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\begin{document}

\title{Microcarrier expansion of c-MycER\textsuperscript{TAM} - modified human
olfactory mucosa cells for neural regeneration}



\author[1]{Ana Valinhas}%
\author[1]{Gerardo Santiago-Toledo}%
\author[2]{Ivan Wall}%
\affil[1]{University College London Faculty of Engineering}%
\affil[2]{Aston University}%


\vspace{-1em}



  
  \date{June 13, 2024}


\begingroup
\let\center\flushleft
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\endgroup





\selectlanguage{english}
\begin{abstract}
Human olfactory mucosa cells (hOMCs) have potential as a regenerative
therapy for spinal cord injury. In our earlier work we derived the PA5
cells, a polyclonal population that retains functional attributes of
primary OMCs. Microcarrier suspension culture is an alternative to
planar 2D culture to produce cells in quantities that can meet the needs
of clinical development. This study aimed to screen the effects of 10
microcarriers on PA5 hOMCs yield and phenotype. Studies performed in
well plates led to a 2.9-fold higher cell yield on Plastic compared to
Plastic Plus microcarriers with upregulation of neuronal markers \selectlanguage{greek}β-\selectlanguage{english}III
tubulin and nestin for both conditions. Microcarrier suspension culture
resulted in concentrations of 1.4x10\textsuperscript{5} cells/mL and
4.9x10\textsuperscript{4} cells/mL for Plastic and Plastic Plus,
respectively, after 7 days. p75\textsuperscript{NTR} transcript was
significantly upregulated for PA5 hOMCs grown on Plastic Plus compared
to Plastic. Furthermore, co-culture of PA5 hOMCs grown on Plastic Plus
with a neuronal cell line (NG108-15) led to increased neurite outgrowth.
This study presents the successful expansion of PA5 cells using
microcarrier suspension culture and it reveals competing effects of
microcarriers on cell expansion versus functional attributes, showing
that designing scalable bioprocesses should not only be driven by cell
yields.%
\end{abstract}%



\sloppy


\emph{Key words: human olfactory mucosa cells, spinal cord injury,
allogeneic cell therapy, microcarrier, suspension culture}



\textbf{Introduction}

250,000 to 500,000 people are affected by spinal cord injury every year
worldwide (Bickenbach et al., 2013). The spinal cord is a part of the
central nervous system (CNS), and unlike the peripheral nervous system
(PNS), has limited regenerative capacity after injury (Ahuja et al.,
2017). The slow regenerative capacity of the CNS is attributed to the
generation of a ``glial scar'' after injury which constitutes a barrier
for axonal regrowth. Human olfactory mucosa cells (hOMCs) have shown
promising results in both pre-clinical and clinical studies for the
treatment of spinal cord injury (SCI) (F\selectlanguage{ngerman}éron et al., 2005; Iwatsuki et
al., 2008; Mackay-Sim et al., 2008; Tabakow et al., 2013). The
regenerative capacity of OMCs is attributed to their unique ability to
support the regeneration of olfactory receptor neurons (ORNs) which
extended their axons from the PNS to the CNS, a property unique to the
olfactory system in mammals (R. Doucette, 1991). Several studies have
attributed the neural regeneration of the hOMC population to the
presence of different cell types within this population, such as neural
stem cells (NSCs), mesenchymal stem cells (MSCs) and olfactory
ensheathing cells (OECs), a type of glia (Delorme et al., 2010; J. R.
Doucette, 1984; Lindsay et al., 2013; Wolozin et al., 1992). Markers
reported in hOMCs primary populations include glial
(p75\textsuperscript{NTR}, GFAP and S100\selectlanguage{greek}β\selectlanguage{english}), neuronal (\selectlanguage{greek}β-\selectlanguage{english}III tubulin and
nestin), mesenchymal, and fibroblast associated markers (CD90/Thy1 and
fibronectin) (Au et al., 2002; Bianco et al., 2004; Hahn et al., 2005;
Kawaja et al., 2009).

Reported clinical trials have used an autologous approach which can lead
to variable outcomes since hOMCs populations are highly variable between
patients, are challenging to expand and there is a lack of consistency
between protocols used for tissue biopsy and preparation of cells for
transplant (F\selectlanguage{ngerman}éron et al., 1999). Therefore, it would be beneficial to
develop an allogeneic or universal ``off-the-shelf'' approach. We
previously reported the generation of a candidate cell therapy from
late-adherent hOMCs, by genetic modification of primary cells with
c-MycER\textsuperscript{TAM} conditional immortalization technology, to
advance a potential allogeneic therapeutic product for the treatment of
SCI (Santiago-Toledo et al., 2019). The translation of such a
therapeutic product to the market would require the development of a
scalable bioprocess, able to yield large amounts of cells which can
reach doses up to 1x10\textsuperscript{7} cells/dose (Casarosa et al.,
2014). Commonly, adherent cells have been grown in two-dimensional
platforms such as tissue-culture flasks, cell factories and roller
bottles. Even though these systems are reliable due to their wide spread
use in the industry, they are not easily scaled-up for producing large
quantities of allogeneic cells, making them unsuitable for market scale
production of cells. Microcarriers are a suitable candidate to perform
expansion of adherent cells in a scalable manner in stirred tank
bioreactors and offer a higher larger surface per unit volume of
bioreactor. It may be possible to manufacture early stage clinical
products using planar systems and then transition to stirred tank
bioreactors for phase III and market, yet this would require significant
validation work to meet regulatory approval, and risk product
specification failure later in development due to manufacturing process
changes. Therefore, early adoption of market scale manufacturing
technology is necessary.

