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Alginate hydrogels for short-term storage and
preservation of human cells for cell therapy
Vitória Rafaela Costa Curto
Thesis to obtain the Master of Science Degree in
Biological Engineering
Supervisors:
Doctor Ivan Wall
Professor Duarte Miguel de França Teixeira dos Prazeres
Examination Committee
Chairperson: Professor Arsénio do Carmo Sales Mendes Fialho
Supervisor: Professor Duarte Miguel de França Teixeira dos Prazeres
Member of the Committee: Professor Cláudia Alexandra Martins Lobato da Silva
September 2016
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To my family and friends
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Acknowledgements
This thesis represents the end of a milestone on my academic life which lasted for 5 years. I am
not the same as I was when I started it, both in professional and personal terms. I have so much
to be thankful for, and so I would now like to take this opportunity to acknowledge at least some
of the many people who helped to shape my path and who helped me grow into what I am today.
First of all I would like to acknowledge my supervisor, Dr. Ivan Wall, who gave me the opportunity
to develop this project at his lab and within his research group. Thanks to this I had the opportunity
to work with and learn from amazing and qualified people, as well as to have access to very
modern and technological facilities. I also want to thank him for the support and knowledge he
passed on to me whilst developing this project along the 5 months I was fortunate to spend at
UCL. His insightful suggestions allowed the development of this original work.
Second, I would like to thank Ganaa for all the hours spent together at the lab when I first got to
UCL. I want to thank her for sharing her thoughts on hydrogels with me and for helping me find
my way on the technical side of things. I will never forget the “Hydrogel team”, as everyone called
us, as well as her friendship.
Thank you to all the other people at UCL and especially everyone from the Regenerative Medicine
Group, for helping me whenever and with whatever I needed and for the little relaxing moments
we had along the day when we talked about something else than work. Also, thank you to the
people from the Microfluidics Group, especially Dr. Nikolay Dimov, for letting me borrow the
temperature logger for my work.
To Phil and Leo, thank you for their friendship. I want to thank them for getting me out of the
house and for letting me know about all the cool places in London. They definitely turned being
away from home and everyone I love much easier to endure and I truly hope our friendship lasts.
To Reema, for her always present kindness and friendship, as well as for expanding my horizons.
A special thank you to my parents for always blindly believing in me while doing everything in
their power to allow me to follow my dreams. To my mother, for always telling me that doing my
best was enough and all that mattered, every time I had an important exam, presentation or
assignment. To my father, for encouraging me to invest in myself every single day and for the
rides to university when I was too exhausted to get the 7 a.m. train. I am so lucky to have them.
To Luísa, my former colleague but more importantly my friend and also sort of a mentor. Thank
you for her support every time I needed it and for visiting me in London. I am sure we will be
friends forever.
To Gonçalo, my friend and colleague, thank you so much for being there for me these last couple
of years. Hope he continues to be for many more.
To Rúben, thank you for showing up in my life when you did and always being there for me. I
cannot start to tell you how much it meant to me your support through this months I was away.
Thank you for visiting me in London and always encouraging me to follow my dreams, wherever
they may take me.
Lastly, a huge thank you to all my professors at Instituto Superior Técnico for all the education
and background I now have, which I am sure will help me succeed on the next step of my
academic and professional life.
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Resumo
Apesar do progresso significativo de toda a indústria das terapias celulares, problemas
relacionados com o armazenamento das células e com a sua distribuição são ainda uma
realidade. Neste trabalho, o potencial de hidrogéis de alginato para o armazenamento e
preservação a curto-prazo de olfactory ensheathing cells (OECs) humanas, as quais são
destinadas a ser utilizadas em terapia celular alogénica para regeneração de lesões do sistema
nervoso central, foi avaliado.
As células foram encapsuladas em hidrogéis de alginato com diferentes concentrações de
alginato (0.1% – 0.4% (m/v)) e armazenadas por períodos de 3 e 5 dias, a uma temperatura de
37ºC ou à temperatura ambiente. O estado de preservação das OECs humanas foi avaliado
através de análises de viabilidade e libertação das células, e foi realizada imunocitoquímica para
p75NTR e S100β em diferentes alturas. Foram obtidas, para todas as condições de
armazenamento em que a temperatura de 37ºC foi usada, viabilidades superiores a 70%. Para
o armazenamento realizado à temperatura ambiente, pelo menos 50% das OECs humanas
encapsuladas apresentavam-se viáveis após os períodos de armazenamento. Portanto, a
temperatura mostrou ter influência na viabilidade das células. No entanto, os diferentes tempos
de armazenamento e as diferentes concentrações de alginato apenas mostraram ser
significativas quando o armazenamento foi realizado à temperatura ambiente. Com respeito à
imunocitoquímica, ambos os marcadores foram expressos por todas as células antes da
encapsulação e depois de se libertarem dos hidrogéis, sugerindo que o fenótipo das OECs é
mantido durante o armazenamento.
Este trabalho apresenta um sistema de armazenamento simples e económico capaz de entregar
pelo menos 50% das OECs encapsuladas viáveis e funcionais, na situação mais desfavorável.
Este método de armazenamento tem um grande potencial e poderá ajudar a transformar a terapia
celular com OECs humanas para a regeneração de lesões no sistema nervoso central numa
realidade do dia-a-dia.
Palavras-chave: Olfactory ensheathing cells, Armazenamento celular, Preservação
celular, Curto-prazo, Regeneração do sistema nervoso central
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Abstract
Despite significant progress within the whole cell therapy industry, logistical issues regarding the
storage of cells and their distribution are still a reality. Here, the potential of alginate hydrogels for
the short-term storage and preservation of human olfactory ensheathing cells (OECs), which are
intended to be used on an allogeneic cell therapy for the regeneration of central nervous system
(CNS) injuries, was assessed.
The cells were encapsulated within alginate hydrogels with different alginate contents (0.1% –
0.4% (w/v)) and stored for periods of 3 and 5 days, under a temperature of 37ºC or at RT. The
preservation condition of the human OECs was assessed through viability and cell release
assays, and immunocytochemistry for p75NTR and S100β was performed at different time points.
Viabilities higher than 70% were obtained for all the storage conditions in which a temperature of
37ºC was used. For the RT situations, at least 50% of the encapsulated human OECs were alive
after the storage periods. Thus, temperature showed to have an impact on the viability of the
cells. However, different times of storage and alginate concentrations were only meaningful when
the storage was performed at RT. Regarding the immunocytochemistry, both markers were
expressed by all the cells before the encapsulation and after releasing from the hydrogels,
suggesting that the OECs phenotype is maintained through storage.
This work presents a simple and economical storage system capable of delivering at least 50%
of the encapsulated OECs alive and functional, in the most unfavourable situation. This storage
method has great potential and it can help to transform cell therapy with human OECs for the
regeneration of CNS injuries into a day-to-day reality.
Keywords: Olfactory ensheathing cells, Cell storage, Cell preservation, Short-term,
Central nervous system regeneration
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Contents
Acknowledgements ................................................................................................................... v
Resumo .................................................................................................................................... vii
Abstract ..................................................................................................................................... ix
List of Tables ........................................................................................................................... xiii
List of Figures ........................................................................................................................... xv
List of Abbreviations ................................................................................................................ xxi
1 Introduction ................................................................................................................................ 1
1.1 The adult mammal central nervous system and its lack of intrinsic regeneration
capacity………………………………………………………………………………………………....1
1.2 Olfactory Ensheathing Cells .......................................................................................... 4
1.3 Hydrogels for cell storage and preservation .................................................................. 8
1.3.1 Cell storage and preservation are needed for regenerative medicine therapies . 8
1.3.2 Current storage methods present issues ............................................................. 8
1.3.3 Hydrogels as candidates for the improved preservation of cells .......................... 9
1.4 Motivation and aim of the project ................................................................................ 11
2 Materials and methods ............................................................................................................ 13
2.1 Cell culture and banking .............................................................................................. 13
2.1.1 Culture medium formulation ............................................................................... 13
2.1.2 Thawing .............................................................................................................. 13
2.1.3 Expansion ........................................................................................................... 14
xii
2.1.4 Freezing .............................................................................................................. 14
2.2 Cell encapsulation and storage ................................................................................... 15
2.3 Viability assays ............................................................................................................ 16
2.4 Cell release assays ..................................................................................................... 16
2.5 Immunocytochemistry.................................................................................................. 18
2.6 Statistical analysis ....................................................................................................... 20
3 Results and discussion ............................................................................................................ 21
3.1 Human OECs encapsulated inside alginate hydrogels ............................................... 21
3.2 Viability of human OECs encapsulated and stored within alginate hydrogels ............ 23
3.2.1 Viability of the encapsulated cells after 3 and 5 days of storage at 37ºC .......... 25
3.2.2 Viability of the encapsulated cells after 3 and 5 days of storage at RT ............. 26
3.2.3 Viability of the encapsulated cells stored at 37ºC vs RT .................................... 28
3.2.4 Viability assessment after the storage at 37ºC of the encapsulated cells, after a
change in the encapsulation protocol .................................................................................. 30
3.3 Release of the encapsulated human olfactory ensheathing cells out of the
hydrogels.................................................................................................................................32
3.4 Immunocytochemistry.................................................................................................. 41
4 Concluding remarks and future perspectives .......................................................................... 43
References .............................................................................................................................. 45
xiii
List of Tables
Table 1 Classifications attributed to the pictures taken of the different alginate content
hydrogels (0.1 – 0.4% (w/v)) highlighting the cell release occurred, after these
being stored in tissue culture grade well-plates for 3 and 5 days at 37ºC and at
RT and after 7 days of being transferred into new tissue culture grade well-
plates and being left at 37ºC. Scale: 0 – no cells released; 1 – only a few
released cell; 2 – marked release of cells; 3 – extensive release of cells………… 40
Table 2 Classifications attributed to the pictures taken of the different alginate content
hydrogels (0.1 – 0.4% (w/v)) highlighting the cell release occurred, after these
being stored in ultra-low attachment well-plates for 3 and 5 days at 37ºC and
at RT and after 7 days of being transferred into new tissue culture grade well-
plates and being left at 37ºC. Scale: 0 – no cells released; 1 – only a few
released cell; 2 – marked release of cells; 3 – extensive release of cells…......... 40
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xv
List of Figures
Figure 1 The structure of the CNS of humans, with the core constitutions of its main
divisions, the brain and the spinal cord………………………………………………. 1
Figure 2 A neuron, with its constituents identified……………………………………………. 2
Figure 3 Illustration of the cells of the CNS, namely neurons, and the glial cells which
support them: oligodendrocytes, microglia and astrocytes………………………… 3
Figure 4 Diagram of the adult mammal olfactory system intending to show the location of
OECs. Olfactory ensheathing cells (OECs, pink areas) of the OM ensheath
bundles of olfactory receptor axons (olfactory nerves, ON) of the olfactory
receptor neurons (ORN) along their course through the lamina propria in the
PNS. Olfactory nerves and their accompanying OECs cross through the
cribriform plate of the skull into the CNS and surround the OB to form the
olfactory nerve layer………………………………………………………………….. 5
Figure 5 Schematics of the 3D tunnel shaped ensheathment of olfactory axons performed
by OECs. In the mucosa, the axons (ax) of the olfactory neurons (ON) are first
captured in bundles. Processes of the OECs penetrate the basal lamina (BL)
and tightly invest bundles of axons, which they then guide through the olfactory
nerves all the way through the OB…………..………………………………………... 5
xvi
Figure 6 Heterogeneity of OECs in vitro. (a) Fibrous OECs with bipolar body; (b) OECs
with flatten body and no apparent process; (c) OECs with multipolar body and
long processes; (d) OECs with long process and exuberant arborizations; (e)
OECs with thin, long processes and poor branching……………………………….. 7
Figure 7 Calcium promotes the solidification of alginate networks. Alginate is a long,
negatively charged polysaccharide. Positively charged sodium ions (Na+)
dissociate from the sodium alginate when this is dissolved. Doubly charged
calcium ions (Ca2+) can bind two different alginate strands simultaneously,
thereby crosslinking and solidifying the solution by a process called ionic-
crosslinking……………………………………………………………………………... 10
Figure 8 Chemical structure of alginate………………………………………………………... 10
Figure 9 Phase contrast microscopic image of the cultured PA7s at passage 11, 4 days
after passaging and right before encapsulation in the alginate hydrogel. Their
morphology can be defined by flat bodies with no apparent processes, much like
fibroblasts. Scale bar: 1000 µm…………………………………….………………… 21
Figure 10 Picture of a 24 well-plate with alginate hydrogels (some of them are highlighted)
right after gelation for 30 min with 10 mM CaCl2 in complete medium…………….. 22
Figure 11 Representative phase contrast microscopic images of hydrogels with
encapsulated human OECs after 3 days of storage at 37ºC on a tissue culture
grade 24 well-plate. Cell seeding density was 107 cells/mL for all the hydrogels
and the cells show to be evenly distributed and with an even density along them.
