microparticles (n = 74) after washed with PBS. Images
of the low MeLam microparticles in oil (D) in PBS (E) obtained by optical microscopy and respective
histogram of the distribution (F) of microparticles size (n = 74) after washed with PBS. SEM image of
monodisperse high (G) and low (H) MeLam microparticles
49
Chapter 4 - Multifunctional laminarin microparticles for cell adhesion and
expansion
3.3.Cumulative release of platelet lysates from MeLam microparticles
Hydrogels microparticles have demonstrated great potential as drug delivery systems due
to facile incorporation and finely tuned release of biomolecules. The MeLam
microparticles are highly porous and exhibit water uptake, that was confirmed by the
swelling degree when in exposure to the PBS solution. In this work, platelet lysates were
used as a source of growth factors to improve cell expansion. The lysates were mixed
with the laminarin solution and this mixture was processed as previously described, using
the microfluidic device. To further assess their potential for the controlled release of
proteins, these particles were incubated in PBS at 37ºC and the protein content in the
supernatant was evaluated after pre-determined time-points. The cumulative release
profile of the total protein content released from the MeLam microparticles is show in
figure 4.5 A. The release profile of encapsulated PL in high and low MeLam
microparticles was followed up to 14 days. High MeLam microparticles exhibit a burst
release of 40.31 ± 0.27% after 12 hours while the low MeLam exhibit a burst release of
22.54 ± 1.13% after 12 hours. At the end of 14 days 56.89 ± 1.03% of the total protein
was released from the high MeLam microparticles, while only 35.31 ± 2.73% of the total
encapsulated protein was released from the low MeLam microparticles. Protein release
rate depends on the solubility, diffusion and biodegradation of the matrix of
encapsulation. The difference in release profile of the microparticles can be related to the
microparticle crosslinking level. According to the previously characterization of the
microparticles, it was expected a high release from the gels with low degree of
substitution due to larger pores than the ones with high degree of substitution.11 However,
our results demonstrated that the high MeLam microparticles had a better release profile.
Due to different levels of substitution of the microparticles, the chemical interactions
between them and proteins will be different, which may explain the different release
profiles. Ngyyen and co-workers studied the influence of different degrees of
methacrylation and the ability to bind and release GFs.16 They demonstrated that
decreasing of the degree of methacrylation increases GFs binding. This corroborates the
results obtained once the release profile is higher for high MeLam microparticles. This
can be explained by covalent or electrostatic interactions between the matrix of the
microgel and proteins. Some authors defend that sometimes nucleophilic double bonds,
including vinylsulfone, methacrylate and maleimide, can be react with the protein amine
groups under physiological conditions. Yu and co-workers demonstrated that in your
works.17 Here, after polymerization some methacrylate groups continuous available to
50
Chapter 4 - Multifunctional laminarin microparticles for cell adhesion and
expansion
react with amine groups of proteins. Also, the kinetics of GFs release in vitro has been
controlled by the types of non-covalent interactions between the microgels and GFs. The
low MeLam microparticles possess more hydroxyl groups available to link the proteins
by hydrogen bonds.18 PLs are rich in several chemokines and growth factors (GFs) such
as platelet derived growth factor isoforms (PDGF-AA, -AB and -BB), transforming
growth factor-β (TGF-β), insulin-like growth factor-1 (IGF-1), vascular endothelial
growth factor (VEGF) and bone morphogenetic protein 2, -4 and -6 (BMP-2, -4, -6).19
Considering the PL composition and the response of the PL in wound healing, several
works have been exploring PLs as a possible substitute to animal derived serum (FBS) in
cell culture. FBS is rich in growth factors like PL, however this shows high risk of
contamination and a potential to promote xenogeneic immune reactions, which make PL
an excellent alternative.20,21 The release of specific GFs, namely VEGF and TGF-β1 was
confirmed by ELISA assay (figure 4.5 C and D). The total amount of these GFs in PL
was quantified and results show 9.78 ± 0.07 pg for VEGF and 73.94 ± 4.44 pg for TGF-
β1 (figure 4.5 B). In this study, we have demonstrated the capacity to produce
microparticles with encapsulated PL capable to release GFs present in PL. The
comparison between different studies using PL is not easy, once the composition of PL is
variable depending of the donor. The release results of this specific growth factors showed
a similar behavior to total protein amount.22 Comparing the total amount of VEGF and
TGF-β1 encapsulated into the microparticles can conclude which the VEGF is practically
all released into the high MeLam. In its turn, the release profile of TGF-β1 confirms the
higher release from the high MeLam microparticles. But instead of VEGF, here the
microparticles release 6.62 ± 1.68 pg of total amount encapsulated in high MeLam
microparticles while the release of TGF-β1 was 45.78 ± 1.20 pg (figure 4.5 C and D).