Microcarrier cell culture platforms have been reported for the
successful expansion of mesenchymal stem cells (MSCs) (dos Santos et
al., 2014; Rafiq et al., 2013), embryonic stem cells (ESCs) (Oh et al.,
2009) and induced pluripotent stem cells (iPSCs) (Badenes et al., 2017;
Carlos AV Rodrigues et al., 2018). Stirred tank bioreactors are well
explored systems that enable the monitoring and control of several
culture parameters such as oxygen tension, pH and stirring regimens.
These platforms also offer a closed bioprocess environment with reduced
operator interference and variability. Therefore, these are platforms
that offer more robust and reproducible bioprocesses that theoretically
can deliver consistent quality products, compliant with Good
Manufacturing Practice (GMP) and Good Clinical Practice (GCP)
requirements.

In this work, we report the microcarrier expansion of a candidate cell
line for the treatment of SCI, namely PA5 hOMCs, on microcarrier stirred
culture using spinner flasks. First, a variety of commercially available
microcarriers were selected based on a screening methodology using ultra
low attachment 96-well plates. Besides the suitability of the
microcarrier, considerations of compliance for good manufacturing
practices (GMP) and adaptability to xeno-free conditions were considered
for microcarriers selection. Then, cells were grown on two types of
microcarriers using spinner flasks, based on protocols previously
reported for the expansion of hMSCs (Santos et al., 2011). In order to
further understand cell growth kinetics, PA5 hOMCs were grown on
microcarriers for 7 days. Cell phenotype was assessed through
immunocytochemistry and RT-qPCR. Potency of PA5 hOMCs was assessed
through the capacity of the harvested cells to promote neurite outgrowth
using a co-culture assay of hOMCs and NG108-15 neurons.

We report the use of a microcarrier-based spinner flasks system for the
expansion of the PA5 hOMCs population of cells, a candidate cell therapy
for the treatment of spinal cord-injury. These are the first steps for
the development of a bioprocess strategy that enables large scale
expansion of a potentially neuroregenerative cell type, taking us closer
to the development of an allogeneic product for the treatment of spinal
cord injury.

\textbf{Materials and methods}

\emph{hOMC monolayer culture}

PA5 hOMCs were expanded in T-flasks, freshly coated with poly-L-lysine
(PLL, Sigma, UK, 100 \selectlanguage{greek}μ\selectlanguage{english}g/mL), using as complete medium DMEM/F12 +
Glutamax\textsuperscript{TM} (Gibco, Life Technologies, UK) supplemented
with 10\% Fetal Bovine Serum (FBS) (Sigma, Germany) and
4-Hydroxitamoxifen (4-OHT, 1:10,000). Cells were maintained in a
standard incubator (370\selectlanguage{english}C, 5\% CO\textsubscript{2} in air). A seeding
density of 6000 cells/cm\textsuperscript{2} was used and feeding was
performed every two days. Cells were passaged when 60-80\% confluency
was reached, through incubation with TryPLE (Gibco, UK) for 5 minutes.
The dissociation reagent was quenched with complete medium and the cell
suspension was centrifuges for 5 minutes at 400 x g.

\emph{Microcarrier screening in well plates}

10 commercially available microcarriers were tested for expansion of PA5
hOMCs in ultra-low attachment 96-well plates: Plastic, Plastic Laminin
Coated (Plastic L), Plastic Plus, PronectinF\selectlanguage{ngerman}\textsuperscript{®},
Collagen, FACT III, Star-Plus, Hillex II, all from Pall, UK.
Synthemax\textsuperscript{®} II (Low and High Concentration) (Corning,
UK) and Cytodex I (GE Healthcare, UK) were also used. Microcarriers
specifications are shown on Table 1. Sterilization of Pall microcarriers
and Cytodex I microcarriers was achieved by resuspension in distilled
water (Gibco, UK) and PBS Ca+ and Mg+ free (Lonza, UK), respectively,
followed by autoclaving at 121 ºC for 15 min. Synthemax II ®
microcarriers were sterilized under a UV lamp for 90 minutes.

A monolayer of microcarriers with a total surface area of 1.28
cm\textsuperscript{2} was used per well, in order to cover the bottom of
a 96-well plate well. For 6-well plates, a monolayer of microcarriers
with a total surface area of 38 cm\textsuperscript{2} was used per well.
Total working volumes were of 200 uL and 6 mL for 96-well plates and
6-well plates, respectively. Plastic L microcarriers were incubated with
a solution of 20 \selectlanguage{greek}µ\selectlanguage{english}g/mL Cultrex\selectlanguage{ngerman}\textsuperscript{®} Mouse Laminin I
(Trevigen, UK) for 1 hour in a standard incubator prior to cell seeding.
All microcarriers were let to equilibrate for 1 hour in a standard
incubator with complete medium. A seeding density of 6000
cells/cm\textsuperscript{2} was used. Medium exchanges were performed
for 50\% of the total volume, every two days. Cells were cultured in
ultra-low 96-well plates for a total of 7 days. For 96 well-plates,
relative viable cell number was measured using the Cell Counting Kit-8
(CCK-8) (Dojindo, Japan) every time medium exchanges were performed.
Briefly, 10 \selectlanguage{greek}µ\selectlanguage{english}L of reagent were added per 100 \selectlanguage{greek}µ\selectlanguage{english}L of microcarrier
suspension. After incubation for 1 hour in a standard incubator, the
supernatant was transferred to a 96-well plate and absorbance readings
at 450 nm were performed in a microplate reader (Tecan, Switzerland).
Quantification of viable cells per well was performed based on a
standard curve performed on the first day of culture.

