Cell culture media | Use of commercial media to recover and plate hepatocytes from cryopreservation and supplemented WME media to maintain cell culture was adequate. |
Media supplementation | Supplementation of culture media with dexamethasone produced higher basal CYP3A4 activity levels. (The additional benefit of other standard media supplements such as nonessential amino acids or glutamine will depend on base culture media used). |
Experimental layout | 24- or 96-well collagen-coated plate well format (prevalidate donors for ability to grow on selected surface). |
Cell culture supplementation | Collagen-coated plates with or without matrigel are acceptable. (Inclusion of a matrigel overlay did not conclusively show benefits to experimental design or outcome). |
Culture/experimental time window | Measureable effects of IL-6 on cytochrome P450 mRNA levels can be detected as early as 24 hours following cytokine incubation; however, 48-hour exposure is often necessary to see substantial effects on cytochrome P450 enzyme activity. It is recommended to hold the maximum dosing period to 48 or 72 hours (5-day total culture time). |
Positive controls | Measure effects of IL-6 on CYP3A4 and CRP to indicate the overall responsiveness of the experimental system for cytokine-mediated repression. |