As opposed to a limited supply of primary human hepatocytes (PHHs), induced pluripotent stem cell-derived human hepatocytes (iPSC-HHs) could provide a nearly unlimited supply of cells needed for screening large compound libraries in the early stages of drug discovery when SAR (structure–activity relationship) approaches are still feasible. With multiple donors of iPSC-HHs, the role of genetics on drug toxicity can also be evaluated as opposed to the limited genetic diversity of available PHH donors. However, like PHHs, iPSC-HHs in pure monolayers suffer from a rapid decline in hepatic functions and morphology, making them inadequate for chronic drug toxicity assessments. We adapted the micropatterned co-culture (MPCC) technology to commercially available iPSC-HHs, whereby these cells were organized onto collagen-coated domains of empirically optimized dimensions and then surrounded by 3T3-J2 murine embryonic fibroblasts in industry-standard multiwell plates. These so-called iMPCCs maintain high levels of hepatic functions, including basal cytochrome P450 activities and drug-mediated enzyme induction, for at least 4 weeks in vitro and reduced expression of fetal markers (i.e., alpha-fetoprotein) as compared to a declining phenotype in confluent iPSC-HH monolayers. Furthermore, iMPCCs correctly classified 24 of 37 hepatotoxic drugs (65 % sensitivity), while all 10 non-toxic drugs tested were classified as such in iMPCCs (100 % specificity). These results for drug toxicity detection in iMPCCs were remarkably similar to published data in PHH-based MPCCs that were treated with the same drugs. In this chapter, we describe the methods for micropatterning collagen in multiwell plates, establishing an iMPCC in those plates with commercially available iPSC-HHs, and conducting an initial drug toxicity screen with the cultures.
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We thank Cellular Dynamics International (Madison, WI) for providing the iPSC-HHs as well as Mitchell Durham and Wendy Sunada for assisting in compiling the protocols. Funding was provided by the National Science Foundation (CAREER CBET-1351909 to S.R.K.) and the National Institute of Allergy and Infectious Diseases (1R03AI115171-01 to S.R.K.).
Competing Interests Statement
S.R.K. consults for and holds stock in Ascendance Biotechnology, which has exclusively licensed the iMPCC technology from Colorado State University for drug development applications. B.R.W. has no potential conflicts of interest to disclose.
Bovine serum albumin
Maximum or peak drug concentration measured in human plasma
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