\emph{Spinner-flask culture}

The protocol used for spinner flask culture was the one followed in
Santos et al 2011. Suspension culture experiments were performed in 100
mL flat bottom spinner-flasks (Bellco) with 80 mL working volume.
Spinner-flasks were siliconized using Sigmacote (Sigma, Germany) to
avoid cell attachment. After applying the reagent to all the glass
surface and aspirating it, vessels were let to dry for 24 hours in a
fume hood. After, vessels were rinsed three times with distilled water
(Gibco, UK). Plastic and Plastic Plus microcarriers were used for
spinner-flask culture with a total surface area of 518.4
cm\textsuperscript{2}. Microcarriers were prepared by autoclaving in
distilled water (Gibco, UK) for 121 \selectlanguage{ngerman}ºC for 15 min. After, the distilled
water was aspirated as much as possible and complete medium was added.
Microcarriers were let to equilibrate for 1 hour in a standard
incubator. 60-80\% confluent PA5 hOMCs were detached as described in
section ``hOMC cell culture'' and cells were added to the tube
containing the microcarriers suspension in complete medium with a
seeding density of 6000 cells/cm\textsuperscript{2}. After transferring
the microcarrier and cell suspension to spinner-flasks containing
adequate amounts of complete medium, an agitation of 40 rpm was started.
Spinner-flasks were maintained on a
Bell-Ennium\textsuperscript{TM}Compact 5 position magnetic stirrer
platform (BellCo) in a standard incubator. In order to allow for air
exchange, a side-arm of the spinner-flask was kept loosened (half a turn
of the cap). Cell expansion was maintained for 7 days. Feeding was
performed every two days by removing 50\% of the expanded medium and
adding the same amount of fresh complete medium. Sampling, also
performed every two days, was achieved by retrieving 2 mL of a
homogeneous microcarriers suspension for manual cell counting and
immunocytochemistry. Cell counting and viability measurements were
performed through the trypan blue method, using a haemocytometer.

\emph{Harvest}

Exhausted medium was retrieved from the vessel and the microcarrier-cell
suspension was washed with 40 mL of HBSS (Gibco, UK) twice. 20 mL of
TryPLE\textsuperscript{TM} Select (Gibco, UK) were incubated with the
microcarrier-cell suspension for 15 minutes, at an agitation of 100 rpm
in a standard incubator. To quench the dissociation reagent, 30 mL of
complete medium were added. The suspension was passed through 40 \selectlanguage{greek}µ\selectlanguage{english}m cell
strainers and centrifuged for 5 minutes at 400 x g. Harvested cells were
counted using the trypan blue exclusion method and were either pelleted
for RT-qPCR or plated at 6000 cells/cm\textsuperscript{2} to perform
several assays such as immunocytochemistry and NG108-15 co-culture
assay.

\emph{Analytical techniques}

\emph{Metabolite analysis}

On the days medium exchange occurred, metabolite analysis was performed
using the the CuBian HT-270 analyzer (Optocell GmbH \& Co. KG, Germany)
to obtain the concentration of glucose, lactate and ammonia.

\emph{Immunocytochemistry}

Samples were fixed using a solution of 4\% PFA in PBS for 20 minutes at
RT. After washing with PBS (Lonza) for 5 minutes, twice,
permeabilization was done through incubation of samples with a solution
of 0.1\% Triton\textsuperscript{TM} X-100 (Sigma) in PBS for 20 minutes
at room temperature. After washing samples twice with PBS for 5 minutes,
samples were incubated with a blocking solution of 5\% Goat Serum (DAKO)
in PBS for 1 hour at room temperature. Primary antibodies against
p75\textsuperscript{NTR} (Millipore), fibronectin (Sigma), nestin
(Millipore), GFAP (Dako), S100\selectlanguage{greek}β \selectlanguage{english}(Dako) and \selectlanguage{greek}β-\selectlanguage{english}III tubulin (Sigma) (Table
2) were used with a dilution of 1:200 in blocking solution and incubated
for 90 minutes at room temperature. After washing with PBS, secondary
antibodies (Goat anti-rabbit IgG (H+L)-Alexa Fluor 594; Goat anti-mouse
(H+L)-Alexa Fluor 488, Invitrogen) and Hoechst 33342 (Molecular Probes)
were diluted in blocking solution using a dilution of 1:200 and 1:1000,
respectively. Images of planar culture were acquired using the
microscope system EVOS\selectlanguage{ngerman}\textsuperscript{®} FL (ThermoFisher Scientific),
while microcarrier culture 16-bit multi-color montage images were
obtained with a Zeiss LSM 880 with Airscan confocal microscope system
(Carl Zeiss) and Zen 2009 acquisition software (Carl Zeiss).

\emph{qRT-PCR}

Cells expanded on microcarriers in ultra-low attachment 6-well plates
were detached as previously described. Cell suspensions were centrifuged
and excess medium was removed prior to storage at
-80\textsuperscript{o}C until further analysis. Pellets were first
re-suspended in RLT Buffer (QIAGEN) before being homogenised in
Qiashredder Columns (QIAGEN) according to the manufacturer's
instructions. Synthesis of complementary deoxyribonucleic acid (cDNA)
and removal of genomic DNA was performed using the QIAGEN RT-PCR kit
following the manufacturer's instructions. Genomic DNA was eliminated
using gDNA Wipeout Buffer (QIAGEN) with up to 1 \selectlanguage{greek}µ\selectlanguage{english}g template RNA and
RNAse free water in a total reaction volume of 14 \selectlanguage{greek}µ\selectlanguage{english}L. The entire 14 \selectlanguage{greek}µ\selectlanguage{english}L
reaction volume was subsequently mixed with a MasterMix (Bio-Rad):
template RNAs or no template for NTC (no template control), RT Primer
Mix Quantiscript RT Buffer and Quantiscript Reverse Transcriptase or
with RNA free water for no-RT (No reverse transcriptase control), in a
total volume of 20 \selectlanguage{greek}µ\selectlanguage{english}L. The qPCR reactions were performed in Bio-Rad
Hard-Shell Low Profile Thin-Wall 96 Well Skirted PCR plates on the
Bio-Rad CFX 96 Connect Real-Time PCR System using the Quantitect SYBR
Green PCR Kits (QIAGEN) according to the manufacturer's instructions.
For each reaction Quantitect Mastermix Buffer, Quantitect pre-validated
primer assay (Table 3), water and cDNA were combined to make final
reaction volumes of 25 \selectlanguage{greek}µ\selectlanguage{english}L. On each 96-well plate samples were analysed
in triplicate with \selectlanguage{greek}β-\selectlanguage{english}actin as an internal reference control and standard
controls No-RT and NTCs to check for cross contamination. The 2-\selectlanguage{greek}ΔΔ\selectlanguage{english}Ct
method was used to analyse the data.

