PREPARATION: Human hepatocytes are isolated by the two-step dissociation method and 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.
RECOMMENDED MEDIA: 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.