The gels have different alginate contents: (a) 0.1%, (b) 0.2%, (c) 0.3% and (d)
0.4% (w/v) and all of them present an approximately circular shape, with their
diameter in these particular cases ranging from about 3.87 mm to 4.74 mm.
Scale bar: 2000 µm…………………………………………………..………………… 22
xvii
Figure 12 Representative fluorescence merged images of the assessment by live (green)
and dead (red) staining of the viability of OECs encapsulated in hydrogels with
different alginate contents (0.1% - 0.4% (w/v)) and stored at (a) 37ºC for 3 days,
(b) 37ºC for 5 days, (c) RT for 3 days and (d) RT for 5 days. Cell seeding density
was 107 cells/mL hydrogel in all conditions. Scale bar: 200 µm…………………….
23
Figure 13 Quantification of total cell number extracted from the images resulting from the
live/dead staining, for each storage condition studied (each image covered an
area of approximately 0.309 mm2 of the hydrogels). Values are expressed as
mean ± standard deviation from the 9 images analysed for each condition………. 24
Figure 14 Quantification of the viability of the OECs stored within hydrogels with different
alginate contents (0.1 – 0.4% (w/v)) at 37ºC and for 3 and 5 days. Values are
expressed as mean ± standard deviation from the 9 images analysed for each
condition………………………………………………………………………………… 25
Figure 15 Quantification of the viability of the OECs stored within hydrogels with different
alginate contents (0.1 – 0.4% (w/v)) at RT and for 3 and 5 days. Values are
expressed as mean ± standard deviation from the 9 images analysed for each
condition, with asterisks representing statistical significant differences (*p<0.05;
**p<0.01; ***p<0.001)…………………………..……………………………………... 27
Figure 16 Temperature fluctuations at which the cells were subjected through the 5 day
period of storage at RT. The temperature was recorded every hour. Tmax = 29ºC;
Tmin = 21.5ºC; Tavg = 21.5ºC ± 0.8ºC (mean ± standard deviation)…………......….. 27
Figure 17 Quantification of the viability of the OECs after 3 days of storage within hydrogels
with different alginate contents (0.1 – 0.4% (w/v)), at 37ºC and at RT. Values are
expressed as mean ± standard deviation from the 9 images analysed for each
condition, with asterisks representing statistical significant differences (**p<0.01;
***p<0.001)……………………………………………………………………………... 29
xviii
Figure 18 Quantification of the viability of the OECs after 5 days of storage within hydrogels
with different alginate contents (0.1 – 0.4% (w/v)), at 37ºC and at RT. Values are
expressed as mean ± standard deviation from the 9 images analysed for each
condition, with asterisks representing statistical significant differences (**p<0.01;
***p<0.001)……………………………………………………………………………... 29
Figure 19 Quantification of the viability of the OECs after 5 days of storage within hydrogels
with different alginate contents (0.1 – 0.4% (w/v)), at 37ºC, having the dilution of
the stock alginate solution and the concentrated cell suspension obtained been
done with either a solution of 150 mM NaCl or cell culture medium. Values are
expressed as mean ± standard deviation from the 9 images analysed for each
condition………………………………………………………………………………… 30
Figure 20 Representative phase contrast microscopic images of the hydrogels with
encapsulated OECs trying to highlight their release and migration out of the
hydrogels. The hydrogels present the range of different alginate contents (0.1%
- 0.4% (w/v)), and the 2D cultured cells are also shown. Storage of these
hydrogels took place in tissue culture grade well-plates. (a) Pictures after 3 days
of storage at 37ºC; (b) Pictures at the 7th day after the transfer of the hydrogels
presented in (a); (c) Pictures after 3 days of storage at RT; (d) Pictures at the 7th
day after the transfer of the hydrogels presented in (c). Cell seeding density was
107 cells/mL hydrogel in all conditions. Scale bar: 1000 µm…………………….…. 35
Figure 21 Representative phase contrast microscopic images of the hydrogels with
encapsulated OECs trying to highlight their release and migration out of the
hydrogels. The hydrogels present the range of different alginate contents (0.1%
- 0.4% (w/v)), and the 2D cultured cells are also shown. Storage of these
hydrogels took place in tissue culture grade well-plates. (a) Pictures after 5 days
of storage at 37ºC; (b) Pictures at the 7th day after the transfer of the hydrogels
presented in (a); (c) Pictures after 5 days of storage at RT; (d) Pictures at the 7th
day after the transfer of the hydrogels presented in (c). Cell seeding density was
107 cells/mL hydrogel in all conditions. Scale bar: 1000 µm……………………….. 36
xix
Figure 22 Representative phase contrast microscopic images of the hydrogels with
encapsulated OECs trying to highlight their release and migration out of the
hydrogels. The hydrogels present the range of different alginate contents (0.1%
- 0.4% (w/v)), and the 2D cultured cells are also shown. Storage of these
hydrogels took place in ultra-low attachment well-plates. (a) Pictures after 3 days
of storage at 37ºC; (b) Pictures at the 7th day after the transfer of the hydrogels
presented in (a); (c) Pictures after 3 days of storage at RT; (d) Pictures at the 7th
day after the transfer of the hydrogels presented in (c). Cell seeding density was
107 cells/mL hydrogel in all conditions. Scale bar: 1000 µm…………………….….
37
Figure 23 Representative phase contrast microscopic images of the hydrogels with
encapsulated OECs trying to highlight their release and migration out of the
hydrogels. The hydrogels present the range of different alginate contents (0.1%
- 0.4% (w/v)), and the 2D cultured cells are also shown. Storage of these
hydrogels took place in ultra-low attachment well-plates. (a) Pictures after 5 days
of storage at 37ºC; (b) Pictures at the 7th day after the transfer of the hydrogels
presented in (a); (c) Pictures after 5 days of storage at RT; (d) Pictures at the 7th
day after the transfer of the hydrogels presented in (c). Cell seeding density was
107 cells/mL hydrogel in all conditions. Scale bar: 1000 µm……….………………. 38
Figure 24 Temperature fluctuations at which the cells were subjected through the 5 day
period of storage at RT, when stored in hydrogels placed in tissue culture grade
well-plates. The temperature was recorded every hour. Tmax = 25ºC; Tmin =
21.5ºC; Tavg = 22.4ºC ± 0.7ºC (mean ± standard deviation)………………...……… 39
Figure 25 Temperature fluctuations at which the cells were subjected through the 5 day
period of storage at RT, when stored in hydrogels placed in ultra-low attachment
well-plates. The temperature was recorded every hour. Tmax = 26.5ºC; Tmin =
21.5ºC; Tavg = 22.1ºC ± 0.9ºC (mean ± standard deviation)…………………..…… 39
Figure 26 Representative images of human olfactory ensheathing cells cultured in 2D for
5 days expressing (a) S100β (b) p75NTR. Scale bar: 200 µm………..........……... 41
xx
Figure 27 Representative images of human olfactory ensheathing cells that were allowed
to release for 7 days from hydrogels transferred into tissue culture grade well-
plates, after being stored for 5 days at 37ºC and in ultra-low adherent well-plates,
expressing (a) S100β (b) p75NTR. Scale bar: 200 µm……………………………... 41
xxi
List of Abbreviations
2D Two-dimensional
3D Three-dimensional
Ax Axons
ANOVA Analysis of variance
BL Basal lamina
BDNF Brain-derived neurotrophic factor
CNS Central nervous system
DAPI 4',6-diamidino-2-phenylindole
DMEM/F-12 Dulbecco’s modified eagle medium/nutrient mixture F-12
DMSO Dimethyl sulfoxide
ECM Extracellular matrix
EDTA Ethylenediaminetetraacetic acid
FBS Fetal bovine serum
FGF Fibroblast growth factor
GDNF Glial cell line-derived neurotrophic factor
GFAP Glial fibrillary acidic protein
GFP Green fluorescent protein
GMP Good manufacturing practice
HBSS Hank’s balanced salt solution
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hASCs Human adipose-derived stem cells
hMSCs Human mesenchymal stem cells
IgG Immunoglobulin G
mESCs Mouse embryonic stem cells
NGF Nerve growth factor
NT-3 Neurotrophin 3
NT-4/5 Neurotrophin 4/5
OB Olfactory bulb
OECs Olfactory ensheathing cells
OM Olfactory mucosa
ON Olfactory neurons/nerves
ORN Olfactory receptor neurons
PA7s Human OECs from the PA7 cell line
P/S Penicillin and streptomycin
p75NTR Low-affinity nerve growth factor receptor p75
PBS Phosphate buffered saline
PFA Paraformaldehyde
PLL Poly-L-lysine
PNS Peripheral nervous system
RT Room temperature
S100β S100 calcium-binding protein β
SCI Spinal cord injury
VEGF Vascular endothelial growth factor
1
Chapter 1
Introduction
1.1 The adult mammal central nervous system and its
lack of intrinsic regeneration capacity
The nervous system of mammals is organized
into two parts: the central nervous system
(CNS), which consists of the brain and the
spinal cord (Fig. 1), and the peripheral nervous
system (PNS), which connects the CNS to the
rest of the body.1,2
There are two broad classes of cells in the
CNS: neurons, excitable nerve cells which
process the information, and neuroglia (or
simply glia), which provide and support the
neurons with mechanical and metabolic
support.1 Neurons are, therefore, the basic
units of signalling in the nervous system,
although glial cells may contribute as well.2
A typical neuron (Fig. 2) possesses a cell body
with the nucleus and neurites, which are
projections from the cell body. The dendrites
are the shorter neurites receiving the input and
the axons are the long tubular ones which carry
the outputs.1,2 Figure 1 – The structure of the CNS of
humans, with the core constitutions of its
main divisions, the brain and the spinal cord.1
2
When compared to other organisms, the CNS of adult
mammals is remarkably poor at regeneration after an
injury, with the failing of the axons to regenerate over
long distances and re-establish connections interrupted
by the traumatic lesion. It is believed that this has to do
with the fact that whenever the CNS is damaged, it
undergoes a complex cellular and molecular response
called glial scar, which environment will vary over time,
particularly in the first two weeks.3–6 In this glial scar,
three main type of cells are involved: oligodendrocytes,
microglia and astrocytes (Fig. 3).4
Oligodendrocytes are responsible for the formation of the
myelin sheath of nerve fibbers in the CNS and provide
metabolic support for the axons by passing them
nutrients through channels. These myelin membranes
wrap up the axons forming a spiral of closely opposed
membranes held together by myelin proteins. Each
oligodendrocyte can wrap several segments of different
axons, being each spiral of wraps called an internode
and separated from the next one by the node of Ranvier.
Myelin membranes are important because they work as
an electrical insulator and therefore greatly increase the speed of nerve conduction along the
axons. It is then easy to understand that remyelination after injury is important for regeneration,
namely for restoring the electrical conduction of axons.1,3,7–9 An injury to the CNS will damage
myelin sheaths directly and will lead to the immediate presence of myelin debris in the injury sites.
Therefore, for some time the glial scar contains myelin debris.10 Some of the proteins found in
myelin membranes were found to exert a strong growth inhibition effect on a variety of neuronal
cells in vitro, which explains part of the inhibitory environment characteristic of a glial scar.11,12
Microglial cells are the smallest of the glial cells and are found scattered throughout the CNS. The
microglia of the normal brain and spinal cord are in a quiescent state, exhibiting various
behaviours following an injury such as activation, cell division and migration to the affected site.