\emph{Co-culture assay}

A co-culture of NG108-15 neurons with PA5 cells after expansion on
microcarriers was used to assess neuron outgrowth. NG108-15 cells (ATCC
HB-12317) are a hybrid rodent glioma-neuroblastoma cell line. As a
positive control, a rat SCL 4.1/F7 (ECACC 93031294) Schwann cell line
was used.

NG108-15 and F7 cells were grown for two passages on T-flasks coated
with poly-L-lysine (PLL, Sigma, 100 \selectlanguage{greek}µ\selectlanguage{english}g/mL) or uncoated, respectively.
When 60-80\% confluency was reached, NG108-15 cells were passaged by
hitting the flask and re-plated at 6000 cells/cm\textsuperscript{2},
while F7 cells were passaged following the same protocol used for PA5
hOMCs and re-plated at 6000 cells/cm\textsuperscript{2}. For the
co-culture assay PA5 hOMCs were plated at 6000
cells/cm\textsuperscript{2} onto freshly coated 24-well plates with
poly-L-lysine (PLL, Sigma, 100 \selectlanguage{greek}µ\selectlanguage{english}g/mL). After 24 hours, NG108-15 cells
were plated on wells with PA5 hOMCs or F7 cells using a seeding density
of 500 cells/well. Medium changes were performed every two days and
samples fixed after 5 days. 4-OHT was not added in the medium for this
assay. Immunocytochemistry was performed as previously described. Images
were acquired with EVOS FL Imaging System (Thermo-Scientific) at a 100x
total magnification. 15 frames were acquired per condition (5 per
technical repeat) and neurite quantification was performed manually
using the NeuronJ (Meijering et al., 2004) plugin in ImageJ.

Statistical methods

The Kolmogorov-Smirnov test was used for data normality and Levene's
test was used for homogeneity of variance. For pairwise-comparisons,
one-way ANOVA was used with post-hoc Tukey's or Games-Howell test. A
difference of p\textless{}0.05 was considered significant. All tests
were performed using OriginPro 2016 (OriginLab, USA).

Formulae

The specific growth rate, \selectlanguage{greek}µ \selectlanguage{english}(days\textsuperscript{-1}):

\begin{equation}
\ln\left(X\right)=\ \mu t+ln(X_{0})\nonumber \\
 \end{equation}

Where \(X\) is the concentration of cells,
\(X_{0}\) is the concentration of cells at \(t_{0}\).
\(\mu\) for the exponential phase, considered to be from day
3 to 7.

Doubling time, \(t_{d}\) (days):

\begin{equation}
t_{d}=\frac{ln(2)}{\mu}\nonumber \\
 \end{equation}

Fold-increase:

\begin{equation}
Fold\ increase=\ \frac{X_{f}}{X_{0}}\nonumber \\
 \end{equation}

Where \(C_{X(f)}\) is the cell concentration at the end of the
culture and \(C_{X(0)}\) is the initial cell concentration.

Specific metabolite consumption rate,
\(q_{\text{met}}\)(pmol.cell\textsuperscript{-1}.day\textsuperscript{-1})

\begin{equation}
q_{\text{met}}=\frac{\mu}{X_{0}}\selectlanguage{greek}\frac{C_{met(f)}-C_{met(0)}}{e^{\text{μt}}-1}\selectlanguage{english}\nonumber \\
 \end{equation}

Where \(C_{met(t)}\) and \(C_{met(0)}\) are the concentration
of metabolite at the end and start of the exponential phase.

Lactate yield from glucose, Y\textsubscript{Lac/Glc}

\begin{equation}
Y_{Lac/Glc}=\frac{[Lac]}{[Glc]}\nonumber \\
 \end{equation}

Where \([Lac]\) is the lactate production
and\([Glc]\) is the glucose consumption during the exponential
phase.

\textbf{Results}

\textbf{Microcarrier screening for PA5 hOMCs growth on well plates}

The growth of PA5 hOMCs was tested on 10 commercially available
microcarriers in ultra-low attachment 96-well plates. The growth of PA5
hOMCs was followed over 7 days as this is the length of time for the
duration of one passage (Figure 1 A). After seeding on day 0, viable
cell number was measured every two days, when media was exchanged. On
day 1, viable cell numbers ranged from 8.1x103 cells/well to 9.6x103
cells/well for Plastic LC, Synthemax II LC, Synthemax II HC, Plastic,
Plastic Plus and FACT II (Figure 1 B). At the same time point, Star-Plus
and Cytodex I microcarriers provided a viable cell number between
3.3x103 to 5.0x103 cells/well. As shown in Figure 1 C, by day 7, three
distinct groups of microcarriers were observed. Plastic and Collagen
microcarriers supported high growth of 8.8x104 and 7.5x104 cells/well.
Plastic L, Synthemax II LC, Synthemax II HC, Collagen, PronectinF, FACT
III and Cytodex I performed similarly between each other, yielding
between 4.5x104 and 6.4x104 cells/well. Plastic Plus and Star-Plus
microcarriers yielded low cell numbers, lower than 3.0x104 cells/well.
All microcarriers except Star-Plus led to increase cell number across
the days of culture, where after day 3 there was an increase in cell
number/well (Figure 1 A).

