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Microscale culture of human liver cells for drug development

Abstract

Tissue function depends on hierarchical structures extending from single cells (10 μm) to functional subunits (100 μm–1 mm) that coordinate organ functions. Conventional cell culture disperses tissues into single cells while neglecting higher-order processes. The application of semiconductor-driven microtechnology in the biomedical arena now allows fabrication of microscale tissue subunits that may be functionally improved1 and have the advantages of miniaturization2. Here we present a miniaturized, multiwell culture system for human liver cells with optimized microscale architecture that maintains phenotypic functions for several weeks. The need for such models is underscored by the high rate of pre-launch and post-market attrition of pharmaceuticals due to liver toxicity3. We demonstrate utility through assessment of gene expression profiles, phase I/II metabolism, canalicular transport, secretion of liver-specific products and susceptibility to hepatotoxins. The combination of microtechnology and tissue engineering may enable development of integrated tissue models in the so-called 'human on a chip'4.

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Figure 1: Soft lithographic process to fabricate microscale liver hepatocyte cultures in a multiwell format.
Figure 2: Functional characterization of microscale liver cultures.
Figure 3: Gene expression profiling of hepatocytes in microscale liver cultures.
Figure 4: Utility of microscale liver cultures for screening of hepatotoxicity and drug interactions.

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Acknowledgements

We are grateful to Emanuele Ostuni and Surface Logix, Inc. for design and fabrication of the PDMS stencils, Howard Green for providing 3T3-J2 fibroblasts, Jennifer Koh for assistance with pilot studies, David Eddington for assistance with microfabrication, Taylor Sittler for helpful discussions regarding compound selection, Elise Liu for assistance with biochemical assays and Sandra March for assistance with RNA isolation. Funding was generously provided by a National Science Foundation (NSF) graduate fellowship (S.R.K.), NSF CAREER, National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases, Deshpande Center at MIT, the David and Lucile Packard Foundation, the Massachusetts Technology Transfer Center, and the Center for Environmental Health Sciences at MIT.

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S.R.K. designed and performed the experiments, analyzed the data and wrote the manuscript. S.N.B. designed the experiments, analyzed the data and wrote the manuscript.

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Correspondence to Sangeeta N Bhatia.

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Competing interests

S.R.K. and S.N.B. have stock in Hepregen.

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Supplementary Figures 1–5, Supplementary Table 1, Supplementary Methods (PDF 379 kb)

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Khetani, S., Bhatia, S. Microscale culture of human liver cells for drug development. Nat Biotechnol 26, 120–126 (2008). https://doi.org/10.1038/nbt1361

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