Therefore, after an injury there will be an influx of microglial cells. These microglial cells will turn
into a more macrophage-like morphology and will stay at the injury site for weeks.1,13,14 With
activated microglia having most of the properties of macrophages, these cells are capable of
producing toxic molecules, which will prevent the CNS from regenerating. When stimulated they
can undergo a respiratory burst and release, for instance, free radicals, nitric oxide and
arachidonic acid derivatives. However, there is evidence of microglia to be both toxic and
neuroprotective in vivo, which provides mixed conclusions.15
Figure 2 – A neuron, with its
constituents identified.1
3
Figure 3 – Illustration of the cells of the CNS, namely neurons, and the glial cells which support
them: oligodendrocytes, microglia and astrocytes.16
Astrocytes are complex cells that exist around the whole CNS and contribute to a lot of functions
in the normal brain and spinal cord, such as regulation of blood flow, provision of energy
metabolites to neurons and maintenance of the extracellular balance of ions.17,18 In the normal
CNS, the majority of astrocytes have part of the cell in contact with a blood vessel and many fine
processes interwoven around neuronal cell bodies and processes. In response to an injury there
will be proliferation of astrocytes, an increase in the size and complexity of their processes and
even scar formation, by a process commonly referred to as reactive astrogliosis. As a
consequence, the great majority of cell surfaces that will be encountered by an axon trying to
penetrate a glial scar, or trying to regenerate anywhere in the CNS, will be astrocytic. More
specifically, the majority of the glial scar consists of tightly interwoven astrocyte processes
surrounded by extracellular matrix, which contribute to the non-permissive microenvironment of
the glial scar to axon regeneration.19,20
It is also known that a lot of molecules present in the glial scar microenvironment at different time
points are inhibitory to axonal regeneration, such as the already mentioned myelin-associated
proteins and chondroitin sulphate proteoglycans, which originate from oligodendrocytes.4
CNS traumas carry a huge impact on life quality. For instance, spinal cord injury (SCI) leads to
chronic paralysis. As it was previously said, this is due to SCI being characterized by massive
cellular and axonal loss, a neurotoxic environment, inhibitory molecules and physical barriers that
hamper nerve regeneration and reconnection. Therefore, seeking an ideal, simple, safe, effective
and viable repair strategy for axonal regeneration, remyelination and functional recovery is critical.
Transplantation of different types of cells is one of the strategies being examined in order to
restore the lost cell populations and to re-establish a permissive environment for nerve
regeneration.
4
1.2 Olfactory Ensheathing Cells
Contrary to what happens with the adult mammal CNS, the mammalian olfactory system
possesses a good regeneration capacity. This is one of the few zones in the body where
neurogenesis occurs during the lifetime of the organism. Damage can occur to the neurons in the
olfactory neuroepithelium due to large-scale infection by bacteria and viruses or trauma. A
bacterial infection can lead to the death of olfactory sensory neurons and subsequently their
axons and injury can directly cause the destruction of axons. In case this happens, new olfactory
neurons are generated from basal cells in the olfactory epithelium. Regeneration is also needed
for the normal turnover of olfactory sensory neurons.21,22 For this regeneration to happen, the
olfactory neurons are replaced, with their axons elongating from the PNS into the CNS to re-
establish functional connections. When nerves from the periphery enter the CNS, the PNS-CNS
transition zone is normally populated by astrocytes. In 1991, it was discovered that the olfactory
nerve differs from other nerves that enter the CNS because this transition zone contains a
specialized type of glial cell, the olfactory ensheathing glial cells (OECs), to which the regenerative
ability of the olfactory system is largely attributed.23
The olfactory system consists of the olfactory mucosa (OM) and the bundles of olfactory nerves
that project into the olfactory bulb (OB) (Fig. 4). To reach their targets in the OB, the axons of the
olfactory sensory neurons project through the lamina propria that underlies the olfactory
epithelium and pass through the bony cribriform plate to enter the nerve fibre layer, which is the
outer layer of the OB and within the CNS. Therefore, new axons will be constantly crossing the
PNS-CNS transition zone into the OB. The ability of constant regeneration of the olfactory neurons
and their ability to extend their axons across the PNS-CNS boundary are attributed to the
presence of the glia of the olfactory system, called OECs.24
OECs arise from the neural crest and are always in contact with the axons of olfactory neurons,
from the nasal epithelium to the outer layer of the OB. OECs ensheath and guide neuronal axons
from the OM to the outer nerve layer by forming a three-dimensional (3D) structure resembling a
tunnel through which the axons extend (Fig. 5).25,26
The axonal regeneration ability of the OECs has proven its usefulness for regenerative therapies
after their transplantation at sites of SCI, by inducing a modest regrowth of ascending and
descending axon tracts.27–29 Thus, cultivation of OECs in laboratory became important for both
experimental and clinical trials. The preparation and purification of primary OECs can be
performed from the OB or the OM.30,31 OECs from the OB and the OM belong to the CNS and the
PNS, respectively. Primary OECs cultures are easy to set up from both OB and OM. However,
these are more easily purified and cultured from adult OB and OM than from both embryonic and
early postnatal animals.32
5
Figure 4 – Diagram of the adult mammal olfactory system intending to show the location of OECs.
Olfactory ensheathing cells (OECs, pink areas) of the OM ensheath bundles of olfactory receptor
axons (olfactory nerves, ON) of the olfactory receptor neurons (ORN) along their course through
the lamina propria in the PNS. Olfactory nerves and their accompanying OECs cross through the
cribriform plate of the skull into the CNS and surround the OB to form the olfactory nerve layer.33
Figure 5 – Schematics of the 3D tunnel shaped ensheathment of olfactory axons performed by
OECs. In the mucosa, the axons (ax) of the olfactory neurons (ON) are first captured in bundles.
Processes of the OECs penetrate the basal lamina (BL) and tightly invest bundles of axons, which
they then guide through the olfactory nerves all the way through the OB.26
6
Besides forming a 3D structure that allows for the regeneration of axons, OECs have also shown
their potential to establish a permissive environment by providing a large amount of neurotrophic
molecules and cellular substrates after being transplanted into damaged CNS.34 It has been
shown that OECs produce platelet-derived growth factor, neuropeptide Y, glia derived nexin and
S100β.35–39 OECs can also produce BDNF, NT-3 and NT-4/5, which promote neuronal survival
and the extension of new axons within regions of the CNS injury.40–42 Furthermore, OECs secrete
nerve growth factor (NGF), glial cell-derived neurotrophic factor (GDNF), VEGF, display the low-
affinity NGF receptor (p75NTR), and FGF receptors.43–46
When first identified, OECs were considered to be Schwann cells of the olfactory system thanks
to their location.47,48 One of the first signs of their unusual characteristics originated from an
immunohistochemical study of olfactory nerves using antibodies to glial fibrillary acidic protein
(GFAP), a marker which generally defines astrocytes. These olfactory nerve Schwann cells, as
they were called at the time, expressed GFAP, which suggested their resemblance to
astrocytes.49 However, later on it was shown that these cells expressed p75NTR, which
suggested their resemblance to Schwann cells. Other studies followed, in which the variation of
the expression of GFAP led to the thought that olfactory glia included both astrocyte-like and
Schwann cell-like cells. In fact, OECs were constantly being described as antigenically and
morphologically very heterogeneous.50,51 Therefore, to the heterogeneity of OECs contributes the
fact that they express a range of different molecules depending on their location in the olfactory
nerve. OECs within the OM express p75NTR and S100β which are used to confirm the identity
and purity of OECs that have been cultured.52–54 Within the nerve fiber layer of the OB there are
two subpopulations of OECs present, which in turn express these markers in different extents. In
the outer nerve fiber layer, OECs express high levels of S100β and p75NTR and in the inner
nerve fiber layer express lower levels of these molecules.55,56 Other studies have shown
differences in the expression of markers between OECs generated from the OM and OECs
generated from the OB, namely it has been shown that as OECs exit from the olfactory epithelium
they display distinct and variable expression of p75NTR, S100β and GFAP, among others.
Specifically, it was demonstrated that there were fewer GFAP positive cells in the OM and it was
even shown that p75NTR positive cells from the OM proliferate for longer than those from the OB
when cultured in the same conditions.57 It has been verified that OECs obtained from these two
different regions do not have the same properties and effects after transplantation in vivo.32,58
This, together with the fact that the role of each of them following transplantation is not well
defined, and despite that OECs were shown to promote axonal regeneration, led to the existence
of controversy about the potential of OECs to promote neuronal regeneration.59–61 In addition,
OECs also show to be heterogeneous in morphology when in culture (Fig. 6).62 This heterogeneity
means it is necessary to prove whether all this different sources of OECs are able to in fact
regenerate SCI injuries and if so, to which extent. One of the most critical issues for whom works
with OECs is the lack of OEC-specific molecular markers.61
7
Figure 6 – Heterogeneity of OECs in vitro. (a) Fibrous OECs with bipolar body; (b) OECs with
flatten body and no apparent process; (c) OECs with multipolar body and long processes; (d)
OECs with long process and exuberant arborizations; (e) OECs with thin, long processes and
poor branching.63
Comparisons between OECs and Schwann cells are much explored in literature, since they
possess many similarities, but also possess differences which distinguish them. As previously
said, originally OECs were referred to as Schwann cells of the olfactory system. In a
developmental perspective, OECs and Schwann cells are both of neural crest origin, contrary to
astrocytes, which arise from radial glia of neuroepithelial origin.25,64 However, despite the obvious
similarities between these two cell types, there are also distinct differences, especially having to
do with the way they interact with astrocytes. Differently to what happens with Schwann cells,
OECs interact freely with astrocytes, without causing harmful effects on the astrocyte population
in question.65 This particularity of OECs has to do with their potential to be used at CNS injury
sites and promote regeneration, since the glial scar is mostly astrocytic and this means both
populations of OECs and astrocytes will interact at injury sites.66
8
1.3 Hydrogels for cell storage and preservation
1.3.1 Cell storage and preservation are needed
for regenerative medicine therapies
Expanded in vitro cells are progressively being used as therapeutic agents for regenerative
medicine purposes, as well as tools to understand an increasing number of diseases.67–69 One
example of this is the use of OECs to regenerate CNS injuries. So far, more efforts have been
put into using OECs in autologous transplantation.53,54 Although autologous therapies have
advantages in being patient tailored and in not presenting an adverse immune response risk, they
present some disadvantages and challenges downstream when more product is required to be
available, such as the difficulty in achieving sufficient and consistent starting material and the
batch-to-batch variability from one patient to the next. Allogeneic cell therapies allow for an easier
bioprocess scale-up, since there is a single source of cells, being more straightforward to define
and standardize the bioprocess in question. An allogeneic cell therapy with OECs, for instance,
would allow for the patients to receive treatment after less time following a CNS trauma, which
might be decisive since the glial scar environment changes a lot during the first two weeks, as
previously said. Since in autologous therapies the cells still have to be harvested from the patient
and after that expanded before the transplantation into the CNS injury site, it takes a higher
amount of time until the patient gets treated.70
In order to have a successful cell therapy, the cellular product has to be produced in good
manufacturing practice (GMP) compliant facilities and the product has to be implanted at hospital
units. This creates a logistics requirement for strategies for cell storage and preservation. One of
this particular logistics requirements is the need to preserve the cellular products in a short-term
storage mode, which will allow to overcome the time lag and the correct transportation of the
cellular product between these two facilities. A global delivery between different continents can
take up to 3 to 5 days.71 The chosen approach for the storage and preservation of the cells has
to guarantee that the cells are delivered both alive and functional and in sufficient number to
generate the required therapeutic response.72 If the cells are correctly preserved prior to their
usage, namely during transportation, the frequency of cellular therapies can increase, while
predictable results are provided. Thus, a correct storage allows for the generation of quality-
controlled cellular product stocks.73,74
1.3.2 Current storage methods present issues
Nowadays the storage and preservation of cells is mostly done by cryopreservation. This comes
with biological and technical issues associated, since it requires the use of cryprotective agents,
such as dimethyl sulfoxide (DMSO). DMSO has shown to have detrimental effects on the stored
cells, such as viability loss after thawing, and has proven costly to remove, making
9
cryopreservation an unreliable preservation method, since this can lead to the final product having
a high variability.75–77
Since cryopreservation is not an ideal option, one might think about doing the transportation of
the cells under the optimum culture conditions, such as a temperature of 37ºC, would be a better
one. That way, the viability of the cells upon arrival to hospital facilities would, in theory, be
maximum, since they would continue to be in culture. One can also imagine, though, that it would
be very costly to do so for 3 to 5 days while the cells are being transported. It would carry a more
reasonable financial cost and it would be easier to achieve the appropriate delivery time window
if the transportation was done under ambient conditions. However, cells have previously shown
to not survive well for considerable time intervals during transfer under ambient conditions.78,79
Therefore, methods that improve the preservation of cell viability, function and therapeutic
potency under ambient conditions would offer a number of benefits throughout the supply chain
of cellular products.