High MeLam microparticles revealed an increased release of proteins and were chosen to
evaluate the ability this system to support cell expansion.
51
Chapter 4 - Multifunctional laminarin microparticles for cell adhesion and
expansion
Figure 4.5: (A) Cumulative protein profile release from low and high methacrylate laminarin
microparticles by incubation in PBS solution up to 14 days quantified by micro-BCA assay. ELISA assay
performance to quantify specific growth factors release from microparticles. (B) Concentration of VEGF
and TGF-β1 presented in PL sample. VEFG release profiled (C) and TGF-β1 release profile (D) up to 14
days from low and high methacrylate laminarin. Error bars represent standard deviation (n = 3).
3.4. RGD functionalization of MeLam microparticles
One of the most common strategies used to conjugate biomolecules on the surface of
biomaterials is through the use of specific interaction between streptavidin (SaV) and
biotin.5,23,24 Biotin is a small molecule with high affinity to SaV. Due to the high stability
and specificity, this complex has been a very powerful tool in the study of biological
systems being used for chemical conjugation of biomolecules (e.g. antibodies, peptides
sequences and GFs.25 Taking advantage of the SaV-biotin pair, the focus in this work was
the immobilization of biotinylated RGD to promote cell attachment to the laminarin
microgels. The MeLam microparticles were first modified with biotin-PEG-SH by
reaction with the alkene groups presents in MeLam microparticles via Michael type
reaction. A second modification step was performed to create a coating of SaV in the
microgels, followed by an incubation with biotin-RGD. One the biggest advantage of the
use of this system, is the possibility to modify different surfaces with a large number of
different biotinylated molecules. The effective modification of the laminarin
52
Chapter 4 - Multifunctional laminarin microparticles for cell adhesion and
expansion
microparticles was assessed by florescence microscopy using fluorescent-labeled SaV
and the unmodified microparticles were used as control. The fluorescence images
demonstrate the efficient conjugation with the microparticles with SaV, confirming the
effective modification of the microparticles with biotin. (figure 4.6 A and B). The last
step of the microparticles functionalization was the addition of the RGD-biotinylated.
RGD is a peptide sequence (Arg-Gly-Asp) that constitute a major recognition system for
cell adhesion (figure S4.1).26 The goal of this work is to use the microparticles as a support
for cell expansion and prove the PL improve the cell adhesion. Taking into account the
results from PL release, the high MeLam showed a better profile release, being expected
which that shows better results in cell culture.
Figure 4.6: Fluorescence imagens showing the functionalization of MeLam microparticles. Images of
MeLam microparticles with biotin-PEG-SH (A) and MeLam microparticles without biotin (control) (B)
after incubation with fluorescently labeled SaV.
3.5. L929 fibroblast capture and expansion on MeLam microparticles
Microcarrier beads of different materials, have been widely used to culture anchorage-
dependent cells. Microcarriers have innumerous advantageous when compared with the
conventional cell culture systems. The low cost and great surface-to-volume ratio allow
the culture of high cell numbers, eliminating multiple trypsinization steps.27 Recently,
Soure and co-workers studied the effect of PL on the expansion of umbilical cord matrix
derived from mesenchymal stem cells (MSCs) in plastic microcarriers under dynamic
conditions.28 Their results demonstrated the advantages of the use of PL for the effective
expansion of MSCs in a xenogeneic-free microcarrier-based system. In this work, we
proposed the fabrication of MeLam microparticles with encapsulated PL to be used as
microplatforms for cell culture. Here, we not use PL to supplement the culture media, we
propose the encapsulation of PL to improve cell adhesion and expansion on the cell
53
Chapter 4 - Multifunctional laminarin microparticles for cell adhesion and
expansion
microcarriers. L929 cells were used to evaluate our hypothesis. L929 cells were seeded
on the RGD functionalized microparticles, with encapsulated PL and cultured for 11 days.
