2017 Progress Report: Organotypic Models of Mammalian Liver as a Platform for Adverse Outcomes Pathway Assessment of Engineered Nanomaterials

EPA Grant Number: R835738C003
Subproject: this is subproject number 003 , established and managed by the Center Director under grant R835738
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Predictive Toxicology Center for Organotypic Cultures and Assessment of AOPs for Engineered Nanomaterials
Center Director: Faustman, Elaine
Title: Organotypic Models of Mammalian Liver as a Platform for Adverse Outcomes Pathway Assessment of Engineered Nanomaterials
Investigators: Kavanagh, Terrance J , Eaton, David
Institution: University of Washington
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:

One of the primary objectives of our project is to develop an organotypic 3D model of human and rodent liver using a microphysiological device, and evaluate its suitability for assessing the adverse effects of engineered nanomaterials and heavy metals. For this project, we will focus on the ENMs (Quantum Dots and AgNPs) that have practical applications in photonics, optics, solar cells, sensors, imaging, and anti-microbial activity.

Our hypotheses for this project are:

1) Differentiation status of organotypic liver cultures will define the response to ENMs.

2) The oxidative stress and endoplasmic reticulum (ER) stress responses within organotypic liver cultures will be key common response pathways associated with exposure to ENMs.

3) Inflammatory pathways within organotypic liver cultures will have significant and specific impacts beyond general oxidative stress.

4) Species and genetic (strain) differences among organotypic liver cultures will be critical factors in interpreting liver specific responses.

5) Using a systems-based Adverse Outcome Pathway (AOP) analysis with toxicokinetic and dynamic models as a part of a risk assessment framework will allow for cross assay and organ interpretation.

In order to address these hypotheses, this project will focus on developing three different organotypic liver model systems derived from mouse, rat, and human liver. We will use a microphysiometer flow-through system (MPS) developed by Nortis Inc., to establish and differentiate long-term cultures of liver cells for this project. Accordingly, our Specific Aims are:

Specific Aim 1. Culture hepatocytes with and without non-parenchymal liver cells obtained from rats, humans and mice in Nortis microphysiometers, and determine optimum conditions for retaining long-term viability and function of each. Viability and function will be assessed using biomarkers of liver toxicity, fluorescence probes, enzyme assays, differentiation-specific mRNA and protein expression analysis.

Specific Aim 2. Assess the in vitro uptake and toxicity of QDs and AgNPs in these three microphysiometer models of liver. This will be done under both short- (1 day) and long-term (3-4 weeks) exposure scenarios, meant to model acute and repeated exposure to these ENMs. We will examine the effects of these ENMs on viability, oxidative stress, endoplasmic reticulum (ER)-based stress, and inflammation pathways within and across the different species being tested.

Progress Summary:

Development and use of human hepatocyte spheroids to evaluate hepatic function in MPS devices.

We have explored utilizing specialized six-well plates (AggreWell™ 400ex from STEMCELL Technologies) that allow the formation of cell spheroids by seeding at specified cell densities in plates treated to prevent cell adhesion to the plate (Figure 1). We found that spheroids at a density of 100 cells/spheroid are easily formed from cryopreserved human hepatocytes and can be directly seeded into MPS devices such as the Nortis MPS or the Barofuse chamber or cryopreserved for use at later times. As we typically grow human hepatocytes in our MPS devices using sandwich culture techniques, this is technically not a three-dimensional (3-D) model and has its limitations. This 3-D spheroid model for hepatocytes provides a method that could be used to more accurately recapitulate what occurs in the intact liver and could provide improved metabolic functions with increased longevity over conventional sandwich cultures. 3-D spheroid models with HepaRG cells have demonstrated that this cell culture format improves tissue functionality and longevity (Ramaiahgari et al., 2017)

We have demonstrated that liver spheroids respond metabolically, as measured by changes in oxygen consumption rates, to ambient glucose concentrations and flow rates within the BaroFuse MPS device (Figure 2). When the hepatocyte spheroids are cultured in a Nortis device under constant perfusion, the spheroids maintained excellent viability as determined by live/dead staining after 5 days in culture and even when the spheroids were cryopreserved and thawed out and cultured for up to 11 days in a Nortis device. These promising preliminary results have encouraged us to follow up on this cell culture technique with subsequent studies to address specific metabolic activities and exploration of the 3-D cellular architecture that is formed when these cells are allowed to interact in this context.

We have continued to evaluate the utility of glutathione deficient immortalized mouse hepatocytes derived from a Gclm null mouse model as a reporter system for metal-induced oxidative stress. These cells were established from a Gclm null mouse that had exon 1 of the Gclm gene replaced by a b-galactosidase/neomycin phosphotransferase (b-Geo) gene cassette. Thus, in this context, b-gal activity reflects the activity of the Gclm gene promoter, which is known to be a Nrf2 target. Accordingly, when these cells were exposed to the model oxidant hydroquinone (HQ) or to silver nanoparticles (AgNPs), there was a dose-dependent increase in the amount of b-gal activity (as determined by the fluorescence of DDAO substrate) in the immortalized Gclm null cells, but not in the Gclm wildtype (WT) cells which lack the inserted b-Geo cassette and therefore serve as a negative control for the assay (Figure 3). These changes were in keeping with the level of b-galactosidase mRNA expression, as determined by real-time PCR in separate assays (data not shown). This assay will allow us to evaluate Nrf2 activation in real time for metals and other chemicals that are suspected to exert toxicity through oxidative stress. Portions of this work were presented at the SOT 2017 Annual meeting in San Antonio, TX.

