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Micropatterned coculture of primary human hepatocytes and supportive cells for the study of hepatotropic pathogens

Nature Protocols volume 10, pages 2027–2053 (2015)Cite this article

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Abstract

The development of therapies and vaccines for human hepatropic pathogens requires robust model systems that enable the study of host-pathogen interactions. However, in vitro liver models of infection typically use either hepatoma cell lines that exhibit aberrant physiology or primary human hepatocytes in culture conditions in which they rapidly lose their hepatic phenotype. To achieve stable and robust in vitro primary human hepatocyte models, we developed micropatterned cocultures (MPCCs), which consist of primary human hepatocytes organized into 2D islands that are surrounded by supportive fibroblast cells. By using this system, which can be established over a period of days, and maintained over multiple weeks, we demonstrate how to recapitulate in vitro hepatic life cycles for the hepatitis B and C viruses and the Plasmodium pathogens P. falciparum and P. vivax. The MPCC platform can be used to uncover aspects of host-pathogen interactions, and it has the potential to be used for drug and vaccine development.

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Figure 1: Schematic of MPCC fabrication, seeding, culture and application for in vitro hepatotropic pathogen infection.
Figure 2: Micropatterned cocultures of hepatocytes and supportive fibroblasts.
Figure 3: Selecting cryopreserved primary human hepatocyte sources for hepatotropic pathogen infection.
Figure 4: Application of MPCC for in vitro infection by Plasmodium parasites.
Figure 5: Application of MPCC for in vitro infection by HCV.
Figure 6: Application of MPCCs for in vitro infection by HBV.

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Acknowledgements

We thank R. Wirtz (Centers for Disease Control and Prevention) and F. Zavala (Johns Hopkins University) for PvCSP and HSP70 monoclonal antibodies, respectively, and the Malaria Research and Reference Reagent Resource Center (MR4) for monoclonal antibodies recognizing PfCSP (clone 2A10, deposited by E. Nardin) and EBA175 (clone R217, deposited by the US National Institute for Allergy and Infectious Disease (NIAID)). We are grateful to S. Hoffman and the Sanaria manufacturing team for the production of P. falciparum sporozoites and P. vivax sporozoites. We also thank J. Sattabongkot Prachumsri and R. Patrapuvich (Mahidol University) for providing fresh P. vivax sporozoites, and H. Green (Harvard University) for providing 3T3-J2 fibroblasts. This work has been supported in part by funding from the NIH (RO1 DK85713), a Skolkovo Institute of Science and Technology Grant 022423-003 and the Bill and Melinda Gates Foundation (OPP1023607). S.N.B is a Howard Hughes Medical Institute Investigator. S.R.K. acknowledges funding from the National Science Foundation (CAREER CBET-1351909) and Colorado State University. This paper is dedicated to the memory of Dr. Howard Green, a visionary and inspiring scientist.

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Authors and Affiliations

  1. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA

    Sandra March, Ani Galstian & Sangeeta N Bhatia

  2. Department of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Sandra March, Vyas Ramanan, Kartik Trehan, Shengyong Ng, Ani Galstian, Nil Gural, Heather E Fleming & Sangeeta N Bhatia

  3. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA,

    Shengyong Ng

  4. Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, New York, USA

    Margaret A Scull, Amir Shlomai & Charles M Rice

  5. Unidade de Malaria, Instituto de Medicina Molecular, Universidade de Lisboa, Lisboa, Portugal

    Maria M Mota

  6. Department of Mechanical Engineering, School of Biomedical Engineering, Colorado State University, Fort Collins, Colorado, USA

    Salman R Khetani

  7. Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, USA

    Sangeeta N Bhatia

  8. Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Sangeeta N Bhatia

  9. Howard Hughes Medical Institute, Cambridge, Massachusetts, USA

    Sangeeta N Bhatia

  10. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Sangeeta N Bhatia

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  1. Sandra March
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S.M., V.R., K.T., M.M.M., S.R.K., C.M.R. and S.N.B. conceived the study; S.M., V.R., K.T., S.N., M.S. and A.S. designed the experiments; malaria studies were conducted by S.M., S.N., N.G. and A.G.; hepatitis experiments were conducted by V.R., K.T., M.S. and A.S.; and the manuscript was prepared by S.M., V.R., K.T., N.G., S.R.K., H.E.F. and S.N.B.

Corresponding author

Correspondence to Sangeeta N Bhatia.

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Competing interests

S.N.B. and S.R.K. are the founders of Hepregen Corporation. The remaining authors declare no competing financial interests.

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March, S., Ramanan, V., Trehan, K. et al. Micropatterned coculture of primary human hepatocytes and supportive cells for the study of hepatotropic pathogens. Nat Protoc 10, 2027–2053 (2015). https://doi.org/10.1038/nprot.2015.128

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