Enhanced Primary Human Hepatocytes – 5 million

Enhanced Primary Human Hepatocytes are expanded primary cells which retains the physiologically relevant profile and phenotype of primary hepatocytes. These cells are ready-to-use for applications in research and development, such as viral infections, screening, co-cultures, transient transfection, and for metabolism studies. The late expansion cells are tested for CYP induction and inhibition. It is not recommended for further extensive passaging after thawing but can be used for long term culture which is necessary for cell-based assays.</p><br>To make complete Hepatocyte Growth Basal Medium add the entire content of Hepatocyte Growth Supplement Mix into Hepatocyte Growth Basal Medium and mix properly in BioSafety Cabinet. Addition of the Hepatocyte Growth Supplement Mix may change medium appearance more opaque.To thaw:1. Pre-warm Hepatocyte Thawing Medium and fully supplemented Hepatocyte Growth Medium to room temperature.2. Carefully remove cryovial from storage tank.3. Thaw cells in 37°C water bath until only a small chunk of ice is left. Do not shake the vial, or take it out of the water during thawing, as this will damage the cells.4. Spray the vial and the tube containing 50 ml of thawing medium with 70% ethanol and transfer to a Biosafety cabinet.5. Transfer the now completely thawed cell suspension from the cryovial into 50 ml Hepatocyte Thawing Medium by gently pouring the cells into the medium.6. Use a 1 ml pipette to transfer 1 ml of the thawing medium back to the cryovial and pour the contents back into the 50 ml tube. Repeat this process twice to completely remove the cells from the cryovial.7. Pellet the cells by centrifuging at 90×g for 5 min at RT.Important note: Higher g-forces will significantly reduce cell recovery.8. Aspirate the supernatant without disturbing the pellet. Leave approximately 200-400 ?l medium on top of the cells. 9. Gently loosen and re-suspend the cells without adding any extra medium by agitating and rotating the tube. Do not vortex or shake the cells as this will compromise cell survival.10. Add an appropriate volume of pre-warmed supplemented Hepatocyte Growth Medium to the pellet (~1ml per million cells thawed) and re-suspend the cells. Avoid pipetting the cells up and down.11. Determine viable cell number by cell count.12. Dilute hepatocytes in pre-warmed, fully supplemented Hepatocyte Growth Medium and seed at ~10,000 cells/cm2 in collagen coated cell culture flasks (e.g. T175) or appropriate cell culture dishes.13. Incubate the cells at 95% humidity, 37°C and 5% CO2.Subculture ProtocolTo Subculture:1. Pre-warm PBS, trypsin/EDTA and Hepatocyte Medium to 37Preservation ProtocolWe do not recommend to freeze down the Enhanced Primary Human Hepatocytes.