In order to study the influence of encapsulated PL in cell adhesion, RGD functionalized
microparticles without encapsulated PL were used as a control. Cell adhesion was
monitored by optical microscopy (figure S 4.2). The images shown an increased in cell
attachment in microparticles with encapsulated PL. This may be due to the initial burst
release of protein from the microcarriers that stimulate cell attachment. Also, phalloidin
and DAPI staining were performed to evaluate cell morphology on the surface of
microparticles at 3, 7 and 11 days (figure 4.7 A to D and figure S 4.3). The dependence
of cell adhesion and morphology from the encapsulated PLs was evident. The L929 cells
adopt an elongated, spreading morphology on the microparticles containing PL. After 11
days, is possible to observe the surface of microparticles completely covered with cells
and a cell matrix connecting the microparticles. In the absence of PLs a few cells adhered
on the surface. Nevertheless, this may be justified by the presence of the RGD moieties
as plain particles did not show any cell attachment.
The cell viability of L929 was assessed at day 3, 7 and 11 (figure 4.7 E). In the first-time
points, were no significant differences between the sample and control were observed.
Only after 11 days of culture we could observe a significant difference of cell viability in
the two conditions. The microparticles with encapsulated PL showed increase cell
viability. Lastly, DNA assay quantification was performed to evaluated the cell
proliferation. Results corroborate the hypothesis that PL have a positive influence in cell
expansion. (figure 4.7 F).
54
Chapter 4 - Multifunctional laminarin microparticles for cell adhesion and
expansion
Figure 4.7: Fluorescence images of the high MeLam microparticles with encapsulated PL (A-D) culture
with L929 cells up to 14 days. These images demonstrate the ability to cell attach, cytoskeleton was stained
with phalloidin (red) and nuclei was stained with DAPI (blue). (E) Cell viability by MTS assay was
determined at 3, 7 and 11 days. (F) DNA quantification of all formulations tested up to 11 days of culture.
Results are present as mean ± standard error of the mean (n = 3).
4. Conclusions
Here, we demonstrated an efficient one-step method to generate monodisperse MeLam
microparticles incorporating PL using a microfluidics device coupled to a source of UV
light. The pendant acrylate groups of MeLam allowed also the conjugation of thiolated
biotin via thiol-Michael addition and further conjugation with RGD peptides.
The size of microparticles was easily controlled by the adjustment of the flow rates of the
aqueous phase and continuous phase. The multifunctional MeLam microparticles were
seeded with L929 cells and the results demonstrate their potential to support cell adhesion
and expansion. MeLam microgels offer a high degree of tunability over both structural
and chemical properties, and can be used to recapitulate highly varied tissue
environments.
Cultured microgels could self-assemble to form structures with packing densities,
suggesting potential applications in tissue engineering and regenerative medicine.
5. References:
1. Discher, D. E., Mooney, D. J. & Zandstra, P. W. Growth Factors, Matrices, and Forces Combine
and Control Stem Cells. Science (80 ). 324, 1673–1677 (2009).
2. Hu, Y. et al. Controlled self-assembly of alginate microgels by rapidly binding molecule pairs.
Lab Chip 17, 2481–2490 (2017).
3. Kim, P. H. et al. Injectable multifunctional microgel encapsulating outgrowth endothelial cells
and growth factors for enhanced neovascularization. J. Control. Release 187, 1–13 (2014).
55
Chapter 4 - Multifunctional laminarin microparticles for cell adhesion and
expansion
4. Cavalieri, F., Postma, A., Lee, L. & Caruso, F. Assembly and Functionalization of DNA -
Polymer Microcapsules. ACS NanoNano 3, 234–240 (2009).
5. Custódio, C. A., Cerqueira, M. T., Marques, A. P., Reis, R. L. & Mano, J. F. Cell selective
chitosan microparticles as injectable cell carriers for tissue regeneration. Biomaterials 43, 23–31
(2015).
6. Mao, A. S. et al. Deterministic encapsulation of single cells in thin tunable microgels for niche
modelling and therapeutic delivery. Nat. Mater. 1, 1–10 (2016).
7. Neto, A. I. et al. Fabrication of Hydrogel Particles of Defined Shapes Using Superhydrophobic-
Hydrophilic Micropatterns. Adv. Mater. 28, 7613–7619 (2016).
