2017 Progress Report: Organotypic Liver Model for Predictive Human Toxicology and Metabolism

EPA Grant Number: R835736C004
Subproject: this is subproject number 004 , established and managed by the Center Director under grant R835736
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Vanderbilt Pittsburgh Resource for Organotypic Models for Predictive Toxicology
Center Director: Hutson, Michael Shane
Title: Organotypic Liver Model for Predictive Human Toxicology and Metabolism
Investigators: Taylor, D. Lansing , Davidson, Jeffrey M. , Gough, Albert , Vernetti, Lawrence
Institution: University of Pittsburgh Main Campus , Vanderbilt University
EPA Project Officer: Klieforth, Barbara I
Project Period: December 1, 2014 through November 30, 2018 (Extended to November 30, 2019)
Project Period Covered by this Report: December 1, 2016 through November 30,2017
RFA: Organotypic Culture Models for Predictive Toxicology Center (2013) RFA Text |  Recipients Lists
Research Category: Safer Chemicals , Health , Human Health

Objective:

In Year 3, our VPROMPT Center goals were focused on seven Milestones covering three areas of development. Two of our Milestones focused on fluorescence-based biosensors. Four of our Milestones for Year 3 concerned the area of integration of our Liver OCM with other OCMs. In addition to the biosensor and integration Milestones, we had the additional task to validate iPSC hepatocytes for inclusion in our OCM.

Progress Summary:

In Year 3, our VPROMPT Center goals were focused on seven Milestones covering three areas of development: (1) Fluorescence-based biosensors, (2) integration of the Liver OCM with other OCMs, and (3) validation of iPSC hepatocytes.

Validation of iPSC hepatocytes. Matured iPSC-derived human hepatocytes were obtained from Dr. Alex Soto-Gutierrez through a collaboration to take advantage of his patented iPSC hepatocyte maturation methodology (Figure 1). The functionality of the iPSC hepatocytes is currently being determined by testing a metabolism probe mixture for 3 specific isoforms of human Cytochrome P450 activity and two glucuronidation probes. The probes and signature metabolites are: A) testosterone → 6β-Hydroxytestos-terone (Cyp 3A4); B) Diclofenac → 4’ Hydroxydiclofenac (Cyp 2C9); C) Phenacetin → Acetaminophen (Cyp 1A2); D) Coumarin → 7-Hydroxycoumarin → 7-Hydroxycoumarin-glucuronide (Cyp 2A6, Uridine 5'-diphospho-glucuronosyltransferase [UGT], and; E) Phenolphthalein → Phenolphthalein-glucuronide. All probes and metabolites are being analyzed by mass spectroscopy. Initial results are promising with testing to be completed early 2018.

ADD Figure 1

Development of Two Real-Time, Fluorescence-Based Biosensors of Key Physiological Functions. In 2017 we developed two new biosensors, the AhR biosensor for xenobiotic metabolism (Figure 2) and a HIF1α biosensor for cellular hypoxia (Figure 3). This brings our total number of biosensors produced to seven.

ADD FIGURES 2 & 3 HERE IF POSSIBLE

Incorporate mechanism-based biosensors into the Projects 1-3 OCMs. Several biosensors, available in multiple colors (Table 1), have been disseminated to the VPROMPT project teams. Incorporation of the biosensors involved working with Projects 1-3 to build sentinel cells for their OCMs (Table 2).

ADD TABLE 1 & 2 HERE

Work with Project 5 to test our Liver OCM on the integrated platform. A preparatory step in coupling two or more organs together was to establish a common pump system that is robust and compatible with the biological and biomaterials of the OCMs. The Vanderbilt Project 5 team have designed and produced a small footprint peristaltic pump which was successfully assembled and used at the University of Pittsburgh Drug Discovery for the Liver OCM (Figure 4A). We demonstrated comparable liver function over 18 days using the Vanderbilt pump system with a 30 μL/hour flow rate (the minimum flow rate), our standard syringe pumps with flow rates of 30 μL/hour and 15 μL/hour (previously our standard flow rate) (Figure 4B).

ADD FIGURES 4A & 4B HERE

Figure 4. Setup and results from VPROMPT pump perfusion of media into two University of Pittsburgh liver MPS devices. (A) The VPROMPT pump unit ① pulls media from reservoir ② for perfusion through the MPS Liver device ③. Efflux media is collected in vial ④ for secretome measurements. (B) Comparison of flow rate and pump type on albumin and urea secretion. The gaps between days 6 – 8 were due to the Vanderbilt pump being inadvertently shut down.

Optimizing the media compatibility with other OCMs. We successfully demonstrated that liver conditioned media can be mixed 1:1 with naïve mammary gland media to support the mammary gland model, and 100% liver conditioned media can support chondrogenesis (see Project 1 mammary gland and Project 2 chondrogenesis reports). In the reverse order we have successfully demonstrated naïve mammary gland media is compatible with liver health and function when tested up to 4 days (Figure 5). However, 100% fetal membrane media is not compatible with liver function and health (Figure 5). Additional testing is required to determine the correct mix of liver-fetal membrane media for integration.

