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.