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Micropatterned Co-Cultures of Induced Pluripotent Stem Cell-Derived Hepatocytes and Stromal Cells for Drug Toxicity Studies

Protocol
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Part of the Methods in Pharmacology and Toxicology book series (MIPT)

Abstract

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.

Key words

Induced pluripotent stem cell-derived hepatocytes Co-culture Polydimethylsiloxane Micropatterning Photolithography High-throughput screening Drug-induced liver injury 

Notes

Acknowledgments

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.

Glossary

BSA

Bovine serum albumin

Cmax

Maximum or peak drug concentration measured in human plasma

CYP450

Cytochrome P450 enzyme

DMEM

Dulbecco’s Modified Eagle’s Medium

DMSO

Dimethyl sulfoxide

FDA

US Food and Drug Administration

HEPES

2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid

iPSC-HHs

Induced pluripotent stem cell-derived human hepatocytes

iMPCC

Micropatterned co-cultures containing iPSC-HHs and stromal cells

ITS+

Insulin/transferrin/selenious acid with BSA and linoleic acid

MPCC

Micropatterned co-culture containing PHHs and stromal cells

PBS

Phosphate buffered saline

PDMS

Polydimethylsiloxane

PHHs

Primary human hepatocytes

TCPS

Tissue culture polystyrene

TC50

Interpolated concentration that reduces functions to 50 % of DMSO-only controls (can be reported as multiples of Cmax)

UV

Ultraviolet

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.School of Biomedical EngineeringColorado State UniversityFort CollinsUSA
  2. 2.Department of BioengineeringUniversity of Illinois at ChicagoChicagoUSA
  3. 3.Department of Mechanical EngineeringColorado State UniversityFort CollinsUSA

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