1.3.3 Hydrogels as candidates for the improved
preservation of cells
Hydrogel could be a useful material to improve the preservation of stored cells. Since hydrogel is
a biocompatible, chemically inert and structurally uniform material, it could be used in a simple
and economical storage method, while subjecting cells to minimal manipulation and preserving
their viability and phenotype.71 Hydrogels are 3D cross-linked polymer networks which store large
amounts of biological fluids, mainly water, and therefore can swell or shrink.80 This allows
hydrogels to respond to the fluctuations of the environment stimuli, such as temperature, pH, ionic
strength, electric field and presence of enzymes. When they are swollen, they are soft and
rubbery, resembling the living tissue and exhibiting excellent biocompatibility. This similarity with
soft biological tissues makes hydrogels materials of great use to the biomaterials research
community.81 Some of the applications associated with hydrogels have been in regenerative
medicine and controlled drug release.82,83
Hydrogels have several unique characteristic features which make them resemble to the
extracellular matrix (ECM) of tissues and which allow hydrogels to support cell proliferation and
migration, controlled release of growth factors, minimal mechanical irritation to surrounding tissue
and nutrient diffusion, which support the viability and proliferation of cells.84–86 These in turn allow
the usage of hydrogels in tissue engineering and regenerative medicine as carriers for growth
factors, cells, drugs and genes.87–89 It makes sense, considering all the characteristics of
hydrogels, that there is a growing interest in using this material for cell storage and preservation.
Alginate is a natural occurring anionic and hydrophilic polysaccharide derived from seaweed,
which polymerizes rapidly in the presence of cations to form a biocompatible hydrogel, being
calcium the conventional crosslinking ion (Fig. 7).90 Alginate contains blocks of (1-4)-linked β-D-
10
mannuronic acid (M) and α-L-guluronic acid (G) monomers (Fig. 8).91 Due to its outstanding
properties in terms of biocompatibility, biodegrability and non-antigenicity, alginate has been
widely used in a variety of biomedical applications.92,93 Specifically, cell encapsulation within
alginate hydrogels is well established in the fields of tissue engineering and regenerative
medicine.94–97 Thanks to all of these features, alginate encapsulation has previously been
explored for its protective effect on the storage of a different number of cell types under
temperatures below the cell culture optimum temperature.71,94,98,99 The potential of alginate
hydrogel to improve the preservation of cells during storage at lower temperatures has been said
to result from the fact that it contributes to the stabilization of the cellular membranes, resulting in
a higher protection to the mechanical stress experienced during storage and transportation.99
Figure 7 – Calcium promotes the solidification of alginate networks. Alginate is a long, negatively
charged polysaccharide. Positively charged sodium ions (Na+) dissociate from the sodium
alginate when this is dissolved. Doubly charged calcium ions (Ca2+) can bind two different alginate
strands simultaneously, thereby crosslinking and solidifying the solution by a process called ionic-
crosslinking.100
Figure 8 – Chemical structure of alginate.91
It is also important to refer that hydrogels, alginate hydrogels included, which also possess the
desirable characteristic of being injectable are gaining importance in the field of tissue engineering
and regenerative medicine. This is so because injectable hydrogels allow for cell delivery at the
injury site while overcoming problems related to conventional scaffolds, such as the ones related
with surgical implantation. Surgical procedures increase the risk of infections and improper
adaptation to the defect site, which might eventually lead to scaffold failure. Injectable hydrogels
have the potential to reach the defects in very deep tissues with a good adaptation to the defect
site and with minimum invasiveness. The importance of injectable hydrogels for the tissue
11
engineering area is therefore a result of their reduced risk of infection, less scarring and less pain
for the patient.101 To perform the transportation of the cells, already as a final product, stored and
preserved within an injectable material into the places where the patients are going to get the
treatment, opens a door for the possibility of direct injection post-storage of the cells within the
hydrogel. Alginate hydrogels have proven to be widely injectable, namely within the field of CNS
injuries regeneration.102
1.4 Motivation and aim of the project
Whenever the CNS of adult mammals is damaged, it does not have the capacity to regenerate
itself again. The axons of neurons cannot re-establish the connections which were interrupted by
the trauma.3–6 This continues to be one of the most unmet medical needs in terms of treatment
for these patients, not existing at the moment a standard and reliable treatment which can reverse
the loss of function that occurs. Given the fact that CNS traumas come with a huge loss of life
quality for the ones affected (for instance, SCI leads to chronic paralysis), it is of critical importance
to search for a safe and effective approach to treat these patients. CNS trauma leads to glial
scaring, a process through which CNS glial cell types such as oligodendrocytes, microglia and
astrocytes undergo certain reactions, which in turn make the glial scar a non-permissive
microenvironment for axon regeneration.4
OECs are a type of glia, specific of the olfactory system, to which the constant regeneration
capacity of the olfactory neurons in mammals is attributed.23 OECs have shown to possess
potential for the regeneration of CNS injuries, by allowing axons to regenerate and extend through
a 3D structure which highly resembles a tunnel.26 They have also shown to establish a permissive
environment for axonal regeneration thanks to the secretion of neurotrophic factors.34
Up until now, OECs have been used for autologous transplantation.53,54 However, this means a
big amount of time will be needed to reach a significant amount of cells to transplant into the CNS
injury site and there will be variability from one patient to the next, since the amount of starting
material taken from the biopsy is not always the same, which can produce variable results. It is,
therefore, of interest to create an off-the-shelf cell therapy with OECs. This allogeneic treatment
would allow for the cells to be available for the patient in less time and to create more predictable
results.70 The need for an off-the-shelf cell therapy creates, in its turn, a logistics need for an
effective strategy to store and preserve the cells while they are transported between the
manufacturing places and the hospitals where the patient will get treated. This can take up to 3
to 5 days in case a long distance delivery has to be performed.71 Even though there has been a
considerable progress within the whole cell therapy industry, these bioprocessing and logistical
problems having to do with the storage and distribution of cells between the manufacturing sites
and the clinical sites still exist, having the actual methods shown to be unfitted.75–77,103
12
Hydrogels, namely alginate hydrogels, have previously shown to have a protective effect on the
storage of a different number of cell types under temperatures below the cell culture optimum
temperature, this is, under the conditions felt in a more realistic situation of storage and
transportation of a cellular product.71,94,98,99 Alginate is biocompatible, biodegradable and non-
antigenic, and it stabilizes the cellular membranes of the cells which are encapsulated within it.99
This study is motivated by the hypothesis that encapsulation and short-term storage within
alginate hydrogels has a protective effect on human cells, and in this particular case will have a
protective effect on human OECs. Thus, this study aims at assessing the potential of the material
alginate hydrogel for the short-term storage and preservation of human OECs, which are intended
to be used in allogeneic cell therapy for regeneration of CNS injuries. In order to do so, human
OECs were encapsulated within alginate hydrogels and different storage conditions were used.
Different concentrations of alginate to encapsulate and store the cells were studied. The
encapsulated cells were stored at different temperatures, namely under a controlled temperature
of 37ºC and at room temperature (RT), for periods of 3 and 5 days. After these storage periods,
the preservation condition of the human OECs was assessed through viability assays, as well as
through cell release assays. Additionally, immunocytochemistry assays were carried out in 2D
cultured human OECs, as well as in encapsulated ones after completion of the storage time, and
also in cells which have released from the hydrogels where they were previously stored.
13
Chapter 2
Materials and methods
2.1 Cell culture and banking
The cell line of human OECs used for this research was called PA7. This is simply because the
sample for the primary culture was collected from the OM of a so called donor number 7. All the
methods for cell culture were performed under sterile conditions in a laminar flow hood.
2.1.1 Culture medium formulation
Human OECs from the PA7 cell line were cultured in Dulbecco’s modified eagle medium/nutrient
mixture F-12 (DMEM/F-12) supplemented with GlutaMAXTM (GibcoTM Invitrogen by Life
Technologies, UK), 10% fetal bovine serum (FBS, Sigma-Aldrich, USA) and 1% penicillin and
streptomycin (P/S, Sigma-Aldrich, Israel). Complete medium was stored at 4ºC and always used
within one month after preparation.
2.1.2 Thawing
A T-75 tissue culture flask (NuncTM EasY FlaskTM 75 cm2 with NunclonTM Delta Surface, Thermo
ScientificTM, Denmark) was previously coated with poly-L-lysine (PLL, Sigma-Aldrich, UK).
Concerning the PLL coating, 3 mL of the PLL solution were left for 5 min inside the T-75 flask,
while making sure that all the culture surface was covered. Then, the solution was aspirated and
the flask was allowed to dry for at least 2 hrs before thoroughly rinsing it with Hank’s balanced
salt solution (HBSS, GibcoTM Invitrogen by Life Technologies, UK) with no calcium and no
magnesium.
One cryogenic storage vial (Greiner Bio One, Germany) containing 1 mL and 1x106 PA7s at
passage 6 was thawed from liquid nitrogen, in order to have starting material for expansion and
also create a bank of cells for the purpose of keeping individual working stocks. The cryogenic
storage vial was retrieved from the liquid nitrogen storage and transported inside a liquid nitrogen
14
container until it was placed into a 37ºC water bath, where the cells were quickly thawed for about
1 min. The thawed cell suspension was transferred to one PLL-coated T-75 tissue culture flask
pre-filled with 10 mL of culture medium at 37ºC. The T-75 tissue culture flask was labelled with
the cell line, passage number and date of thawing. The cells were then incubated at 37ºC and 5%
CO2 and on the next day, culture medium was exchanged to get rid of the dimethyl sulfoxide
(DMSO, Sigma-Aldrich, UK) coming from the freezing of the cells.
Centrifugation is not used to get rid of the DMSO during the thawing of PA7s in order to reduce
the mechanical stress applied into the cells. This protocol was repeated with cryogenic storage
vials from the individual working stock whenever new cells for culture were needed. The number
of cryogenic storage vials with cells thawed corresponded to the number of T-75 flasks needed
for this proceeding.
2.1.3 Expansion
Every day, cell cultures were checked and the cells were passaged when they presented about
70% confluency. This assures cells are growing exponentially, and therefore are the healthiest
and more metabolically active as possible. Usually this would occur at day 4 of culture. At each
cell passage and for each T-75 tissue culture flask, the spent medium was discarded and a wash
of the culture surface of the T-75 tissue culture flasks was performed with 5 mL of HBSS. Then,
cells were enzymatically detached from the culture surface through the exposure for 5 min, at
37ºC and 5% CO2 to 5 mL of a 0.25% (v/v) trypsin/0.02% (w/v) EDTA solution (Sigma-Aldrich,
USA). Cell detachment from the culture surface was confirmed by light microscopy and the flasks
were gently tapped to aid the process. Enzymatic inactivation was performed with 15 mL of
complete medium. The content of the flasks was then transferred to 50 mL centrifugation tubes
(Greiner Bio One, Germany) and centrifuged at 400 g for 5 min at room temperature. Following
centrifugation, the supernatant was aspirated and the remaining cell pellet was dislodged by
manually taping the centrifugation tube and then resuspended in an appropriate volume of culture
medium. Viable cell number was quantified and cells were seeded at a density of 5,000 cells/cm2
into new PLL-coated T-75 tissue culture flasks, each one pre-filled with 10 mL of culture medium
at 37ºC. The T-75 tissue culture flasks were labelled with the cell line, passage number and date
of passaging. The Trypan blue (Sigma-Aldrich, UK) exclusion assay was used for viable cell
quantification, with a hemocytometer (Hawksley, UK) and a cell counter (Fisher Scientific,
Taiwan). Culture medium exchange was performed every 48 hrs.
2.1.4 Freezing
Freezing medium, composed of 90% (v/v) FBS and 10% (v/v) DMSO, was prepared and stored
at 4ºC. Then, cells were enzymatically detached from the tissue culture flasks following the
protocol described on 2.1.3. After resuspension of the cells with complete medium, viable cell
number was quantified. Each 1x106 viable cells were to be frozen in 1 mL of freezing medium and
15
inside one cryogenic storage vial. The cell suspension was centrifuged at 400 g for 5 min and the
supernatant was discarded without disturbing the cell pellet. The cells were then resuspended
with the appropriate volume of cold freezing medium and the resulting cell suspension was
aliquoted. The cryogenic storage vials were labelled with the species, cell line, passage number
and date of freezing. The cells were frozen by placing the cryogenic storage vials inside a Mr.
