Microcarrier screening in well plates
10 commercially available microcarriers were tested for expansion of PA5
hOMCs in ultra-low attachment 96-well plates: Plastic, Plastic Laminin
Coated (Plastic L), Plastic Plus, PronectinF®,
Collagen, FACT III, Star-Plus, Hillex II, all from Pall, UK.
Synthemax® II (Low and High Concentration) (Corning,
UK) and Cytodex I (GE Healthcare, UK) were also used. Microcarriers
specifications are shown on Table 1. Sterilization of Pall microcarriers
and Cytodex I microcarriers was achieved by resuspension in distilled
water (Gibco, UK) and PBS Ca+ and Mg+ free (Lonza, UK), respectively,
followed by autoclaving at 121 ºC for 15 min. Synthemax II ®
microcarriers were sterilized under a UV lamp for 90 minutes.
A monolayer of microcarriers with a total surface area of 1.28
cm2 was used per well, in order to cover the bottom of
a 96-well plate well. For 6-well plates, a monolayer of microcarriers
with a total surface area of 38 cm2 was used per well.
Total working volumes were of 200 uL and 6 mL for 96-well plates and
6-well plates, respectively. Plastic L microcarriers were incubated with
a solution of 20 µg/mL Cultrex® Mouse Laminin I
(Trevigen, UK) for 1 hour in a standard incubator prior to cell seeding.
All microcarriers were let to equilibrate for 1 hour in a standard
incubator with complete medium. A seeding density of 6000
cells/cm2 was used. Medium exchanges were performed
for 50% of the total volume, every two days. Cells were cultured in
ultra-low 96-well plates for a total of 7 days. For 96 well-plates,
relative viable cell number was measured using the Cell Counting Kit-8
(CCK-8) (Dojindo, Japan) every time medium exchanges were performed.
Briefly, 10 µL of reagent were added per 100 µL of microcarrier
suspension. After incubation for 1 hour in a standard incubator, the
supernatant was transferred to a 96-well plate and absorbance readings
at 450 nm were performed in a microplate reader (Tecan, Switzerland).
Quantification of viable cells per well was performed based on a
standard curve performed on the first day of culture.