8. Wang, Q., Liu, S., Wang, H., Zhu, J. & Yang, Y. Alginate droplets pre-crosslinked in
microchannels to prepare monodispersed spherical microgels. Colloids Surfaces A Physicochem.
Eng. Asp. 482, 371–377 (2015).
9. Hu, Y., Azadi, G. & Ardekani, A. M. Microfluidic fabrication of shape-tunable alginate
microgels: Effect of size and impact velocity. Carbohydr. Polym. 120, 38–45 (2015).
10. Wang, D. et al. The first bacterial β-1,6-endoglucanase from Saccharophagus degradans 2-40T
for the hydrolysis of pustulan and laminarin. Appl. Microbiol. Biotechnol. 101, 197–204 (2017).
11. Custódio, C. A., Reis, R. L. & Mano, J. F. Photo-Cross-Linked Laminarin-Based Hydrogels for
Biomedical Applications. Biomacromolecules 17, 1602–1609 (2016).
12. Juhl, M. et al. Comparison of clinical grade human platelet lysates for cultivation of
mesenchymal stromal cells from bone marrow and adipose tissue. Scand. J. Clin. Lab. Invest. 76,
93–104 (2016).
13. Che, H. et al. CO 2 -switchable drug release from magneto-polymeric nanohybrids. Polym. Chem.
6, 2319–2326 (2015).
14. Cha, C. et al. Microfluidics-Assisted Fabrication of Gelatin-Silica Core − Shell Microgels for
Injectable Tissue Constructs. Biomacromolecules 15, 283–290 (2014).
15. Zhao, X. et al. Injectable Stem Cell-Laden Photocrosslinkable Microspheres Fabricated Using
Microfluidics for Rapid Generation of Osteogenic Tissue Constructs. Adv. Funct. Mater. 26,
2809–2819 (2016).
16. Nguyen, A. H., McKinney, J., Miller, T., Bongiorno, T. & McDevitt, T. C. Gelatin Methacrylate
Microspheres for Growth Factor Controlled Release. Acta Biomater. 13, 101–110 (2015).
17. Yu, Y. & Chau, Y. Formulation of in situ chemically cross-linked hydrogel depots for protein
release: From the blob model perspective. Biomacromolecules 16, 56–65 (2015).
18. King, W. J. & Krebsbach, P. H. Growth factor delivery: How surface interactions modulate
release in vitro and in vivo. Adv. Drug Deliv. Rev. 64, 1239–1256 (2012).
19. Burnouf, T., Strunk, D., Koh, M. B. C. & Schallmoser, K. Human platelet lysate: Replacing fetal
bovine serum as a gold standard for human cell propagation? Biomaterials 76, 371–387 (2016).
20. Bieback, K. Platelet lysate as replacement for fetal bovine serum in mesenchymal stromal cell
cultures. Transfus. Med. Hemotherapy 40, 326–335 (2013).
21. Turner, P. A., Thiele, J. S. & Stegemann, J. P. Growth factor sequestration and enzyme-mediated
release from genipin-crosslinked gelatin microspheres. J. Biomater. Sci. Polym. Ed. 28, 1826–
56
Chapter 4 - Multifunctional laminarin microparticles for cell adhesion and
expansion
1846 (2017).
22. Santo, V. E. et al. Enhancement of osteogenic differentiation of human adipose derived stem cells
by the controlled release of platelet lysates from hybrid scaffolds produced by supercritical fluid
foaming. J. Control. Release 162, 19–27 (2012).
23. Li, C. Y., Wood, D. K., Hsu, C. M. & Bhatia, S. N. DNA-templated assembly of droplet-derived
PEG microtissues. Lab Chip 11, 2967 (2011).
24. Riccardi, C. et al. Fluorescent Thrombin Binding Aptamer-Tagged Nanoparticles for an Efficient
and Reversible Control of Thrombin Activity. ACS Appl. Mater. Interfaces 9, 35574–35587
(2017).
25. Chivers, C. E., Koner, A. L., Lowe, E. D. & Howarth, M. How the biotin–streptavidin interaction
was made even stronger: investigation via crystallography and a chimaeric tetramer. Biochem. J.
435, 55–63 (2011).
26. Ruoslahti, E. RGD and other recognition sequences for integrins. Annu. Rev. Cell Dev. Biol. 12,
697–715 (1996).
27. Sun, L. et al. Novel konjac glucomannan microcarriers for anchorage-dependent animal cell
culture. Biochem. Eng. J. 96, 46–54 (2015).
