Development of a xeno-free cell culture platform for novel candidate human cell therapies for neural regeneration

Abstract

The olfactory mucosa is a source of cell types that are associated with neural regeneration such as olfactory ensheathing cells (OECs), mesenchymal stromal cells (MSCs), and neural stem cells (NSCs). Although cells from this region have been used for autologous transplantation onto damaged spinal cord injury, there is a gap for an allogeneic cell therapy approach. The aim of this thesis was to establish key bioprocessing parameters for the expansion of c-MycERTAM-derived populations of human late-adherent olfactory mucosa cells (hOMCs). First, growth kinetics, identity and potency were characterised in both monolayer and suspension culture platforms. Profiles of cell growth kinetics were obtained for seeding densities ranging from 3,000 cells/cm² to 12,000 cells/cm² by manual counting and confluence measurements. PA5 hOMCs achieved between 35-41 population doublings in culture when seeded at 6,000 cells/cm² in long term culture. Moreover, removal of 4-hydroxytamoxifen (4-OHT) did not have an effect on cell growth kinetics. Replicative senescence was observed due to undesired silencing of c-MycERTAM. Next, alternative media compositions were examined as a means of optimising the process and transitioning to xeno-free culture. hOMCs showed a high dependence on fetal bovine serum (FBS) supplementation, and substitution with commercially available supplements did not sustain growth in serum-free conditions. However, human platelet lysate (hPL) was found to be a comparable to FBS when used to supplement basal media at concentrations of 2% and 5% (v/v). In this xeno-free media, hOMCs showed comparable cell growth kinetics to FBS supplemented media. Additionally, identity and potency marker expression in hOMCs was comparable between the FBS and the xeno-free hPL conditions. Finally, to progress towards scalable production of hOMCs, microcarrier screening and subsequent cell expansion on microcarriers in spinner flasks was undertaken. PA5 hOMCs were successfully expanded as a suspension culture on microcarriers at 80-mL scale in spinner flasks. A total of 8.40 ± 0.54 ×10⁶ viable cells were harvested when grown in medium supplemented with 5% hPL, and a similar number of 6.70 ± 1.05 ×10⁶ cells when grown in 10% FBS using Plastic microcarriers. PA5 hOMCs expanded on microcarriers conserved markers of identity post-harvest. In conclusion, by addressing bioprocessing fundamentals, advances towards scalable production of hOMCs using xeno-free culture reagents have been achieved