Human hepatocytes are isolated by the two-step dissociation methodand seeded on collagen I coated supports at a density of 0.16-0.18 million of cells per cm2 in a Williams E medium.One day later, the medium is changed for a long-term culture medium.
CONTROLS AND SHIPMENT
Cells are sent at room temperature after a maximum of 10 days of culture. Each cell batch is controlled for phenacetin deethylase and nifedipine oxidase activities and for paracetamol glucuronidation and sulfation.
SAFETY OF THE MATERIAL
Absence of hepatitis B, hepatitis C and HIV1 viruses checked by serology or by PCR on a liver fragment or on the cells. Notice: although controls are performed, human material has to be considered as potentially dangerous. Take maximum care in order to protect yourself and your colleagues. Handle the cells in a level 2 of biology safety room.
For the maintenance of the culture
Use long term culture medium (maintenance medium” ) for human hepatocytes, provided by BIOPREDIC International. Renewal of the medium every 2-3 days. The cells can be maintained in this medium for several weeks.
For the use of the cultures
Incubation medium (named “use medium” hereafter): Williams E with Glutamax-I (or added with 2 mM of glutamine) added with 100 IU/ml of penicillin, 100 μg/ml of streptomycin, 4 μg/ml of bovine insulin and 5 10-5 M of hydrocortisone hemisuccinate, provided by BIOPREDIC International. Renewal of the medium every 24 hours