Collagen coating 48 well plate

After Medium A inside the chamber was removed, 0.3 mL of a cell suspension of PXB-cells in medium A was poured in to the CVM chamber at a density of 2.1 × 10 5 viable cells/well on the liquid-liquid interface in accordance with the vitrigel culture procedure (Oshikata-Miyazaki and Takezawa, 2016), and the PXBcells in CVM chamber were cultured for 29 days at 37°C in a humidified atmosphere of 5% CO 2 and 95% air (vitrigel culture). The both media inside and outside the chamber were changed on Days 1, 4, 7, 10, 14, 17, 21, 24, and 27 with fresh culture medium (dHCGM medium: DMEM supplemented with 10% FBS, 20 mmol/L HEPES, 15 μg/mL L-proline, 0.25 μg/mL insulin, 50 nmol/L Dexamethasone, 44 mmol/L NaHCO 3 , 5 ng/mL EGF, 0.1 mmol/L ascorbic acid 2-phosphate, 100 IU/mL penicillin G, 100 μg/mL streptomycin, and 2% DMSO). Simultaneously, 0.2 mL of a cell suspension of PXB-cells in medium A was seeded at a density of 1.6 × 10 5 cells/well in the well of a collagen coating 48-well plate (BD Biosciences, Bedford, MA, USA), and the PXB-cells on the collagen coating 48-well plate were cultured for 21 days at 37°C in a humidified atmosphere of 5% CO 2 and 95% air. The medium in the well was replaced with fresh dHCGM medium on Days 1, 4, 7, 10, 14, and 17 (conventional monolayer culture).