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.