A second experiment was performed using ultra-low attachment 6-well
plates using the an increased working volume but maintaining scalable
parameters. The number of microcarriers per well surface and the same
initial seeding density of 6000 cells/cm2 were used. Haemocytometer
counts were performed to measure viable cell density on day 7 and
phenotypic changes were assessed through RT-qPCR. Plastic microcarriers
led to the highest cell numbers (1.7\selectlanguage{ngerman}±0.1x106 cells/mL), while Plastic
Plus led to the lowest numbers (7.5±0.4x105 cells/mL) (Figure 2 A). Cell
viability analysis revealed \textgreater{}95\% viability of PA5 hOMCs on
all microcarriers tested (Figure 2 B). Metabolite analysis of glucose,
lactate and ammonium were performed on day 7 (Figure 2 C, D and E).
Glucose concentrations were all between 4 - 8.5 mM, with Plastic showing
the lower concentration and Plastic Plus the highest. The opposite trend
was seen for lactate concentration with values between 12 - 16.5 mM. The
concentration of ammonia was similar between all conditions with 0.8 mM
for all microcarriers, except for Plastic microcarriers which produced a
concentration of 0.65±0.1 mM. The phenotype of PA5 hOMCs was
investigated using RT-qPCR. It is known that the olfactory mucosa
population of cells harbours a mix of different cell types including
mesenchymal stem cells, neural stem cells, fibroblasts and olfactory
ensheathing cells (OECs). The OEC phenotype has been characterised by
the expression of the glial markers p75NTR, S100\selectlanguage{greek}β, \selectlanguage{english}GFAP and the neural
stem cell markers nestin and \selectlanguage{greek}β-\selectlanguage{english}III tubulin. Fibronectin has been used as
a marker of `contaminating' cell types as it can indicate the presence
of fibroblasts. Results show the upregulation of neural stem cell
markers \selectlanguage{greek}β-\selectlanguage{english}III tubulin for Plastic L, Plastic and Plastic Plus
microcarriers by 2-fold, and nestin for Plastic and Plastic Plus by
1.6-fold (Figure 2 F). Given the phenotypic traits obtained for cells
grown in ultra-low attachment in 6 well-plates, with the objective to
create a scalable bioprocess, Plastic and Plastic Plus were subsequently
used to grow PA5 hOMCs in agitated cell culture conditions in spinner
flasks.

\textbf{Expansion of hOMCs in microcarriers suspension culture using
spinner-flasks}

\textbf{PA5 hOMCs growth and metabolic profile in cell cultures using
Plastic and Plastic Plus microcarriers}

A method previously developed by Santos et al 2011 was adopted to
performed PA5 hOMCs microcarrier cell culture in spinner flasks. A total
working volume of 80 mL in 100 mL Belco spinner flasks was used with
50\% of media change every two days, for a total of 7 days. An initial
seeding density of 6000 cells/cm\textsuperscript{2} was used, which is
equivalent to 3.4x10\textsuperscript{4} cells/mL. Figure 3 A shows that
both Plastic and Plastic Plus microcarrier cultures experienced a
decrease in cell concentration after day 1 from
3.4\selectlanguage{ngerman}±0.03x10\textsuperscript{4} cells/mL to
1.4±0.3x10\textsuperscript{4}cells/mL and 9.4±1.3x10\textsuperscript{3}
cells/mL, respectively. Cell number started increasing after day 3. On
day 7, the concentration of cells in cultures employing Plastic
microcarriers was 1.4±1.1x10\textsuperscript{5} cells/mL and for Plastic
Plus microcarriers was 4.9±1.8x10\textsuperscript{4} cells/mL
(statistically significant, p\textless{}0.05). Cell viability remained
above 90\% (Figure 3 A) throughout the culture period. Metabolic
analysis of glucose, lactate and ammonia revealed similar concentration
of these nutrient and metabolites for both conditions (Figure 3 B-D). On
day 7, glucose concentration was of 10.5±1.7 mM and 11.5±1.6 mM, while
lactate concentration was of 7.3±2.3 mM to 5.2±2.5 mM, for PA5 hOMCs
grown on Plastic and Plastic Plus, respectively. The concentration of
ammonia was of 0.7 mM for both conditions. Considering an exponential
phase starting on day 3, specific consumption of glucose, specific
production of lactate and ammonia, as well as yield of lactate per
glucose were calculated. PA5 hOMCs grown of Plastic Plus led to a
specific glucose consumption rate of 24.4±2.3
pmol.day\textsuperscript{-1}cell\textsuperscript{-1}, similar to the one
measure for PA5 hOMCs grown on Plastic which was 20.4±4.7
pmol.day\textsuperscript{-1}cell\textsuperscript{-1}. Specific
production rates for lactate and ammonia was 47.9±6.3
pmol.day\textsuperscript{-1}cell\textsuperscript{-1} and 0.6±0.1
pmol.day\textsuperscript{-1}cell\textsuperscript{-1}, respectively for
PA5 hOMCs grown on Plastic Plus. These results were on average 2 times
higher than the specific production rates observed for PA5 hOMCs grown
on Plastic microcarriers (24.6±7.2
pmol.day\textsuperscript{-1}cell\textsuperscript{-1} and 0.3±0.05
pmol.day\textsuperscript{-1}cell\textsuperscript{-1} for lactate and
ammonia, respectively). The yield of lactate per glucose was 1.4±0.4 and
2.0±0.3 for PA5 hOMCs grown on Plastic and Plastic Plus microcarriers,
respectively.

