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WINCHESTER, MA, United States

Jones C.T.,Rockefeller University | Catanese M.T.,Rockefeller University | Law L.M.J.,Rockefeller University | Khetani S.R.,Massachusetts Institute of Technology | And 10 more authors.
Nature Biotechnology | Year: 2010

Hepatitis C virus (HCV), which infects 2-3% of the world population, is a causative agent of chronic hepatitis and the leading indication for liver transplantation. The ability to propagate HCV in cell culture (HCVcc) is a relatively recent breakthrough and a key tool in the quest for specific antiviral therapeutics. Monitoring HCV infection in culture generally involves bulk population assays, use of genetically modified viruses and/or terminal processing of potentially precious samples. Here we develop a cell-based fluorescent reporter system that allows sensitive distinction of individual HCV-infected cells in live or fixed samples. We demonstrate use of this technology for several previously intractable applications, including live-cell imaging of viral propagation and host response, as well as visualizing infection of primary hepatocyte cultures. Integration of this reporter with modern image-based analysis methods could open new doors for HCV research.


Trask O.J.,Hamner Institutes for Health Sciences | Moore A.,Hepregen, Inc. | Lecluyse E.L.,Hamner Institutes for Health Sciences
Assay and Drug Development Technologies | Year: 2014

The current landscape of in vitro models used to identify drug-or chemical-induced hepatotoxicity relies heavily on cell culture models consisting of HepG2, induced pluripotent stem cell-derived, or primary hepatocytes. While these in vitro models offer powerful approaches for predicting toxicity, each system has challenges, including variable metabolic capacity, brief ex vivo life span in culture, and adoption with standard automated microscopy high-content screening (HCS) systems to measure reproducibility data at the single-cell level. In this report we introduce a novel primary hepatocyte coculture model, HepatoPac™, as an alternative to current model systems for evaluation of in vitro hepatotoxicity in 96-well microtiter plate format examined by HCS. The coculture model consists of primary hepatocytes that are micropatterned to form a discrete microarchitecture or "hepatocyte islands" that are surrounded by supporting fibroblasts resulting in long-term viability and metabolic function of primary hepatocytes. Using multiple HCS image capture and image analysis strategies, we established methods to interrogate various morphometric parameters, such as size, shape, and intensity, at the island or single-cell level. We applied these approaches to identify subpopulations of both fibroblasts and hepatocytes that exhibited alterations in nuclear parameters, cell permeability, mitochondria function, and apoptosis using known reference control compounds and an eight-point dose curve. Subpopulation analysis with additional bioprobe sets can provide a powerful means of addressing differential cell and tissue susceptibilities during compound profiling. Our data show that the HepatoPac is amendable for HCS imaging applications and provides a unique approach for studying hepatotoxicity over prolonged periods of time. © 2014, Mary Ann Liebert, Inc.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 100.00K | Year: 2008

DESCRIPTION (provided by applicant): Liver toxicity is the leading cause of pre-launch and post-market attrition of pharmaceutical compounds (i.e. Rezulin, Prexige). Significant species-specific differences in organ functions now necessitate supplementatio n of animal data with assays designed to assess human responses to drugs. Adherent cultures of primary human hepatocytes are considered to be the gold standard for evaluating preclinical drug metabolism, enzyme induction, and liver-specific toxicity. Howev er, hepatocytes display a rapid (hours) decline in liver-specific functions under conventional culture conditions. Recently, a robust model of human liver tissue has been developed with optimized microscale architecture in an industry-standard multiwell fo rmat that retains liver-specific functions for 4-6 weeks in vitro. Hepatocytes in this microscale platform secrete liver- specific products, display functional CYP450 and conjugation enzymes, secrete molecules into the bile canaliculi, and maintain high le vels of expression of liver-specific genes relevant for evaluating drug disposition. The primary objective of this Small Business Innovation Research (SBIR) Phase I project is to assess the utility of microscale tissues for evaluating drug metabolism and d rug-drug interactions in vitro with better clinical predictivity than that afforded by conventional, declining cultures. In particular, the time course of un-induced (baseline) activities of a broad range of Phase I and II enzymes will first be characteriz ed in microscale tissues to determine the optimal time window for drug metabolism applications. Then, the utility of microscale tissues for evaluating time-dependent modulation of Phase I, II, and III transcripts and protein activities by pharmaceutical co mpounds will be evaluated towards screening drug-drug interactions in drug development. Lastly, the utility of microscale liver tissues to predict in vivo metabolic clearance and identify all major and minor metabolites better than conventional systems wil l be explored. The results of this SBIR feasibility study will enable the small business to form strategic partnerships with major pharmaceutical companies towards dissemination of microscale human liver tissues into the marketplace. In the future, microsc ale liver tissues may be used to eliminate problematic compounds much earlier in drug development towards substantially reducing development costs ( 1 billion per successfully marketed drug), increasing the likelihood of clinical success, and limiting pati ent exposure to unsafe drugs. Microscale human liver tissues may also enable the investigation of mechanisms of drug action, allow identification of new biomarkers, and enable studies to assess the risk associated with exposure to mixtures of drugs. PUBLIC HEALTH RELEVANCE: Toxicity to the liver is the leading cause of drug withdrawals (i.e. Rezulin, Prexige) from the marketplace by regulatory agencies. The studies proposed in this SBIR Phase I project can establish utility of microscale human liver tissues for evaluating liver-specific metabolism of candidate drugs in drug development, and significantly more clinically predictive than allowed in existing conventional model systems. Therefore, in the future, microscale human liver tissues may eliminate probl ematic compounds much earlier in the drug development pipeline towards reducing patient exposure to unsafe drugs in clinical trials and in the marketplace.


Patent
Hepregen, Inc. | Date: 2013-03-12

The present disclosure provides compositions, systems, and tools for modeling liver inflammation and methods of using the same. The disclosure provides micropatterned hepatocyte co-cultures where individual cell populations remain functionally stable during long-term culture. The in vitro liver inflammation models of the present disclosure may be useful for evaluating inflammation-mediated toxicities of compounds in a pre-clinical setting.


Trademark
Hepregen, Inc. | Date: 2013-08-08

Microliver platforms, namely, mini mammalian liver models consisting of human and animal cells, for use in laboratory, scientific and medical research.

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