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.