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.