\textbf{PA5 hOMCs phenotype and promotion of neuron outgrowth after
growth on Plastic and Plastic Plus microcarrier suspension culture}

RT-qPCR results (Figure 4 A and B), show the upregulation of several
markers for PA5 hOMCs grown on Plastic Plus compared to cells at day 0.
Fibronectin (3.5-fold), \selectlanguage{greek}β-\selectlanguage{english}III tubulin (2.3-fold), KDR1 (2.9-fold) and
Sox9 (2.9-fold) are upregulated for PA5 hOMCs grown on Plastic Plus
microcarriers compared to PA5 hOMCs grown on Plastic microcarriers by
day 7. Most importantly, p75\textsuperscript{NTR}, a neurotrophic marker
expressed by OECs is expressed 2.3-fold more on day 7 by PA5 hOMCs grown
on Plastic Plus microcarriers compared with cells on day 0, while PA5
hOMCS grown on Plastic microcarriers maintained the same level of
expression of this marker (1.0-fold) (p\textless{}0.05). Furthermore, a
co-culture assay with NG108-15 cells was used as an assay indicative of
the capacity of PA5 hOMCs to promote neurite outgrowth, using F7 Schwan
cells as positive control and NG108-15 cell only as a negative control.
Representative images used to quantify neurite outgrowth are shown in
Figure 4 C. The average neurite length was 36\selectlanguage{ngerman}±1.3 \selectlanguage{greek}µ\selectlanguage{english}m for Plastic Plus
microcarriers, significantly higher (p\textless{}0.05) than the average
neurite length obtained for Plastic (29.1\selectlanguage{ngerman}±1.5 \selectlanguage{greek}µ\selectlanguage{english}m) microcarriers and the
positive control (27.9\selectlanguage{ngerman}±0.1 \selectlanguage{greek}µ\selectlanguage{english}m) (Figure 4 D). The maximum neurite length
obtained for Plastic Plus was 96\selectlanguage{ngerman}±7.0 \selectlanguage{greek}µ\selectlanguage{english}m, significantly higher
(p\textless{}0.05) than the maximum neurite length obtained for Plastic
microcarriers (81.8\selectlanguage{ngerman}±3.2 \selectlanguage{greek}µ\selectlanguage{english}m) and the positive control (57.9\selectlanguage{ngerman}±3.3 \selectlanguage{greek}µ\selectlanguage{english}m)
(Figure 4 E). The average neurite per neuron was of 0.38\selectlanguage{ngerman}±0.05,
significantly higher (p\textless{}0.05) than the one obtained for cells
grown on Plastic microcarriers (0.23±0.02) and the positive control
(0.22±0.04) (Figure 4 F).

\textbf{Discussion}

\textbf{Microcarrier screening in ultra-low attachment well plates}

PA5 hOMCs is a candidate therapy for an allogeneic cell therapy for the
treatment of spinal cord injury (Santiago-Toledo et al., 2019). High
cell doses of up to 1 billion cells will be required to produce cell
banks to be supplied to patients (Casarosa et al., 2014). It has been
shown that stirred tank bioreactor cell culture using microcarriers is a
reliable, reproducible method to achieve high numbers of cells in a
scalable manner (Qiu et al., 2016; Rafiq et al., 2013; Carlos AV
Rodrigues et al., 2018; Santos et al., 2011). The first step to develop
such a production method would be to select the right type of
microcarrier and several authors have shown the successful screening of
microcarriers for the growth of MSCs and NSCs (Chen et al., 2011; Rafiq
et al., 2016; Carlos A. V. Rodrigues et al., 2011). Therefore, a similar
strategy to those already presented for other cell types was used and in
addition cell phenotype analysis was performed. The growth of PA5 hOMCs
was followed for 7 days on microcarriers using static 96-well plates. In
terms of cell concentration after 7 days, while Plastic, Plastic L,
Synthemax II LC, Synthemax II HC and PronectinF showed the most
promising results, FACT III, Plastic Plus and Star-Plus microcarriers
led to the lowest cell yields. Interestingly, it was observed that in
spite of PA5 hOMCs efficiently attached to Plastic Plus microcarriers,
the yield of cells on day 7 was lower than all other microcarriers with
similar attachment efficiency (Plastic, Plastic L, Synthemax II LC,
Synthemax II HC, and Collagen). This indicates that PA5 hOMCs grown on
Plastic Plus microcarriers are a slower growing cell population. PA5
hOMCs were found to grow to higher yields on neutrally charged
microcarriers compared to their positively charged counterparts and this
aligns with previous observations for BM-MSCs (Rafiq et al., 2016).

In order to further investigate the phenotype of PA5 hOMCs after 7 days
of culture, static microcarrier cultures on ultra-low attachment 96-well
plates were scaled up to ultra-low 6-well plates. Interestingly, RT-qPCR
results show the upregulation of neural stem cell markers such as nestin
and \selectlanguage{greek}β-\selectlanguage{english}III tubulin on Plastic and Plastic Plus microcarriers. Neural stem
cells are a cell type present in the olfactory mucosa (Delorme et al.,
2010; Murrell et al., 2005; Nash et al., 2001) and may therefore be
regenerative constituents of the PA5 hOMC populations.

These results led to the selection of Plastic and Plastic Plus
microcarriers for suspension culture in spinner flasks following a
previously described method to grow hMSCs (Santos et al., 2011). Plastic
L microcarriers were not taken into further studies due to the
impracticality of in-house coating of microcarriers which is an
additional expense and manipulation which would have to be considered in
the manufacturing process.

