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Micropatterned coculture of primary human hepatocytes and supportive cells for the study of hepatotropic pathogens
Sandra March, Vyas Ramanan, Kartik Trehan, Shengyong Ng, Ani Galstian, Nil Gural, Margaret A Scull, Amir Shlomai, Maria M Mota, Heather E Fleming, Salman R Khetani, Charles M Rice & Sangeeta N Bhatia
Affiliations
Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
Sandra March, Ani Galstian & Sangeeta N Bhatia
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
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Shengyong Ng
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
Unidade de Malaria, Instituto de Medicina Molecular, Universidade de Lisboa, Lisboa, Portugal.
Maria M Mota
Department of Mechanical Engineering, School of Biomedical Engineering, Colorado State University, Fort Collins, Colorado, USA.
Salman R Khetani
Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, USA.
Sangeeta N Bhatia
Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Sangeeta N Bhatia
Howard Hughes Medical Institute, Cambridge, Massachusetts, USA.
Sangeeta N Bhatia
Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Sangeeta N Bhatia
Published online
19 November 2015
Abstract
Abstract• Introduction• Materials• Procedure• Troubleshooting• Timing• Anticipated results• References• Acknowledgments• Author information• Comments
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.
For HBV
If high-titer patient-derived virus (starting titer of ~108–109 GE per ml before clot removal; yield after clot removal is about tenfold lower, and then diluted 1:10–1:20 in culture medium) is used to infect a highly permissive hepatocyte donor, HBV infection results in sustained production of HBsAg, robust transcription of viral RNAs and establishment of a cccDNA pool (Fig. 6). As discussed more thoroughly in a recent publication58, inhibition of the hepatocyte interferon-inducible innate immune response, by treating cultures with a pan-JAK inhibitor (JAKi), results in even more robust infection, including sustained production of HBV E antigen (a clinical marker of active viral replication), and growth in the cccDNA pool over time. Upon JAKi treatment, ~20–25% of hepatocytes stain positive for HBV Core protein, which generally exhibits nuclear localization as expected, given its incorporation into the HBV cccDNA 'minichromosome'.
HBV-infected MPCCs respond to antiviral drug treatment in ways that match clinical outcomes, providing a useful platform for testing new antivirals. Upon pretreatment of MPCCs with either entecavir (a reverse transcriptase inhibitor) or IFN, HBV titers are markedly reduced, along with all other viral readouts including cccDNA. However, when drug treatment is started after an infection is established (e.g. at 7 d after infection), responses to the two drugs differ. IFN, a multifunctional innate immune stimulator that can generate cures in a small minority of prolonged HBV infections, effectively decreases the amount of viral transcription and the levels of cccDNA in infected cells. Entecavir, in contrast, is very effective at reducing the viral titer by preventing reverse transcription, but it does not affect forward transcription or cccDNA levels. This mimics the clinical scenario, in which entecavir prevents viral rebound but cannot cure chronically infected patients.
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发表于 2015-11-20 14:58