28. de Soure, A. M. et al. Integrated culture platform based on a human platelet lysate supplement for
the isolation and scalable manufacturing of umbilical cord matrix-derived mesenchymal
stem/stromal cells. J. Tissue Eng. Regen. Med. 11, 1630–1640 (2017).
57
Supporting Information
Multifunctional laminarin microparticles for cell adhesion and expansion
Martins CR,1 Custódio CA,1 Mano JF1
1-Department of Chemistry, CICECO, Aveiro Institute of Materials, University of Aveiro Campus
Universitário de Santiago, 3810-193 Aveiro - Portugal
Figure S4.1: Images of MeLam microparticles with biotin-PEG-SH bioconjugate with pure SaV (A) and
without bioconjugation with pure SaV (control) (B) after incubation with fluorescence biotin
58
Figure S4.2: Images assessed by optical microscopy with encapsulated PL (A and B) and without PL (C
and D) for 24 hours and 7 days.
Figure S4.3: Fluorescence images of the high MeLam microparticles without encapsulated PL (A-C)
culture with L929 cells up to 14 days. These images demonstrate the ability to cell attach, cytoskeleton was
stained with phalloidin (red) and nuclei was stained with DAPI (blue)
CHAPTER 5
General Conclusions
59
Chapter 5 – General Conclusions
Chapter 5 – General Conclusions
Tissue engineering (TE) combines mainly three different areas: biology, chemistry and
engineering to mimic or replace the native architecture of tissues. TE uses two approaches
to recreate a native tissue: the top-down approach in which cells are seeded in a scaffold
and the bottom-up approach in which small microunits are used to construct a complex
3D structure. Hydrogels are a versatile class of polymeric networks that have been used
for a wide variety of cell culture and TE applications. Additionally, hydrogels can be
selectively functionalized to present specific chemical or mechanical cues to trigger
desired cellular responses. Hydrogels can also be spatially confined as microscopic
shapes (microgels). Microgels are attractive for TE applications because of their
biological and physical properties (i.e., well defined shapes, mechanical strength, and
biodegradability and resemblance to the natural extracellular matrix (ECM)).
Chapter 1 presents general concepts in hydrogels and microfabrication and the thesis
motivation. Chapter 2 reviews the efforts developed regarding microfabrication of
hydrogels, their applications, strategies for microgels assembly and future perspectives in
the field. This chapter also highlights the advantages in the use of hydrogel
microstructures to engineer hierarchically organized 3D structures. The 3D structures
produced by bottom-up approaches demonstrate better vascularization and diffusion of
nutrients and oxygen when compared with traditional scaffolds. These microstructures
can be used to many applications, such as cell microcarriers for cell expansion, drug
delivery or cell encapsulation.
Detailed materials and methods using during the development of this work are presented
in Chapter 3 of this thesis.
Aiming the design of innovate multifunctional microparticles for cell expansion, in
chapter 4, we propose a method to produce laminarin microgels using a microfluidics
device coupled to a UV lamp. This technique allowed to produce highly monodisperse
microgels with perfect spherical shape. A subsequent reaction with pendent alkene groups
was then utilized in a thiol-ene addition reaction to obtain functionalized microparticles
with thiolated biotin. Here was demonstrated the ability of functionalized particles to
promote cell adhesion by conjugation with biotin-RGD molecules. However, the
versatility of this method allows the combination of the biotin-SaV conjugated microgels
with any biotinylated molecules of interest. Here, we demonstrate that this technique can
also be used for the direct encapsulation of platelet lysates (PL) within the microgels, in
a one-step approach. PL are a cocktail of proteins and growth factors and have been
60
Chapter 5 – General Conclusions
studied as a substitute of animal serum in cell culture. This work offers a novel system
encompassing MeLam microgels functionalized with adhesive peptides to promote cell
adhesion that provide simultaneously a controlled release of PL that should be useful in
cell expansion and cell differentiation protocols. To validate this system, the MeLam
microgels were cultured with L929 cells. The cells cultured with microparticles with
encapsulated PL demonstrated clear enhancement in cell attachment and expansion.
Cultured microgels could assemble to form structures with packing densities, suggesting
potential applications in tissue engineering and regenerative medicine.
Further studies will validate the effective potential of released PL by culturing the
microgels in media without FBS supplementation.