Suspension microcarrier culture of PA5 hOMCs was carried out for 7 days.
After day 1, a drop in viable cell concentration was observed due to
only around 70-75\% of cells attaching for both conditions, indicating
inefficient cell attachment. After day 3, cells in both conditions start
to grow, however, lower cell expansion was observed on Plastic Plus
microcarriers. Thus Plastic microcarriers may encourage expansion of
faster growing clones. By day 7 significantly higher viable cell
concentration is obtained for PA5 hOMCs grown on Plastic microcarriers
compared to Plastic Plus microcarriers, showing comparable results to
studies performed in ultra-low attachment well-plates. Even though lower
amount of PA5 hOMCs was obtained on Plastic Plus microcarriers on day 7,
compared to Plastic microcarriers, cultures produced similar
concentrations of glucose, lactate and ammonia over time. The
concentrations of ammonium and lactate were below the reported
inhibitory levels for hMSCs of 35.4 mM and 5.8 mM, respectively (Schop
et al., 2009). Therefore, it is unlikely that the concentration of
lactate and ammonia had a negative impact on PA5 hOMCs growth, assuming
that hOMCs and hMSCs may share similar metabolic response properties.
The specific consumption of glucose was similar for both cultures, while
the production of lactate and ammonia was double the amount for PA5
hOMCs grown on Plastic Plus microcarriers. In this study, the yield of
lactate from glucose is close to 2 for PA5 hOMCs grown in both
conditions, suggesting a metabolic shift from OXPHOS to aerobic
glycolysis. In aerobic glycolysis, in the presence of oxygen, glucose
metabolism is shifted from OXPHOS to glycolysis (Jones \& Bianchi,
2015). These results can be explained by the upregulation of LDH
activity promoted by the presence of the oncoprotein c-Myc (Shim et al.,
1998). The PA5 hOMCs population is genetically modified with a fusion
gene, c-MycER\textsuperscript{TAM}, activated by the synthetic
estrogen-like agonist 4-hydrotamoxifen (Santiago-Toledo et al., 2019;
Wall et al., 2016).

Phenotype analysis was also assessed through RT-qPCR for PA5 hOMCs
harvested at day 7 from Plastic and Plastic Plus microcarriers.
p75\textsuperscript{NTR} expression has been attributed to the presence
of OECs, a slow adherent cell type present in the olfactory mucosa,
which has been reported to promote neural regeneration (Franceschini \&
Barnett, 1996; Pixley, 1992; Ramon-Cueto et al., 1993). Phenotypic
analysis performed through RT-qPCR, revealed the upregulation of
p75\textsuperscript{NTR} more than 2-fold for PA5 hOMCs grown on Plastic
Plus microcarriers compared to Plastic microcarriers, at day 7. Factors
related to microcarrier surface such as charge and stiffness may have
led to an increase in the expression of p75\textsuperscript{NTR} for PA5
hOMCs grown on Plastic Plus microcarriers (Yang et al., 2017). The
upregulation of \selectlanguage{greek}β-\selectlanguage{english}III tubulin, although not statistically significant,
might indicate the presence of a higher number of neural stem cell-like
cells on Plastic Plus microcarriers compared to Plastic microcarriers,
on day 7 of culture.

PA5 hOMCs potency was assessed in terms of ability to promote neurite
outgrowth in a co-culture assay with NG108-15 cells. Results reveal that
PA5 hOMCs grown on Plastic and Plastic Plus microcarriers have promising
functional activity, specially the latter. Studies of PNS repair which
employed this assay shown that neurite outgrowth \emph{in vitro} can be
associated with Schwann cell potency to enhance neurite length \emph{in
vivo} (Daud et al., 2012; Jonsson et al., 2013).

In conclusion, we have demonstrated that expansion of PA5 hOMCs on
Plastic and Plastic Plus microcarriers in suspension culture using
spinner flasks is permissive. This study shows the possibility of
expanding c-Myc-ER\textsuperscript{TAM}-derived hOMCs in a scalable
manner with evidence of maintenance of
p75\textsuperscript{NTR}expression and subsequent potency after cell
detachment from the microcarriers. The work presented in this manuscript
may lead not only to the development of future strategies for the
production of an off-the-shelf advanced therapy for the treatment of CNS
injuries such as those for the spinal cord using cells such as PA5 hOMCs
or its derivatives, but also to studies focusing on xeno-free and
serum-free processing.

\textbf{Acknowledgements}

This work was supported by grants from the National Council of Science
and Technology of Mexico (CONACyT, Gerardo Santiago-Toledo), the
Engineering and Physical Sciences Research Council (Gerardo
Santiago-Toledo), and the Peter Dunnill Scholarship (Ana Valinhas).

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\par\null

\textbf{Tables}

Table 1 - List of microcarriers used and respective properties.\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\textbf{Microcarrier}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\textbf{Manufacturer}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\textbf{Diameter} \selectlanguage{greek}\textbf{(µm)}\selectlanguage{english}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\textbf{Matrix}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\textbf{Surface coating/charge}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\textbf{Average} \textbf{density}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Plastic\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Pall\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
125-212\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Polystyrene\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
None\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.02\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Plastic Laminin coated (Plastic L)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Pall\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
125-212\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Polystyrene\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Cultrex \selectlanguage{ngerman}\textsuperscript{®} Mouse Laminin I\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.02\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Plastic Plus\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Pall\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
125-212\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Polystyrene\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Cationic surface\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.02\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
PronectinF \selectlanguage{ngerman}\textsuperscript{®}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Pall\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
125-212\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Polystyrene\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Recombinant Fibronectin\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.02\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Collagen\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Pall\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
125-212\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Polystyrene\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Type I porcine collagen\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.02\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
FACT III\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Pall\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
125-212\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Polystyrene\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Type I porcine collagen\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.02\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Star-Plus\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Pall\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
125-212\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Polystyrene\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Cross-linked polystyrene\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.02\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Hillex II\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Pall\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
160-200\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Polystyrene\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Cross-linked polystyrene Cationic surface\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
1.09\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Synthemax II \selectlanguage{ngerman}\textsuperscript{®} (Low and High Concentration)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Corning\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
125-212\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Polystyrene\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Synthemax II \selectlanguage{ngerman}\textsuperscript{®}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
1.02\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Cytodex I\textsuperscript{TM}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
GE Healthcare\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
60-87\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Dextran\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Positively charged N,N-diethylaminoethyl groups\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.03\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

