Microcarrier screening for PA5 hOMCs growth on well plates
The growth of PA5 hOMCs was tested on 10 commercially available microcarriers in ultra-low attachment 96-well plates. The growth of PA5 hOMCs was followed over 7 days as this is the length of time for the duration of one passage (Figure 1 A). After seeding on day 0, viable cell number was measured every two days, when media was exchanged. On day 1, viable cell numbers ranged from 8.1x103 cells/well to 9.6x103 cells/well for Plastic LC, Synthemax II LC, Synthemax II HC, Plastic, Plastic Plus and FACT II (Figure 1 B). At the same time point, Star-Plus and Cytodex I microcarriers provided a viable cell number between 3.3x103 to 5.0x103 cells/well. As shown in Figure 1 C, by day 7, three distinct groups of microcarriers were observed. Plastic and Collagen microcarriers supported high growth of 8.8x104 and 7.5x104 cells/well. Plastic L, Synthemax II LC, Synthemax II HC, Collagen, PronectinF, FACT III and Cytodex I performed similarly between each other, yielding between 4.5x104 and 6.4x104 cells/well. Plastic Plus and Star-Plus microcarriers yielded low cell numbers, lower than 3.0x104 cells/well. All microcarriers except Star-Plus led to increase cell number across the days of culture, where after day 3 there was an increase in cell number/well (Figure 1 A).
A second experiment was performed using ultra-low attachment 6-well plates using the an increased working volume but maintaining scalable parameters. The number of microcarriers per well surface and the same initial seeding density of 6000 cells/cm2 were used. Haemocytometer counts were performed to measure viable cell density on day 7 and phenotypic changes were assessed through RT-qPCR. Plastic microcarriers led to the highest cell numbers (1.7±0.1x106 cells/mL), while Plastic Plus led to the lowest numbers (7.5±0.4x105 cells/mL) (Figure 2 A). Cell viability analysis revealed >95% viability of PA5 hOMCs on all microcarriers tested (Figure 2 B). Metabolite analysis of glucose, lactate and ammonium were performed on day 7 (Figure 2 C, D and E). Glucose concentrations were all between 4 - 8.5 mM, with Plastic showing the lower concentration and Plastic Plus the highest. The opposite trend was seen for lactate concentration with values between 12 - 16.5 mM. The concentration of ammonia was similar between all conditions with 0.8 mM for all microcarriers, except for Plastic microcarriers which produced a concentration of 0.65±0.1 mM. The phenotype of PA5 hOMCs was investigated using RT-qPCR. It is known that the olfactory mucosa population of cells harbours a mix of different cell types including mesenchymal stem cells, neural stem cells, fibroblasts and olfactory ensheathing cells (OECs). The OEC phenotype has been characterised by the expression of the glial markers p75NTR, S100β, GFAP and the neural stem cell markers nestin and β-III tubulin. Fibronectin has been used as a marker of ‘contaminating’ cell types as it can indicate the presence of fibroblasts. Results show the upregulation of neural stem cell markers β-III tubulin for Plastic L, Plastic and Plastic Plus microcarriers by 2-fold, and nestin for Plastic and Plastic Plus by 1.6-fold (Figure 2 F). Given the phenotypic traits obtained for cells grown in ultra-low attachment in 6 well-plates, with the objective to create a scalable bioprocess, Plastic and Plastic Plus were subsequently used to grow PA5 hOMCs in agitated cell culture conditions in spinner flasks.