FrostyTM Freezing Container (Thermo ScientificTM, UK) filled with isopropanol (Fisher Scientific,
USA) and storing them at −80ºC overnight. On the next day, the frozen cells were transferred to
liquid nitrogen storage, transporting the cryogenic storage vials inside a liquid nitrogen container.
2.2 Cell encapsulation and storage
The method used for encapsulation of the PA7s inside alginate hydrogel was adapted from
Palazzolo et al.104 with some modifications.
The alginate powder (PRONOVA UP LVG, Novamatrix, Norway) was dissolved in 150 mM NaCl
(Sigma-Aldrich, USA) and filtered with 0.22 µm filters (Millex-GP syringe filter unit, Merck Millipore
Ltd., Ireland), in order to get a 1% (w/v) stock alginate solution. Cells were recovered recurring to
enzymatic detachment as previously described in 2.1.3 when they were at about 70% confluency.
After centrifugation, viable cells were quantified using the Trypan blue exclusion assay. After new
centrifugation, the resulting pellet was resuspended with the right amount of culture medium in
order to form a concentrated cell suspension of 105 cells/µL. The resulting concentrated cell
suspension and the stock alginate solution were then mixed and diluted on the right proportions
with a 150 mM NaCl solution, in order to get different alginate contents (from 0.1 to 0.4% (w/v))
and cells always at a density of 104 cells/µL. On a parallel experiment, culture medium was used
instead of the 150 mM NaCl solution to dilute the concentrated cell suspension and the stock
alginate solution. This aimed at assessing if this very small and easy to make change in the
protocol would increase the viability of the encapsulated cells, since these would not spend so
much time under nutrient deprivation.
For each single hydrogel, 30 µL of this cell suspension in alginate were pipetted into a well of a
culture tissue grade 24-well plate (NunclonTM Delta Surface, Thermo Scientific, Denmark). Next,
450 µL of a solution of 10 mM CaCl2 (Sigma-Aldrich, USA) in complete medium were gently
pipetted along the walls of each well, so that this solution covered the whole hydrogel and gelation
was allowed to occur for 30 min at RT. Once the cross-linking solution had been removed, 2 mL
of culture medium were added to each well if the hydrogels were to be stored on this tissue culture
grade well-plate. If hydrogels were to be stored into an ultra-low attachment surface 24 well-plate
(Costar®, Corning, USA), the hydrogels were first transferred with a cell scraper (Costar®, Corning,
Mexico) following gelation. Half of the culture medium was exchanged every 48 hrs.
16
The resulting hydrogels were stored for different time periods of 3 and 5 days. These were the
chosen time periods for the storage of the hydrogels since a global delivery between different
continents of the cellular product could take between 3 to 5 days. The well-plates containing the
hydrogels were either stored in an incubator at 37ºC or at RT inside the laminar flow hood, to
ensure sterility. In the latter, the temperature was recorded every hour with a temperature data
logger (EL-USB-1, Labfacility, UK) for 5 days, the maximum time for which the hydrogels were
stored. Using the software EasyLog USB (Version 7.4.0.0, Lascar electronics), the maximum and
minimum temperature at which the hydrogels were subjected was recorded, as well as the
average temperature and the respective standard deviation. Graphics showing the temperature
fluctuations with time were also extracted from the software.
2.3 Viability assays
The method used for the viability assays was adapted from Palazzolo et al.104 with some
modifications.
After 3 and 5 days of the hydrogels being stored into tissue culture grade 24-well plates at either
37ºC or RT, the exhausted medium was removed from the wells and fresh complete medium was
used to wash the hydrogels once. After this, the LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen,
USA) was used to perform live and dead staining of the encapsulated PA7s. The hydrogels were
incubated with 1.07 µM calcein and 1.73 µM ethidium homodimer in complete medium for 30 min
at 37ºC and 5% CO2. After this, the hydrogels were washed twice with complete medium for 15
min. Finally, fresh medium was added into the wells for imaging.
From each of the tri-replicates prepared for each tested condition, three random images were
obtained with the combination of both channels, GFP and Texas Red, in order to obtain 9 images
for each condition and therefore significant numbers for statistical analysis. Fluorescent images
were acquired using an EVOS FL cell imaging system (ThermoFisher Scientific, UK). Live (green)
and dead (red) cells were counted using the image processing and analysis software ImageJ,
namely the plugin Cell Counter. Cell viability for each image was calculated using equation 1.
2.4 Cell release assays
These assays aimed at assessing the migratory potential of the encapsulated PA7s to the outside
of the hydrogels, and therefore their capacity to release out of the hydrogels with different alginate
contents, after being stored for 3 and 5 days. This is relevant to evaluate if the human OECs
Viability (%)= number of live cells
number of total cells×100 (1)
17
would have the capacity to release from the hydrogels and migrate out into the tissues where they
are needed, in the case these hydrogels were to be used also as a delivery method into the
affected site, immediately after the storage time. These assays are, therefore, also a measure of
the quality of the cells after these being stored for 3 and 5 days in the different previously
explained conditions and also aims at assessing which storage condition results in the highest
release potential.
In order to assess this release capacity, the cells were encapsulated inside the hydrogels and
stored, as previously explained, in either tissue culture grade 24 well-plates or ultra-low
attachment surface 24 well-plate and either in temperature controlled conditions of 37ºC or at RT.
Cells were also cultured in 2D conditions in these well-plates, as a control, since the cells which
will release from the hydrogels will be adherent to the tissue culture surface of the wells. For this,
the concentrated cell suspension obtained during the cell encapsulation protocol and containing
105 cells/µL was diluted on a proportion of 1:10 with cell culture medium and 3 µL of this new cell
suspension were plated into each well previously coated with PLL, totalizing 3×104 cells per well.
Then, 2 mL of fresh cell culture medium were placed into each well. At the 3rd and 5th day of
storage, phase contrast microscopy pictures from the hydrogels and the 2D cultured cells were
taken. Immediately after, all the hydrogels were transferred into a new tissue culture grade 24
well-plate, by using a cell scraper. This step is supposed to mimic the transfer of the hydrogel
with the cells from the storage location into the affected site of the patient receiving the treatment.
To transfer the cells in 2D that had been plated into tissue culture grade well-plates, the spent
medium was discarded and a wash of the wells surface was performed with 200 µL of HBSS.
TrypLETM Express (200 µL) (ThermoFisher Scientific, UK) was used to detach the cells, since no
subsequent centrifugation step was performed to separate the detached cells and this reagent is
gentler than trypsin. Also, unlike trypsin, this can be inactivated only by dilution. The detachment
step took 5 min to occur at 37ºC and cell detachment from the culture surface was confirmed by
light microscopy. The inactivation step was performed by adding 800 µL of fresh culture medium.
Then, the resulting 1 mL of cell suspension was pipetted into the new well and another 1 mL of
fresh culture medium was added, to totalize the 2 mL of volume. To transfer the cells in 2D that
were in ultra-low attachment well-plates, and before perform any other step with this well-plate,
the “top” 1 mL of spent medium was discarded while trying not to agitate it. The left 1 mL, where
most of the cells should be, was simply transferred into the new well after resuspension with the
pipette. Finally, 1 mL of fresh culture medium was added into each well. All the new well-plates
containing the 2D and 3D cultures were placed at 37ºC. The hydrogels which were stored at RT
were also then placed at 37ºC, since this better mimics the physiological conditions at which these
hydrogels would be submitted in the case of them being used also for the delivery of the cells.
Half of the complete medium was exchanged every 48 hrs. 7 days after the transfers, new phase
contrast microscopy pictures of each well were taken, trying to highlight the cell release and
migration occurring out from the hydrogels.
18
This assay was first performed only by storing the hydrogels with the encapsulated cells in tissue
culture grade well-plates and it was possible to observe that after 3 and 5 days of the hydrogels
with the encapsulated cells being stored in tissue culture grade well-plates at 37ºC, a lot of cells
had already released (Fig. 20 (a); Fig. 21 (a); Results section), especially the ones within the
0.1% alginate hydrogels, where the cell release was extensive. This was not the ideal, since the
goal is to deliver as many cells as possible into the receiver patients, and in the case these
hydrogels are used to deliver the cells into the patients after the storage time, the aim will be to
keep as many cells alive and within the hydrogel matrix as possible. It was this observation that
led to the decision to also use ultra-low attachment well-plates, in parallel, to perform the storage
step, since this way there should be no cell release in this phase. Another thing that can be
questioned is whether if the cell release will increase after the transfer of hydrogels previously
stored in ultra-low attachment well-plates, when comparing with hydrogels previously stored in
tissue culture grade well-plates. In order to assess this it is necessary to have a way to compare
the cell release and migration out of the hydrogels that is occurring. This is also necessary in
order to allow for the assessment and comparison of other things, as for instance if there was a
different extent of cell release from the hydrogels with lower alginate contents comparing to the
ones with higher alginate contents and if there is a different extent of cell release after transfer of
hydrogels which were stored for 3 days comparing to 5 days. Finally, it is also interesting to assess
if there is release of cells from the hydrogels which were previously stored at RT, and if so, at
which extent.
The strategy used to make possible a comparison of the cell release extent between the different
conditions studied was based on the creation of a scale that goes from zero to 3, where zero
means “no cells released”, 1 means “only a few released cells”, 2 means “marked release of cells”
and 3 means “extensive release of cells”. Each picture taken from the hydrogels was then rated
with a number from this scale.
2.5 Immunocytochemistry
This assay aimed at assessing and comparing the expression by the human OECs of a panel of
markers on the different stages of the former experiments, to assess if their phenotype shifts or if
it is kept the same. Therefore, the human OECs were stained: when cultured in 2D at 37ºC in
tissue culture grade well-plates, after 5 days of plating, as a control; when encapsulated, in 3D,
after 5 days of storage in ultra-low attachment well-plates at 37ºC; and finally, the cells that
released from the hydrogels which were transferred into tissue culture grade well-plates after 5
days of storage were stained with the 2D staining protocol, after having 7 days to release. Tri-
replicates were performed for every staining.
19
The chosen markers to stain the cells for were S100β and p75NTR, since these markers are
expressed by OECs from the OM, the source location of the cells here cultured, and they are
usually used to confirm their identity and purity.52–54
The protocol for the 2D staining started in the laminar flow hood by removing the spent medium
and wash the wells with HBSS once. Then, a solution of 4% paraformaldehyde (PFA, Sigma-
Aldrich, UK) was placed in the wells for 20 min at RT, after which it was replaced with a solution
of phosphate buffered saline (PBS, Lonza, UK). The next steps were performed in non-sterile
conditions. Each well was washed with PBS three times for 5 min each time. A solution of 0.25%
(w/v) Triton X-100 (Sigma-Aldrich, UK) in PBS was then applied for 30 min at RT and three more
washes with PBS of 5min each were done. After this, the samples were blocked with a solution
of 5% (w/v) goat serum (Dako, UK) in PBS for 30min at RT, followed by three more washes with
PBS. Then, the samples were incubated for 90 min at RT with the primary antibodies diluted in
PBS on a proportion of 1:200. The primary antibodies were rabbit anti-S100β (Dako, UK) and
mouse anti-p75NTR (Millipore, UK). After three washes with PBS the samples were incubated
with the secondary antibodies diluted in PBS on a proportion of 1:200 and Hoechst (Sigma-
Aldrich, UK) diluted on a proportion of 1:1000 for 45 min at RT with the well-plates wrapped up in
foil, since the secondary antibodies are light sensitive. The secondary antibodies were goat anti-
mouse IgG Alexa Fluor® 488 conjugate (ThermoFisher Scientific, UK) and goat anti-rabbit IgG
Alexa Fluor® 594 conjugate (ThermoFisher Scientific, UK). After three more washes with PBS
the samples were ready for imaging. From each of the tri-replicates prepared for each tested
condition, three random images were obtained with the combinations of the DAPI and Texas Red
channels. The number of positive and negative cells for the markers tested were counted.
Fluorescent images were acquired with an EVOS FL cell imaging system. For the staining of the
cells which released from the hydrogels, the remaining hydrogel had to be removed from each
well before performing the 2D staining protocol.
For the immunostaining of the encapsulated cells after 5 days of storage in the ultra-low
attachment well-plates, the method used was adapted from Palazzolo et al.104 with some
modifications. These modifications were: the blocking solution, which was 5% goat serum instead;
the fact that the primary antibodies were diluted only in the buffer; Hoechst having been used
instead of DAPI; and still the fact that only 0.2% alginate hydrogels were used at this stage. This
was the chosen concentration for a first experiment since these are easier to handle than the
0.1% alginate hydrogels but still softer than the 0.3 and 0.4% alginate hydrogels, having therefore
shown to allow for cell release, which was needed in order to stain released cells.