Optimizing our Liver OCM for integration with Project 1-3 OCMs. The liver and the mammary gland will be functionally coupled by media transfer and then physically coupled. The mammary gland OCM was chosen for this test because it was ready for integration. The functional coupling experiments are scheduled to start January 2018 as presented in Figure 6. Undiluted liver conditioned media was found to impact mammary gland growth (see Project 1 report) so a configuration diluting liver media 1:1 in naïve mammary gland media was chosen for the coupling experiments. The fetal membrane and chondrogenesis OCMs are still in the final stage of development but expected to be completed in Q1, 2018.

Validation of the liver readouts on the integrated platform. This milestone will be completed in early 2018 following completion of the mammary gland and liver model functional coupling. We will be testing albumin and urea production and LDH leakage throughout the functional coupling. We will couple the liver to the fetal membrane and chondrogenesis OCMs when their developments are finalized.

ADD FIGURES 5 & 6 HERE

Future Activities:

The liver and the mammary gland will be functionally coupled by media transfer and then physically coupled. The mammary gland OCM was chosen for this test because it was ready for integration. The functional coupling experiments are scheduled to start January 2018. The fetal membrane and chondrogenesis OCMs are still in the final stage of development but expected to be completed in Q1, 2018.

We will be testing albumin and urea production and LDH leakage throughout the functional coupling. We will couple the liver to the fetal membrane and chondrogenesis OCMs when their developments are finalized.

In Year 4, we plan to transfer liver modules to Vanderbilt, adapt the liver OCM to the Vanderbilt moderate throughtput platform and validate the platform with all tissue models, and screen several ToxCast compounds through the platform and all organoids.


Journal Articles on this Report : 5 Displayed | Download in RIS Format

Other subproject views: All 34 publications 10 publications in selected types All 10 journal articles
Other center views: All 149 publications 39 publications in selected types All 39 journal articles
Type Citation Sub Project Document Sources
Journal Article Gough A, Vernetti L, Bergenthal L, Shun TY, Taylor DL. The MicroPhysiology Systems Database for analyzing and modeling compound interactions with human and animal organ models. Applied In Vitro Toxicology 2016;2(2):103-117. R835736 (2015)
R835736 (2016)
R835736C004 (2017)
  • Full-text from PubMed
  • Abstract from PubMed
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  • Full-text: University of Pittsburg-Full Text PDF
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  • Abstract: Mary Ann Liebert-Abstract
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  • Journal Article Soto-Gutierrez A, Gough A, Vernetti LA, Taylor DL, Monga SP. Pre-clinical and clinical investigations of metabolic zonation in liver diseases: the potential of microphysiology systems. Experimental Biology and Medicine 2017;242(16):1605-1616. R835736 (2017)
    R835736 (2018)
    R835736C004 (2017)
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  • Abstract: Sage Publishing-Abstract
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  • Journal Article Vernetti LA, Senutovitch N, Boltz R, DeBiasio R, Shun TY, Gough A, Taylor DL. A human liver microphysiology platform for investigating physiology, drug safety, and disease models. Experimental Biology and Medicine 2016;241(1):101-114. R835736 (2015)
    R835736 (2016)
    R835736 (2017)
    R835736 (2018)
    R835736C004 (2015)
    R835736C004 (2016)
    R835736C004 (2017)
    R835736C004 (2018)
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  • Journal Article Vernetti LA, Vogt A, Gough A, Taylor DL. Evolution of experimental models of the liver to predict human drug hepatotoxicity and efficacy. Clinics in Liver Disease 2017;21(1):197-214. R835736 (2015)
    R835736 (2016)
    R835736 (2017)
    R835736 (2018)
    R835736C004 (2017)
    R835736C004 (2018)
  • Abstract from PubMed
  • Abstract: Clinics in Liver Disease-Abstract
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  • Other: ScienceDirect-First Page Preview
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  • Journal Article Vernetti L, Gough A, Baetz N, Blutt S, Broughman JR, Brown JA, Foulke-Abel J, Hasan N, In J, Kelly E, Kovbasnjuk O, Repper J, Senutovitch N, Stabb J, Yeung C, Zachos NC, Donowitz M, Estes M, Himmelfarb J, Truskey G, Wikswo JP, Taylor DL. Functional coupling of human microphysiology systems: intestine, liver, kidney proximal tubule, blood-brain barrier and skeletal muscle. Scientific Reports 2017;7:42296 (14 pp.). R835736 (2018)
    R835736C004 (2017)
    R835738 (2017)
    R835738C002 (2016)
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  • Full-text: Scientific Reports-Full Text HTML
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  • Abstract: Scientific Reports-Abstract
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  • Supplemental Keywords:

    SQL-SAL, sentinel cells, liver model, organotypic liver model 

    Relevant Websites:

    https://mps.csb.pitt.edu/ Exit ; www.vanderbilt.edu/vprompt Exit

    Progress and Final Reports:

    Original Abstract
  • 2015 Progress Report
  • 2016 Progress Report
  • 2018 Progress Report

  • Main Center Abstract and Reports:

    R835736    Vanderbilt Pittsburgh Resource for Organotypic Models for Predictive Toxicology

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R835736C001 Mammosphere Bioreactor For Life-Stage Specific Toxicology
    R835736C002 Organotypic Culture Model to Analyze Developmental LimbMalformationsResulting from Toxicant/Teratogen Exposure
    R835736C003 Validating a fetal membrane on a chip model for characterizing reproductive toxicant exposure risks
    R835736C004 Organotypic Liver Model for Predictive Human Toxicology and Metabolism
    R835736C005 Systems Engineering & Analysis for Organotypic Culture Models