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 β-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-MycERTAM, 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.
p75NTR 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
p75NTR 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 p75NTR for PA5
hOMCs grown on Plastic Plus microcarriers (Yang et al., 2017). The
upregulation of β-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 in vitro can be
associated with Schwann cell potency to enhance neurite length 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-ERTAM-derived hOMCs in a scalable
manner with evidence of maintenance of p75NTRexpression 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.