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) (Féron 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 (p75NTR, GFAP and S100β), neuronal (β-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 (Féron 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-MycERTAM 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 1x107 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.