For the controls for the 2D and 3D staining, all the steps mentioned were done except for the
incubation with the primary antibodies, in order to make sure there were no false positives upon
imaging.
20
2.6 Statistical analysis
Both for the encapsulated cells stored at 37ºC and at RT and for the same time of storage, a one-
way ANOVA analysis of variance was performed to check if at least one of the means obtained
for the viability of the cells was different from the rest. Since this test does not tell where the
difference lies, when the one-way ANOVA showed that at least one of the means was different,
a Student’s t-Test was necessary to compare two of the means at each time. In such cases, the
latter was then used in order to try to find significant differences between the viability values taken
from populations with different alginate contents in the hydrogels that had been stored for the
same time at the same temperature. Two-tailed Student’s t-Tests were used, since the test aimed
at assessing if any of the means of the viability for the two compared population was higher or
lower than the other. The Student’s t-Test was also used to check for significant differences
between viabilities obtained for cells encapsulated and stored at the same temperature and within
hydrogels with the same alginate content, varying the storage time and between viabilities
obtained for cells encapsulated and stored for the same time and within hydrogels with the same
alginate content, varying the storage temperature.
In order to perform the Student’s t-Tests correctly and choose the proper test, each time this test
was to be used, a previous F-test was necessary to know if the populations in question were of
equal or unequal variance.
Statistical significance was established with p-value < 0.05.
All the statistical analysis was performed using the tool Data Analysis in Excel.
21
Chapter 3
Results and discussion
3.1 Human OECs encapsulated inside alginate
hydrogels
In the present work, human OECs (Fig. 9) were successfully encapsulated and stored inside
alginate hydrogels (Fig. 10), with the aim of assessing the potential of this material to preserve
them. This was decisive for the progression and success of the following work. The encapsulated
OECs showed to be evenly distributed within the alginate hydrogels (Fig. 11 (a – d)).
Figure 9 – Phase contrast microscopic image of the cultured PA7s at passage 11, 4 days after
passaging and right before encapsulation in the alginate hydrogel. Their morphology can be
defined by flat bodies with no apparent processes, much like fibroblasts. Scale bar: 1000 µm.
22
Figure 10 – Picture of a 24 well-plate with alginate hydrogels (some of them are highlighted) right
after gelation for 30 min with 10 mM CaCl2 in complete medium.
Figure 11 – Representative phase contrast microscopic images of hydrogels with encapsulated
human OECs after 3 days of storage at 37ºC on a tissue culture grade 24 well-plate. Cell seeding
density was 107 cells/mL for all the hydrogels and the cells show to be, for the most part, evenly
distributed and with an even density along them, especially in hydrogels with higher alginate
contents. The gels have different alginate contents: (a) 0.1%, (b) 0.2%, (c) 0.3% and (d) 0.4%
(w/v) and all of them present an approximately circular shape, with their diameter, in these
particular cases, ranging from about 3.87 mm to 4.74 mm. Scale bar: 2000 µm.
(a)
(c) (d)
(b)
23
3.2 Viability of human OECs encapsulated and stored
within alginate hydrogels
In order to evaluate the suitability of alginate hydrogels for the storage and preservation of human
OECs, the viability of the cells encapsulated and stored within this material in different conditions
was assessed. These conditions differed in the alginate content of the hydrogels (0.1% - 0.4%),
time of storage (3 and 5 days) and temperature of storage (37ºC and RT), with tracking of the
temperature fluctuations for the RT storage.
After calcein/ethidium homodimer staining, the live/dead encapsulated OECs within the hydrogels
were imaged by fluorescent microscopy (Fig. 12 (a – d)). Again in these images (as in the ones
of figure 11), the distribution of the encapsulated cells within the hydrogels shows to be
homogeneous for all the storage conditions studied, regardless from the cell numbers.
Figure 12 – Representative fluorescence merged images of the assessment by live (green) and
dead (red) staining of the viability of OECs encapsulated in hydrogels with different alginate
contents (0.1% - 0.4% (w/v)) and stored at (a) 37ºC for 3 days, (b) 37ºC for 5 days, (c) RT for 3
days and (d) RT for 5 days. Cell seeding density was 107 cells/mL hydrogel in all conditions. Scale
bar: 200 µm.
24
The total cell number extracted from the images for each storage condition was plotted (Fig. 13),
with the means ranging from 81 to 337 cells, considering the whole range of different storage
conditions tested. It is possible to observe that there was variation between the numbers of total
cells counted within the same area of hydrogel, for the different conditions assessed. This
difference was already noticeable just from looking at the images obtained during the viability
assessment of the encapsulated OECs (Fig. 12).
Figure 13 – Quantification of total cell number extracted from the images resulting from the
live/dead staining, for each storage condition studied (each image covered an area of
approximately 0.309 mm2 of the hydrogels). Values are expressed as mean ± standard deviation
from the 9 images analysed for each condition.
It was not possible to observe a valid trend for the variation of the cell numbers corresponding to
the different storage conditions. For instance, for the storage at 37ºC, the alginate contents of
0.1% and 0.2% present higher means for the number of cells counted within hydrogels stored for
3 days than for the ones stored for 5 days. However, for the hydrogels with an alginate content of
0.3% and 0.4%, the opposite happens. For the storage at RT, with the exception of the alginate
content of 0.1%, the means of the cell number is always lower on hydrogels stored for 3 days
than for 5 days. Higher cell numbers within the hydrogels stored for 5 days, when compared with
3 days, might suggest that the cells are proliferating within the hydrogels. However, proper
methods and a proper experiment design would be necessary in order to assess such possibility.
The variation observed in the counted number of cells within the hydrogels after storage under
different conditions might also be due to the migration of the cells that occurs out of the hydrogels
during the storage time, something discussed more in detail later on. The following presented
viability results for the different storage conditions would be more valid if the total cell number
within the hydrogels had been kept constant. However, only later on and after having carried out
the different viability assessments, it was realized that by using well-plates with an ultra-low
attachment surface this cell release from the hydrogels could be prevented. If more time had been
available, these viability assessments would have been repeated with this well-plates instead of
the culture grade ones here used and with which the following results were obtained.
25
3.2.1 Viability of the encapsulated cells after 3
and 5 days of storage at 37ºC
After a 3 and 5 day storage period at 37ºC, the percentage of viable OECs encapsulated within
hydrogels with different alginate contents was compared (Fig. 14), in order to assess the effect of
the alginate content of the hydrogels on the preservation of the cells. The viability values of the
OECs within hydrogels with the same alginate content stored for either 3 or 5 days were also
compared, in order to assess the effect of the storage time on the preservation of the cells.
Figure 14 – Quantification of the viability of the OECs stored within hydrogels with different
alginate contents (0.1% – 0.4% (w/v)) at 37ºC and for 3 and 5 days. Values are expressed as
mean ± standard deviation from the 9 images analysed for each condition.
The viability of the OECs stored within the hydrogels for 3 days (0.1%: 76.7% ± 4.02%; 0.2%:
77.2% ± 5.76%; 0.3%: 74.5% ± 7.36%; 0.4%: 76.5% ± 5.36%) was slightly higher than the viability
of the cells stored within the hydrogels for 5 days (0.1%: 75.4% ± 4.10%; 0.2%: 74.7% ± 3.53%;
0.3%: 74.4% ± 3.98%; 0.4%: 71.6% ± 4.51%) for all the alginate contents studied. However, these
differences were not statistically significant. This finding suggests that it is possible to extend the
storage of OECs within the alginate hydrogels under a temperature of 37ºC for a period of up to
5 days without a significant loss on OECs viability, which in its turn has implications on the ease
of the logistics associated with a cell therapy with OECs.
Also for the storage at 37ºC, it was not possible to find statistically significant differences between
viability values for the cells stored within hydrogels with different alginate contents for the same
period of time. The literature suggests that the viability of cells, in this case neurons, encapsulated
within this exact same alginate hydrogel moderately decreases with the increase of the alginate
macromer content.104 It explains so with the possibility of several factors affecting the viability,
26
such as the softness of the hydrogel and its porosity, which in its turn may have implications on
the way the cells molecularly interact with each other.
Overall, all the cells stored within hydrogels kept at 37ºC presented a good maintenance of
viability (always higher than 70%). In a study where primary cortical neurons were encapsulated
within the same material and with the same range of alginate contents, similar results were
obtained when these were kept at 37ºC. Viabilities between 70% and 80% were obtained after 3
days.104 However, another study where corneal epithelial cells were encapsulated and stored
within alginate hydrogels provided conflicting results. When the storage temperature of 37ºC and
an alginate content of 0.3% (w/v) were used, a cell viability no greater than 50% was obtained
after 5 days of storage.94 Nonetheless, the findings here presented show that it is possible to store
human OECs within hydrogels with a range of alginate from 0.1% (w/v) to 0.4% (w/v), at a
temperature of 37ºC and for a period of up to 5 days, with a maintenance of viability always higher
than 70%, which has positive implications on the logistics associated with the use of these cells
for CNS regeneration therapies.
3.2.2 Viability of the encapsulated cells after 3
and 5 days of storage at RT
Similarly to what was done for the storage at 37ºC, after a 3 and 5 day storage period at RT, the
percentage of viable OECs encapsulated within hydrogels with different alginate contents was
compared (Fig. 15), in order to assess the effect of the alginate content on the preservation of the
cells stored under RT. The viability values of the OECs within hydrogels with the same alginate
content stored for either 3 or 5 days were also compared, in order to assess the effect of the
storage time on the preservation of the cells stored under RT.
The temperature was recorded every hour for the storage at RT (Fig. 16), for the maximum studied
time of storage. The maximum and minimum temperatures at which the cells were subjected
through these 5 days were, respectively, 29ºC and 21ºC and the average temperature during the
full recording time was 21.5ºC ± 0.8ºC (mean ± standard deviation).
Contrary to what happened for the storage at 37ºC, the viability of the OECs stored in the
hydrogels for 3 days (0.1%: 56.6% ± 7.74%; 0.2%: 54.7% ± 8.50%; 0.3%: 62.0% ± 7.44%; 0.4%:
58.2% ± 6.09%) was lower than the viability of the cells stored in the hydrogels for 5 days (0.1%:
65.2% ± 4.78%; 0.2%: 71.5% ± 5.92%; 0.3%: 68.8% ± 3.76%; 0.4%: 63.5% ± 5.40%) for the
whole range of alginate concentrations studied. This difference was statistically significant for the
hydrogels which contained 0.1% (w/v) and 0.2% (w/v) of alginate. Since that at RT the cells did
not seem to release out from the hydrogels (data shown later on), one possible explanation for
this would be if the cells were proliferating. However, there is no data to prove such possibility.
Even though this finding is unexpected, it suggests, much like it happened for the storage at 37ºC,
that it is possible to extend the storage of OECs within the alginate hydrogels for a period of up
27
to 5 days without hindering the viability of the cells, when compared with a storage done for a
period of only 3 days.
Figure 15 – Quantification of the viability of the OECs stored within hydrogels with different
alginate contents (0.1% – 0.4% (w/v)) at RT and for 3 and 5 days. Values are expressed as mean
± standard deviation from the 9 images analysed for each condition, with asterisks representing
statistical significant differences (*p<0.05; **p<0.01; ***p<0.001).
Figure 16 – Temperature fluctuations at which the cells were subjected through the 5 day period
of storage at RT. The temperature was recorded every hour. Tmax = 29ºC; Tmin = 21.5ºC; Tavg =
21.5ºC ± 0.8ºC (mean ± standard deviation).
Days 0 1 2 3 4 5
28
Also for the storage at RT, it was possible to find statistically significant differences between
viability values for the OECs stored within hydrogels with different alginate contents for the same
period of time. Namely, for the cells stored for a period of 5 days, significant differences were
found for the viability of the cells stored within hydrogels containing 0.1% vs 0.2% (w/v), 0.2% vs
0.4% (w/v) and 0.3% vs 0.4% (w/v) of alginate. However, no defined trend can be observed, since
the viability increases from 0.1% (w/v) to 0.2% (w/v) alginate hydrogels, to only decrease again
from the later to the 0.3% (w/v) alginate hydrogel, and again to the 0.4% (w/v) alginate hydrogel.
No significant differences were observed between the viability values of the cells encapsulated in
hydrogels with different alginate contents and stored for a period of 3 days.
