Huh7.5 differentiation using HS has been described previously4 . For our studies we use pooled human AB serum (Valley Biomedical (Winchester, Virginia) Sigma-Aldrich, or any other reliable provider). Pooled human serum is prepared from the serum from 80–130 healthy donors, which removes the variability we observe with individual serum samples. Since the use of human serum results in growth arrest4 , cell cultures were normally maintained in FBS-containing media as described above4 . At the time of transfer to human serum, cells were trypsinized, trypsin was inactivated with DMEM/10% FBS/penicillin/streptomycin and cells were centrifuged at 300 g. Cell pellets were then resuspended in DMEM/2% HS/penicillin/streptomycin, and plated at a density of 30–50%4 on Eppendorf cell culture plates. At confluency cells were trypsinized once more, and plated at a density of 50%. They then form confluent layers of undividing cells4 .
The differentiation process takes approximately 21 days. Cells can be trypsinized/sub-cultured for approximately 7–10 days, but continued sub-culturing beyond that leads to cell death, as the cells do not attach to cell culture plastic any more4 .
The differentiation process takes approximately 21 days. Cells can be trypsinized/sub-cultured for approximately 7–10 days, but continued sub-culturing beyond that leads to cell death, as the cells do not attach to cell culture plastic any more4 .