Cell maintenance supplement pack

Cells were seeded into isolated bioreactors within the LiverChip platform (CNBio Innovations, Welwyn Garden City, Hertfordshire, UK) with media flow in the downward direction on top of collagen-coated scaffolds for 8 h at a flow rate of 1.0 µL/s. Following cell attachment within the scaffold, the flow was changed to the upward direction and maintained at 1.0 µL/s for the remainder of the culture. Hepatocyte monocultures were seeded at a density of 0.6 × 10⁶ viable cells in 1.6 mL of medium per well. The cells were maintained in WEM supplemented with Thawing/Plating Supplement Pack (Invitrogen) for the first 24 h of culture and in WEM supplemented with Cell Maintenance Supplement Pack (Invitrogen) thereafter for the duration of the culture. The media was replaced every 48 h. PHH/KC co-cultures were seeded simultaneously, at a ratio of 10:1. Co-cultures were seeded in Advance DMEM supplemented with Thawing/Plating Supplement Pack without dexamethasone (Invitrogen) for the first 72 h of culture and then in WEM supplemented with Cell Maintenance Supplement Pack without dexamethasone and with 100 nM hydrocortisone (Invitrogen). The media was replaced every 48 h.

Fetal liver cell cultures were performed as previously described (75 (link)). Cultures were grown in 6 wells plates with William’s E Medium (Gibco) and epidermal growth factor 0.2 ug/ml and Cell Maintenance Supplement Pack (#CM4000, Thermo Fisher Scientific) providing 0.1 uM dexamethasone, 6.25 ug/ml human recombinant insulin, 6.25 ug/ml human transferrin, 6.25 ng/ml selenous acid,1.25 mg/ml bovine serum albumin, 5.35 ug/ml linoleic acid, 2 mM GlutaMAX and 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. Cells were harvested with TrypLE Enzyme solution (Life Technologies), stained, and analyzed by flow cytometry.