Getting closer to the patient: Upgrade of hepatitis C virus infection in primary human hepatocytes

Microscale primary human hepatocyte co-cultures to study hepatitis C virus (HCV) infection. Schematic representation of micro-patterned co-cultures (MPCC) of primary human hepatocytes (PHH) for the study of HCV infection. (A) A polydimethylsiloxane (PDMS) stencil consisting of membranes with through-holes is applied at the bottom of cell culture wells. (B) A solution of collagen is then added to the wells to allow collagen to adsorb to the substrate via the through-holes. (C) Micro-patterned collagen is left on the substrate upon stencil removal. (D) PHH are seeded in the wells and selectively adhere to collagen-coated domains. (E) Subsequently, mouse embryonic fibroblasts (3T3-J2) are seeded into the remaining bare areas as feeder cells. (F) This generates MPCC PHH co-cultures. (G) PHH may be transduced with a fluorescent RFP-NLS-IPS reporter to visualize serum-derived HCV or recombinant cell culture-derived HCV (HCVcc) infection of PHH. HCV infection is indicated by nuclear translocation of the reporter. (H) Alternatively, Gaussia luciferase reporter viruses may be used to study HCVcc infection of PHH. Infection is detected by quantitation of luciferase secretion into cell culture supernatants.