Alginate hydrogels for short-term storage and preservation of ......3.2 Viability of human OECs encapsulated and stored within alginate hydrogels ..... 23 3.2.1 Viability of the encapsulated

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

ii

iii

To my family and friends

iv

v

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

viii

ix

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

xiv

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


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


condition, with asterisks representing statistical significant differences (**p<0.01;

***p<0.001)……………………………………………………………………………... 29

xviii


with different alginate contents (0.1 – 0.4% (w/v)), at 37ºC and at RT. Values are


condition, with asterisks representing statistical significant differences (**p<0.01;

***p<0.001)……………………………………………………………………………... 29


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


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





hydrogels took place in tissue culture grade well-plates. (a) Pictures after 5 days




107 cells/mL hydrogel in all conditions. Scale bar: 1000 µm……………………….. 36

xix





hydrogels took place in ultra-low attachment well-plates. (a) Pictures after 3 days




107 cells/mL hydrogel in all conditions. Scale bar: 1000 µm…………………….….

37





hydrogels took place in ultra-low attachment well-plates. (a) Pictures after 5 days




107 cells/mL hydrogel in all conditions. Scale bar: 1000 µm……….………………. 38


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


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

xxii

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).


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).


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



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



37



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



38



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



39


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).


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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