Table 2 - List of antibodies used for immunocytochemistry.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\textbf{Reactivity} & \textbf{Antibody} & \textbf{Host} &
\textbf{Company} & \textbf{Code}\tabularnewline
p75\textsuperscript{NTR} & IgG polyclonal & Rabbit & Millipore &
ab1554\tabularnewline
Fibronectin & IgG monoclonal & Mouse & Sigma & F6140\tabularnewline
Nestin & IgG monoclonal & Mouse & Millipore & MAB5326\tabularnewline
S100\selectlanguage{greek}β & IgG polyclonal & Rabbit & Dako & Z0311\tabularnewline
GFAP & IgG polyclonal & Rabbit & Dako & Z0334\tabularnewline
\selectlanguage{greek}β \selectlanguage{english}III Tubulin & IgG monoclonal & Mouse & Sigma & T8660\tabularnewline
Rabbit IgG (H+L) & IgG DyLight 594 & Goat & Thermo Scientific &
35560\tabularnewline
Mouse IgG (H+L) & IgG DyLight 488 & Goat & Thermo Scientific &
35510\tabularnewline
\bottomrule
\end{longtable}

Table 3 - List of Primers used for qRT-PCR.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\textbf{Biomarker} & \textbf{Description} & \textbf{NCBI Accession} &
\textbf{QuantiTect Primer Assay} & \textbf{Catalog
Number}\tabularnewline
p75\textsuperscript{NTR} & Glial Marker & NM\_002507 & Hs\_NFGR\_1\_SG &
QT00056756\tabularnewline
S100\selectlanguage{greek}β & Glial Marker & NM\_006272 & Hs\_S100B\_1\_SG &
QT00059164\tabularnewline
GFAP & Glial Marker & NM\_001131019 & Hs\_GFAP\_1\_SG &
QT00081151\tabularnewline
Nestin & Neural stem cell marker & NM\_006617 & Hs\_NES\_1SG &
QT00235781\tabularnewline
\selectlanguage{greek}β-\selectlanguage{english}III-tubulin & Neural differentiation marker & NM\_006086 &
Hs\_TUBB3\_vb.1\_SG & QT02399950\tabularnewline
Fibronectin & Cell adhesion marker & NM\_002026 & Hs\_FN1\_1\_SG &
QT00038024\tabularnewline
\selectlanguage{greek}β-\selectlanguage{english}actin & Housekeeping gene & NM\_001101 & Hs\_ACTB\_2\_SG &
QT01680476\tabularnewline
\bottomrule
\end{longtable}

\textbf{Figure Legends}

Figure 1 - PA5 hOMCs were grown on microcarriers in 96-well plates for 7
days on 10 different microcarriers. (A) Growth curve showing viable
cells/well obtained through measurements of metabolite activity using
the CCK-8 viability reagent. (B) Total viable cell/well achieved for
each microcarrier on day 1. (C) Total viable cell/well achieved for each
microcarrier on day 7. Data represented as mean \selectlanguage{ngerman}± SEM, n=3. Significant
differences were noted with (*) for p\textless{}0.05.

Figure 2 -- PA5 hOMCs were grown on Plastic L, Synthemax II LC,
Collagen, Plastic, PronectinF and Plastic Plus microcarriers for 7 days
with 50\% media change every 2 days and using an initial seeding density
of 6000 cells/cm\textsuperscript{2}. Viable cell concentration (A) and
cell viability (B) were measured. Metabolite analysis for glucose (C),
lactate (D), and ammonium (E) were performed. (F) Expression of
p75\textsuperscript{NTR}, S100\selectlanguage{greek}β, \selectlanguage{english}GFAP, \selectlanguage{greek}β-\selectlanguage{english}III tubulin, nestin and
fibronectin by PA5 hOMCs grown on Plastic L, Synthemax II LC, Collagen,
Plastic, PronectinF and Plastic Plus, assessed through RT-qPCR on day 7.
PA5 hOMCs before seeding were used as a reference.Data represented as
mean \selectlanguage{ngerman}± SEM, n = 3. Significant differences were noted with (*) for
p\textless{}0.05.

Figure 3 - Comparison of PA5 hOMCs growth on Plastic and Plastic Plus
microcarriers, in spinner flasks for 7 days. (A) Viable cell
concentration (cells/mL) and cell viability was measured via
haemocytometer counts. Metabolite analysis was performed to determine
concentration of (B) glucose (mM), (C) lactate (mM) and (D) ammonium
(mM). (E) Representative images of cells on Plastic and Plastic Plus
microcarriers, for each day of sample. Hoechst was used as a nucleic
dye. Data is presented as mean ± SEM, n=3. Significant differences were
noted with (*) for p\textless{}0.05. Scale bar = 200 \selectlanguage{greek}µ\selectlanguage{english}m.

Figure 4 --The expression of p75\textsuperscript{NTR}, GFAP, S100\selectlanguage{greek}β
\selectlanguage{english}(glial markers), \selectlanguage{greek}β-\selectlanguage{english}III tubulin, nestin (neural markers), fibronectin
(fibroblastic marker), CD90, CD105, CD73 (positive MSCs markers), CD34,
CD45 (negative MSCs markers), PPARg (adipogenesis marker), SPP1, Runx2
(osteogenesis markers), Sox9 (chondrogenesis marker) and VEGFR2
(endothelial cell marker). (A) Heat map for gene expression variation
relative to cells before seeding. (B) Values of expression relative to
cells before seeding. NG108-15 co-culture assay to assess the potential
for neural regeneration of PA5 hOMCs. F7 Schwann cells were used as a
positive control, and NG108-15 neurons only were used as negative
control. (C) Representative images used to quantify neurite outgrowth.
(D) Average neurite length, (E) maximum neurite length (F) average
neurite per neuron, were higher for Plastic and especially Plastic Plus
microcarriers (scale bar = 400 \selectlanguage{greek}µ\selectlanguage{english}m). Data represented as mean \selectlanguage{ngerman}± SEM
(n=3). Significant differences were noted with (*) for p\textless{}0.05.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure2/Figure2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure3/Figure3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure4/Figure4}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