Overall, all the cells stored within hydrogels kept at RT presented a maintenance of viability higher
than 50%, reaching the range of about 70% in certain conditions. These results were better than
the ones found by a study where corneal epithelial cells were encapsulated and stored within
alginate hydrogels and under ambient temperature. When a concentration of 0.3% (w/v) alginate
was used, the viability of the recovered cells was slightly higher than 20% after 3 days of
encapsulation and only of about 10% after 5 days of encapsulation.94 Other similar works, but
where more concentrated alginate hydrogels were used, presented better results. In a study
where human adipose-derived stem cells (hASCs) were stored within 1.2% (w/v) alginate
hydrogels and under different temperatures, the viability of the cells recovered after a 3-day
encapsulation period was assessed. Here, the authors obtained a viability of about 70% for the
encapsulated hASCs stored under a temperature of 21ºC, a temperature very close to the
average recorded temperature of the present study, 21.5ºC ± 0.8ºC (mean ± standard
deviation).103 In another study in which also 1.2% (w/v) alginate hydrogels were used, the storage
temperatures ranged from 18ºC to 22ºC, a range in which the average temperature felt by the
encapsulated OECs falls. Here, the authors obtained viability values as high as 80% for human
mesenchymal stem cells (hMSCs) and mouse embryonic stem cells (mESCs) after a 5-day
storage period.71
3.2.3 Viability of the encapsulated cells stored
at 37ºC vs RT
In order to better understand the effect of the storage temperature on the preservation of the
encapsulated OECs, the obtained viability values at 3 (Fig. 17) and 5 days (Fig. 18) after storage
were now plotted on a way that better allows to compare the differences for cells stored at either
37ºC or RT, for the same amount of time and within hydrogels with the same alginate content.
The viability of the OECs encapsulated and stored within the hydrogels for 3 days at 37ºC was
always significantly higher than the ones stored for the same amount of time at RT, for all the
alginate contents studied. For the OECs encapsulated and stored within the hydrogels for 5 days,
the obtained viability values showed to be significantly higher for the ones stored at 37ºC, for all
the alginate contents studied, except 0.2%. However, even in this case the viability for the storage
29
at 37ºC shows to be slightly higher than the one obtained with storage at RT. These results
suggest that, in this case, the protective effect given by the alginate hydrogel to the encapsulated
cells was not enough to maintain their viability values comparable to the ones obtained when the
optimum culture temperature is used for the storage. This may be due to the lower alginate
concentrations which were used for the hydrogels in the present work, when comparing with other
studies which present better results.71,103
Figure 17 – Quantification of the viability of the OECs after 3 days of storage within hydrogels
with different alginate contents (0.1% – 0.4% (w/v)), at 37ºC and at RT. Values are expressed as
mean ± standard deviation from the 9 images analysed for each condition, with asterisks
representing statistical significant differences (**p<0.01; ***p<0.001).
Figure 18 – Quantification of the viability of the OECs after 5 days of storage within hydrogels
with different alginate contents (0.1% – 0.4% (w/v)), at 37ºC and at RT. Values are expressed as
mean ± standard deviation from the 9 images analysed for each condition, with asterisks
representing statistical significant differences (**p<0.01; ***p<0.001).
30
It would be very significant to achieve similar viability results for the encapsulated human OECs
for the storage at RT as for the storage under a controlled temperature of 37ºC. This would greatly
ease the logistics required for the cell storage and transportation within the appropriate delivery
time windows (3 to 5 days) and it would help to increase the frequency of the therapies using
these cells by giving them a more reasonable financial cost.71
3.2.4 Viability assessment after the storage at
37ºC of the encapsulated cells, after a change in the
encapsulation protocol
As it was mentioned before in the methods section for the storage conditions used, a parallel
experiment was carried out, where cell culture medium was used instead of a solution of 150 mM
NaCl to dilute the stock 1% (w/v) alginate solution, as well as the concentrated cell suspension
obtained previously to the encapsulation. Given that: the cells do not spend so much time on
nutrient deprivation with this method; the change in protocol is very easy to make; and it does not
add significant costs or time of experiment, it is interesting to assess if the viability of the cells
increases. To evaluate this, the viability of the OECs encapsulated within hydrogels with the same
alginate content was quantified and compared after their encapsulation with the conventional and
the new methods, following storage for 5 days at 37ºC (Fig. 19).
Figure 19 – Quantification of the viability of the OECs after 5 days of storage within hydrogels
with different alginate contents (0.1% – 0.4% (w/v)), at 37ºC, having the dilution of the stock
alginate solution and the concentrated cell suspension obtained been done with either a solution
of 150 mM NaCl or cell culture medium. Values are expressed as mean ± standard deviation from
the 9 images analysed for each condition.
31
The results showed that there were no significant differences between the viabilities of the human
OECs stored within hydrogels with the same alginate content and at the same temperature for
the same period of time, when either cell culture medium (0.1%: 76.2% ± 4.40%; 0.2%: 73.5% ±
4.18%; 0.3%: 71.8% ± 2.03%; 0.4%: 71.8% ± 4.62%) or a solution of 150 mM NaCl (0.1%: 77.8%
± 2.31%; 0.2%: 76.4% ± 3.62%; 0.3%: 71.8% ± 2.62%; 0.4%: 70.5% ± 7.89%) were used.
It is also noted that the results found for the viability at day 5 where the saline solution was used
are in accordance with the results previously obtained for the same conditions (Fig. 14) (always
higher than 70%), once more reinforcing the previous statement that it is possible to store human
OECs within hydrogels with a range of alginate from 0.1% (w/v) to 0.4% (w/v), at a temperature
of 37ºC and for a period of up to 5 days, with a maintenance of viability always higher than 70%,
which has positive implications on the use of these cells for CNS regeneration therapies.
32
3.3 Release of the encapsulated human olfactory
ensheathing cells out of the hydrogels
Cell release assays were carried out with the purpose of assessing the capacity of the
encapsulated OECs to release and to migrate out of the hydrogels with different alginate contents
(0.1% - 0.4% (w/v)) into the surrounding environment. This is useful in case these hydrogels were
to be used also for the delivery of the cells after the storage period. For this, phase contrast
pictures of the hydrogels were taken after 3 and 5 days of storage at 37ºC and at RT, either in
tissue culture grade or ultra-low attachment well-plates (Fig. 20 (a & c); Fig. 21 (a & c); Fig. 22 (a
& c); Fig. 23 (a & c)). After 7 days of having transferred these hydrogels into new tissue culture
grade well-plates and having placed these at 37ºC, new pictures were taken with the aim of
showing the cell release occurred (Fig. 20 (b & d); Fig. 21 (b & d); Fig. 22 (b & d); Fig. 23 (b & d)).
The human OECs were also cultured in 2D for 3 and 5 days under the same conditions, and
transferred, having been imaged at the same time points as the hydrogels. Ultra-low attachment
well-plates were also used for the storage of the OECs, besides the tissue culture grade well-
plates, because ideally during the storage time all the encapsulated cells will be maintained within
the hydrogels. After performing this experiment with storage of the hydrogels within tissue culture
grade well-plates, it was evident that part of the cells were releasing from the hydrogel and were
not being kept inside it. The maintenance of the cells within the hydrogels upon their storage or
transportation would, in theory, ensure that a higher number of OECs are transplanted into the
CNS injury site of the patient upon a possible therapy, therefore contributing to a higher chance
of success.
The temperature was recorded every hour for the storage at RT for the maximum studied time of
storage (5 days), when this was done in tissue culture grade well-plates (Fig. 24) and in ultra-low
attachment well-plates (Fig. 25). The maximum temperature at which the cells were subjected
was, respectively, 25ºC and 26.5ºC and the minimum temperature was 21.5ºC for both cases.
The average temperature during the full recording time was, respectively, 22.4ºC ± 0.7ºC and
22.1 ± 0.9ºC (means ± standard deviation). Since these last two values were so similar, the results
were considered to be comparable. The means for these temperatures were also similar to the
one recorded for the viability assays previously performed, 21.5ºC ± 0.8ºC (means ± standard
deviation).
Just from looking at the figures, it is clear that in certain conditions the cells show to be able to
release from the hydrogels, as for instance when stored within 0.1% alginate hydrogels at 37ºC
for 3 and 5 days in tissue culture grade well-plates (Fig. 20 (a) & Fig. 21 (a)) and 7 days after
being transferred from these conditions into new tissue culture grade well-plates (Fig. 20 (b) &
Fig. 21 (b)). This suggests that the cells show to be in good preservation conditions. Even though
it is possible to see the occurred cell release with the images obtained, a way to compare the
extent of the occurred cell release between the various conditions studied was needed. The way
33
found to achieve this consisted on ranking each picture taken for each condition of storage, using
a defined scale of different cell release extents (Tables 1 & 2). Only cells that presented a spindle
shape and showed to be adherent to the well surface were considered to have migrated out from
the hydrogels and released from them. This method presents obvious limitations, such as being
a purely subjective one, with no actual quantification. However, when the differences between the
extents of the cell release are clearly noticeable, it is a useful and simple way to summarize and
structure the observations made, allowing for the comparison of the results obtained for the many
different conditions studied.
The cells that were stored at RT within the hydrogels did not show to release from them while in
storage (Fig. 20 (c); Fig. 21 (c); Fig. 22 (c); Fig. 23 (c)), regardless of the type of surface of the
well-plates used for the storage. This is why a classification of zero, on the scale of the cell release
extent, was attributed to all the corresponding pictures (Tables 1 & 2). However, the cells cultured
in 2D at RT, show to have an unusual morphology (Fig. 20 (c) 2D; Fig. 21 (c) 2D; Fig. 22 (c) 2D;
Fig. 23 (c) 2D), rounder than the one normally observed, which might be the cause of the fact that
there seems to be no cell release from the hydrogels in these situations.
Still regarding the atypical morphology of the 2D cultured cells kept at RT, the fact that the cells
regained the spindle shape when, after 3 days, were transferred into tissue culture grade well-
plates and placed at 37ºC, (Fig. 20 (d) 2D; Fig. 22 (d) 2D), suggests that they are not dead.
However, when the same was done for the 2D cultured cells which were in culture for 5 days at
RT, the cells did not regain the spindle shape (Fig. 21 (d) 2D; Fig. 23 (d) 2D). This suggests that
5 days in these conditions might be too much for the cells to be able to keep their viability and
phenotype. Interestingly, after being stored within hydrogels at RT for 3 days in either tissue
culture grade or ultra-low attachment well-plates, the cells show to release from the hydrogels
after being transferred (Fig. 20 (d); Fig. 22 (d)), but the same does not happen for the ones stored
in the same conditions for 5 days (Fig. 21 (d); Fig. 23 (d)). This, in its turn, suggests that the
encapsulation of the OECs within the hydrogels did not offer a protective effect to the cells when
compared with the control situation, where they are in 2D culture. All of this is reflected by the
ratings given to the pictures (Tables 1 & 2). A zero was always given to the pictures with the
hydrogels after 7 days of transferring them from the well-plates where they were stored for 5 days
at RT. After 7 days of having transferred the hydrogels from the well-plates where they were
stored for 3 days at RT, the ratings vary from zero to 2.
It is possible to observe from the pictures that in most cases, and for the conditions where there
is cell release happening, the hydrogels with lower alginate contents (such as 0.1% (w/v) and
0.2% (w/v)) allow for a higher cell release than the ones with a higher alginate concentration (such
as 0.3% (w/v) and 0.4% (w/v)), with some exceptions. The classifications given to the pictures
are also in accordance with this observation, since in most of the cases the number of the scale
attributed to the pictures decreases with the increase of alginate content (Tables 1 & 2). The
exceptions for this might be due to the fact that upon the transfer of the hydrogels from one well-
34
plate to the other, and because the hydrogels have low concentrations of alginate and are
therefore quite soft, damages often occurred. Especially the hydrogels with an alginate content
of 0.1% (w/v) would often get fragmented into smaller pieces. The lack of homogeneity that comes
with having hydrogels with different sizes on different time points of the experiment might
influence the extent of the cell release that is occurring, making a true comparison not really
feasible. Thus, a better method that allows to simulate the delivery into the patient is necessary.
Ideally, models of CNS injury would be used and the hydrogels containing the OECs would be
delivered by injection.