Future Activities:

We will continue to characterize hepatocyte spheroids in the 3D MPS system and assess their functional phenotypes. We will evaluate the effects of silver nanoparticles (AgNPs), quantum dot nanoparticles, silver and cadmium ions and other heavy metals in hepatocytes cultured in 2D monolayers vs. spheroids 3D MPS. These will include quantitative measures of viability, function, the induction of glutathione pathway genes and proteins, metallothionein (MT) expression, and oxidative stress biomarker expression. In a manner similar to that we have described for tandem organ on a chip evaluation of AA toxicity, we will examine the ability of hepatocyte spheroid expression of MT to deliver Cd and Ag to PTECS and determine if such delivery influences kidney cell viability and function. We also continue to develop and assess the utility of the mouse IM Gclm hepatocytes grown in 2D and 3D spheroids as a model for comparison to the human hepatocyte cultures, especially to evaluate Nrf2 responsiveness in each system.

References:

Ramaiahgari et al, Three-Dimensional (3D) HepaRG Spheroid Model With Physiologically Relevant Xenobiotic Metabolism Competence and Hepatocyte Functionality for Liver Toxicity Screening. Toxicological Sciences 2017;59(1);124–136.

Rountree A et al., BaroFuse, a novel pressure-driven, adjustable-throughput perfusion system for tissue maintenance and assessment. Heliyon Article No-e00210, 2016.


Journal Articles on this Report : 4 Displayed | Download in RIS Format

Other subproject views: All 10 publications 4 publications in selected types All 4 journal articles
Other center views: All 134 publications 39 publications in selected types All 38 journal articles
Type Citation Sub Project Document Sources
Journal Article Chang S-Y, Weber EJ, Van Ness KP, Eaton DL, Kelly EJ. Liver and kidney on chips: microphysiological models to understand transporter function. Clinical Pharmacology & Therapeutics 2016;100(5):464-478. R835738 (2016)
R835738 (2017)
R835738 (2018)
R835738C002 (2016)
R835738C003 (2016)
R835738C003 (2017)
R835738C005 (2017)
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  • Journal Article Chang S-Y, Weber EJ, Sidorenko VS, Chapron A, Yeung CK, Gao C, Mao Q, Shen D, Wang J, Rosenquist TA, Dickman KG, Neumann T, Grollman AP, Kelly EJ, Himmelfarb J, Eaton DL. Human liver-kidney model elucidates the mechanisms of aristolochic acid nephrotoxicity. JCI Insight 2017;2(22):e95978 (15 pp.). R835738C003 (2017)
    R835738C005 (2017)
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  • Journal Article Chang S-Y, Voellinger JL, Van Ness KP, Chapron B, Shaffer RM, Neumann T, White CC, Kavanagh TJ, Kelly EJ, Eaton DL. Characterization of rat or human hepatocytes cultured in microphysiological systems (MPS) to identify hepatotoxicity. Toxicology In Vitro 2017;40:170-183. R835738 (2016)
    R835738 (2017)
    R835738 (2018)
    R835738C002 (2016)
    R835738C002 (2017)
    R835738C003 (2016)
    R835738C003 (2017)
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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 (15 pp.). R835738 (2016)
    R835738C003 (2017)
    R835738C005 (2017)
    R835736 (2015)
    R835736 (2016)
    R835736C004 (2016)
    R835736C005 (2016)
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  • Supplemental Keywords:

    3D organotypic cultures, microphysiological systems, hepatocytes, mouse, human, nanoparticles, quantum dots, aristolochic acid, cadmium, silver, cytotoxicity, redox status, cellular stress response, Nrf2 reporter assay

    Relevant Websites:

    http://deohs.washington.edu/ptc Exit

    Progress and Final Reports:

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

  • Main Center Abstract and Reports:

    R835738    Predictive Toxicology Center for Organotypic Cultures and Assessment of AOPs for Engineered Nanomaterials

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R835738C001 Airway Epithelium Organotypic Culture as a Platform for AdverseOutcomesPathway Assessment of Engineered Nanomaterials
    R835738C002 Organotypic Model of Human Kidney as a Platform for Adverse Outcomes Pathway Assessment of Engineered Nanomaterials
    R835738C003 Organotypic Models of Mammalian Liver as a Platform for Adverse Outcomes Pathway Assessment of Engineered Nanomaterials
    R835738C004 Organotypic Model of Testis as a Platform for Adverse Outcomes Pathway Assessment of Engineered Nanomaterials
    R835738C005 Integrating Liver, Kidney and Testis Nanomaterial Toxicity using the Adverse Outcome Pathway Approach