L-ECM films were prepared as described in a previous report [14] (link). Briefly, 0.03 mg/mL solution of L-ECM was prepared using sterile water, and 200 mL/well was added to a 48-well cell culture plate (bottom area: 1.1 cm2, product no. CN-150687, Thermo Fisher Scientific, Kanagawa, Japan). The solution was air-dried on a clean bench for approximately 2 days, after which, the film was cross-linked with 1% glutaraldehyde (Wako) for 3 h for stabilization. Residual glutaraldehyde was removed by washing with CMF-PBS. Finally, the film was washed with basal medium (DMEM) to inactivate any residual glutaraldehyde. Films of collagen (Cellmatrix Type I-C: 3 mg/mL; Nitta Gelatin, Osaka, Japan) were also prepared using the same method.
Freshly isolated hepatocytes were inoculated onto L-ECM films under standard conditions (37 °C, 5% CO2, 95% air) at a seeding density of 2.5 × 104 cells/mL. Two hundred microliters of D-HDM was added to each well of a 48-well cell culture plate. The culture medium was changed after 1, 3, 5, and 7 days. During the 7-day culture, morphology of the hepatocytes was studied using a cell culture microscope (Olympus CKX53, Nagano, Japan).
Freshly isolated hepatocytes were inoculated onto L-ECM films under standard conditions (37 °C, 5% CO2, 95% air) at a seeding density of 2.5 × 104 cells/mL. Two hundred microliters of D-HDM was added to each well of a 48-well cell culture plate. The culture medium was changed after 1, 3, 5, and 7 days. During the 7-day culture, morphology of the hepatocytes was studied using a cell culture microscope (Olympus CKX53, Nagano, Japan).