Regarding the already mentioned differential extents of cell release observed between hydrogels
with different alginate contents, this might be explained with the also differential storage modulus
observed. In a work where the mechanical and physical characterization of the exact same
alginate hydrogel used in this study was performed, authors concluded that the storage modulus
was greatly dependent on the alginate concentration, increasing as a function of the alginate
macromer content. More specifically, for the hydrogels obtained by using the same gelation
conditions as in this study, the measured storage modulus was of about: 6 Pa for an alginate
concentration of 0.1% (w/v); 60 Pa for an alginate concentration of 0.2% (w/v), 110 Pa for an
alginate concentration of 0.3% (w/v) and 145 Pa for an alginate concentration of 0.4% (w/v).104
Therefore, the forces the cells have to overcome in order to release from the hydrogels with the
different alginate concentrations are bigger when this is higher, which might explain the
differences observed in cell release.
Regarding to the existence or not of a higher cell release from the hydrogels which were
transferred and placed at 37ºC after having been in storage for 3 days comparing to 5 days, this
is more difficult to assess from looking at the pictures. The ratings attributed to the pictures for
the storage at 37ºC seem to show that for the OECs which were stored for only 3 days the extent
of cell release is higher, since the classifications attributed at the 7th day after the transfer are
always equal or smaller for the ones stored for 5 days when comparing, for both tissue culture
grade (Table 1) or ultra-low attachment (Table 2) well-plate storage. This finding suggests that
the cells stored for only 3 days are in a better preservation condition than the ones stored for 5
days. For the storage at RT, since the classification is always zero for the pictures taken at day 7
post-transfer after 5 days of storage, it is not possible to compare.
It is also possible to observe that despite the inexistence of cell release from the hydrogels stored
in ultra-low attachment well-plates, the cell release occurred after the storage time and after the
transfer of the hydrogels was not noticeable higher than the cell release occurring from hydrogels
previously stored in tissue culture grade well-plates, being this corroborated by the ratings given
to the pictures (Table 1 & 2). This observation suggests that even though it seems that the use of
ultra-low attachment surface well-plates prevents the premature release of cells, it does not seem
to improve the preservation of the cells when it comes to their ability to release from the hydrogels
after the storage period.
35
Figure 20 – Representative phase contrast microscopic images of the hydrogels with encapsulated OECs trying to highlight their release and migration out of
the hydrogels. The hydrogels present the range of different alginate contents (0.1% - 0.4% (w/v)), and the 2D cultured cells are also shown. Storage of these
hydrogels took place in tissue culture grade well-plates. (a) Pictures after 3 days of storage at 37ºC; (b) Pictures at the 7th day after the transfer of the hydrogels
presented in (a); (c) Pictures after 3 days of storage at RT; (d) Pictures at the 7th day after the transfer of the hydrogels presented in (c). Cell seeding density
was 107 cells/mL hydrogel in all conditions. Scale bar: 1000 µm.
36
Figure 21 – Representative phase contrast microscopic images of the hydrogels with encapsulated OECs trying to highlight their release and migration out of
the hydrogels. The hydrogels present the range of different alginate contents (0.1% - 0.4% (w/v)), and the 2D cultured cells are also shown. Storage of these
hydrogels took place in tissue culture grade well-plates. (a) Pictures after 5 days of storage at 37ºC; (b) Pictures at the 7th day after the transfer of the hydrogels
presented in (a); (c) Pictures after 5 days of storage at RT; (d) Pictures at the 7th day after the transfer of the hydrogels presented in (c). Cell seeding density
was 107 cells/mL hydrogel in all conditions. Scale bar: 1000 µm.
37
Figure 22 – Representative phase contrast microscopic images of the hydrogels with encapsulated OECs trying to highlight their release and migration out of
the hydrogels. The hydrogels present the range of different alginate contents (0.1% - 0.4% (w/v)), and the 2D cultured cells are also shown. Storage of these
hydrogels took place in ultra-low attachment well-plates. (a) Pictures after 3 days of storage at 37ºC; (b) Pictures at the 7th day after the transfer of the hydrogels
presented in (a); (c) Pictures after 3 days of storage at RT; (d) Pictures at the 7th day after the transfer of the hydrogels presented in (c). Cell seeding density
was 107 cells/mL hydrogel in all conditions. Scale bar: 1000 µm.
38
Figure 23 – Representative phase contrast microscopic images of the hydrogels with encapsulated OECs trying to highlight their release and migration out of
the hydrogels. The hydrogels present the range of different alginate contents (0.1% - 0.4% (w/v)), and the 2D cultured cells are also shown. Storage of these
hydrogels took place in ultra-low attachment well-plates. (a) Pictures after 5 days of storage at 37ºC; (b) Pictures at the 7th day after the transfer of the hydrogels
presented in (a); (c) Pictures after 5 days of storage at RT; (d) Pictures at the 7th day after the transfer of the hydrogels presented in (c). Cell seeding density
was 107 cells/mL hydrogel in all conditions. Scale bar: 1000 µm.
39
Figure 24 – Temperature fluctuations at which the cells were subjected through the 5 day period
of storage at RT, when stored in hydrogels placed in tissue culture grade well-plates. The
temperature was recorded every hour. Tmax = 25ºC; Tmin = 21.5ºC; Tavg = 22.4ºC ± 0.7ºC (mean ±
standard deviation).
Figure 25 – Temperature fluctuations at which the cells were subjected through the 5 day period
of storage at RT, when stored in hydrogels placed in ultra-low attachment well-plates. The
temperature was recorded every hour. Tmax = 26.5ºC; Tmin = 21.5ºC; Tavg = 22.1ºC ± 0.9ºC (mean
± standard deviation).
Days 0 1 2 3 4 5
Days 0 1 2 3 4 5
40
Table 1 – Classifications attributed to the pictures taken of the different alginate content hydrogels
(0.1% – 0.4% (w/v)) highlighting the cell release occurred, after these being stored in tissue
culture grade well-plates for 3 and 5 days at 37ºC and at RT and after 7 days of being transferred
into new tissue culture grade well-plates and being left at 37ºC. Scale: 0 – no cells released; 1 –
only a few released cell; 2 – marked release of cells; 3 – extensive release of cells.
Table 2 – Classifications attributed to the pictures taken of the different alginate content hydrogels
(0.1% – 0.4% (w/v)) highlighting the cell release occurred, after these being stored in ultra-low
attachment well-plates for 3 and 5 days at 37ºC and at RT and after 7 days of being transferred
into new tissue culture grade well-plates and being left at 37ºC. Scale: 0 – no cells released; 1 –
only a few released cell; 2 – marked release of cells; 3 – extensive release of cells.
Ultra-low attachment well-plates Tissue culture grade well-plates
3 days 5 days 7 days after transfer
(3 days storage)
7 days after transfer
(5 days storage)
Alginate
content
(% w/v)
37ºC RT 37ºC RT 37ºC RT 37ºC RT
0.1 0 0 0 0 3 1 2 0
0.2 0 0 0 0 3 2 2 0
0.3 0 0 0 0 2 2 1 0
0.4 0 0 0 0 0 1 0 0
Tissue culture grade well-plates Tissue culture grade well-plates
3 days 5 days 7 days after transfer
(3 days storage)
7 days after transfer
(5 days storage)
Alginate
content
(% w/v)
37ºC RT 37ºC RT 37ºC RT 37ºC RT
0.1 3 0 3 0 3 1 3 0
0.2 2 0 2 0 3 2 2 0
0.3 1 0 2 0 2 1 0 0
0.4 0 0 1 0 0 0 0 0
41
3.4 Immunocytochemistry
With the aim of comparing the expression of the markers S100β and p75NTR by the human OECs
when in either 2D culture for 5 days, encapsulated and stored for 5 days at 37ºC in ultra-low
attachment well-plates, or after releasing from the hydrogels, immunocytochemistry was
performed on the samples. These assays aimed at assessing and comparing the expression by
the PA7s of both markers on the different stages of the former experiments, to assess if their
phenotype shifts or if it is kept the same. The cells cultured in 2D (Fig. 26) and the cells which
released from the hydrogels (Fig. 27) were immunostained for both markers. It was not possible
to image the cells encapsulated within the hydrogels, since these got washed away with all the
washing steps and thanks to the prolonged time of the incubation steps described in the methods
section, despite all the caution taken while handling the samples.
The controls for the experiments (data not shown) showed no signal for any of the markers. Only
the Hoechst stain presented a positive signal, so the experiments were considered to be valid.
Figure 26 – Representative images of human olfactory ensheathing cells cultured in 2D for 5
days expressing (a) S100β (b) p75NTR. Scale bar: 200 µm.
Figure 27 – Representative images of human olfactory ensheathing cells that were allowed to
release for 7 days from hydrogels transferred into tissue culture grade well-plates, after being
stored for 5 days at 37ºC and in ultra-low adherent well-plates, expressing (a) S100β (b) p75NTR.
Scale bar: 200 µm.
It was possible to observe in all the images obtained that all the cells, with no exception, which
were cultured in 2D for 5 days at 37ºC expressed both markers, as expected, as well as the cells
released after 7 days of transferring the hydrogels into tissue culture grade well-plates. However,
42
in this situation, and despite presenting a signal, the S100β marker shows to be faded in the cells
which released from the hydrogel. Also, at the time of imaging it proved difficult to properly focus
the images for this condition.
These findings suggest that the phenotype of the OECs is maintained after having been
encapsulated within alginate hydrogels and stored under a temperature of 37ºC for 5 days, which
has positive implications on the possibility to use this storage technique for therapy with human
OECs. The preservation of the phenotype of the encapsulated OECs is important, since the
function which is expected from the cells will depend on it. Even though it was not possible to
stain the OECs for both markers while encapsulated and stored within the hydrogels, the
assessment of the cells which released after the storage period provided favourable results.
However, only two of the markers for which OECs stain for where used in this assessment.
Therefore, this can be considered as a preliminary assessment, where the study of the expression
of more markers, such as GFAP, would allow for more solid conclusions about whether the
phenotype of the human OECs is kept or not under storage in these conditions.
43
Chapter 4
Concluding remarks and future
perspectives
Even though there has been a considerable progress within the overall of the cell therapy industry,
the problems to do with logistics of cell storage and preservation still exist, with current methods
such as cryopreservation presenting serious limitations.75–77,103 Within the field of regenerative
medicine, OECs have been extensively studied for their capacity to help regenerate CNS
injuries.52,105
This work aimed at assessing the potential of alginate hydrogels for the short-time storage and
preservation of encapsulated human OECs. If proven to be able to do so, this would have a great
impact on helping to turn a day-to-day cell therapy for CNS injuries regeneration with OECs into
a reality.
It was possible to successfully encapsulate the human OECs within the alginate hydrogels and
the assays here performed reveal that under certain circumstances, encapsulation within alginate
hydrogels is capable of preserving most of the cells’ viability, with more than 70% of the
encapsulated OECs being alive after the storage periods in some conditions. In some cases the
encapsulation of the OECs also show to preserve their migration capacity, with the cells showing
to be able to release out from the hydrogels after storage. Additionally, the immunocytochemistry
results suggest that the phenotype of the cells is being kept constant throughout the storage
process, which corroborates the high biocompatibility for which alginate hydrogel is known for.
As future work, it would be interesting to perform immunocytochemistry assays for all the storage
conditions studied. Namely, it would be interesting to investigate the expression of the tested
markers during and after storage at RT, and compare it with the one obtained for the storage at
37ºC. Since the morphology of the 2D cultured OECs changes, the expression of the markers
might be changing as well. Maybe the observed changes in the expression of these markers
would provide some insight on what is happening to the cells under these conditions.
44
Additionally, it would be important as future work to find a way to quantify the cell release that is
occurring out from the hydrogels. This would allow for better comparisons between different
storage conditions of the encapsulated OECs. Something that could be tried would be to select
several different random squares on the images obtained, each with the same area, where the
released, and now attached, cells are shown and count them, in order to statistically get to a
number of released cells. While doing this, it would have to be taken in account the fact that the
number and distribution of released cells closer or farer from the limit of the hydrogel is not
homogeneous.
Moreover, In order to try to give more validity to the preservation and storage of human OECs
with alginate hydrogel encapsulation, cell viability and immunocytochemistry assays could be
performed on human OECs which were stored using cryopreservation, the method most used
nowadays, and compare the results with the ones obtained with alginate hydrogel. If the
preservation condition of the OECs after storage within alginate hydrogel is as good as or better
than the one obtained by using cryopreservation, that would give even more strength to the use
of this technology.
In sum, the here established storage system will hopefully help to create an efficient and
economical way to ease the logistics necessary for the generalized use of regenerative medicine
therapies with OECs.
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