Monocrotaline Toxicity in 3D-Bioprinted Human Liver TissueUmesh M. Hanumegowda1, Yang Wu1, Timothy R. Smith2, Lois Lehman-McKeeman1

1Bristol-Myers Squibb, Wallingford, CT, 2Organovo, San Diego, CA

ABSTRACT: Monocrotaline (MCT), a pyrrolizidine alkaloid causes liver injury in animals similar to that of hepatic venoocclusivedisorder in humans. MCT induced liver injury occurs through a complex set of cellular insults involving multiple cell types which can ultimately lead to fibrotic changes. In this study, we evaluated the effects of MCT in 3D-bioprinted human liver tissue comprising of primary hepatocytes, hepatic stellate cells, and endothelial cells (exVive3D™; Organovo, San Diego CA). The bioprinted tissues were treated with MCT at concentrations of 0.02, 0.2 or 2 mM for seven days. MCT treatment led to time- and dose-dependent decreases in tissue health as measured by LDH leakage and albumin synthesis and by histopathologic changes in the tissues. A dose-dependent increase in soluble LDH and a decrease in albumin production was observed as early as 3 days of treatment with MCT. Additionally, on treatment day 3, increases in the production of the pro-inflammatory cytokines IL-1b, IL-4,IL-8 and IL-10 were observed. Histologic assessment of formalin-fixed, paraffin-embedded tissue sections on treatment day 7 revealed early signs of tissue damage, including dissociation of the network of hepatocytes and reduced cellularity within the tissues. Immunohistochemical analyses revealed a dose-dependent increase in CD31+ cells and a marked increase in the appearance of large, CD31+ bright cells, often forming clusters or complex multi-cellular structures and cells co-expressing desmin and alpha smooth muscle actin (aSMA). Changes in organization of CD31 expressing endothelial cells and appearance of desmin and aSMA coexpressing cells are indicative of remodeling and initiation of fibrotic events. These observations capture the spectrum of changes induced by MCT ranging from reduced hepatocellular function and vascular remodeling, which may involve endothelial cell migration, organization, proliferation, stellate cell activation and initiation of early fibrotic events.

Late Breaking Poster Abstract # 3562Poster #: P254

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FIGURE 1. Biochemicalanalysis of media and3D bioprinted humanliver tissue exposed tomonocrotaline foreither 7 or 14 days. (A)LDH release (B)Albumin production (C)Whole tissue ATPlevels. Monocrotalinetreatment did notsignificantly affectviability of liver tissueas measured ATP levels,however, a transientincrease in LDH wasobserved on treatmentday 3. Monocrotalinetreatment significantlyaffected the function ofliver tissue as noted bydecreases in albuminsynthesis.

FIGURE 3. H&E stained tissues exposed to vehicle or 2mMmonocrotaline. Monocrotaline treatment was associated withdissociation of cellular network and what appears to be a mildreduction in cellularity overall. Top row 100x, bottom row 200xmagnification.

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FIGURE 4. Alpha smooth muscle actin (aSMA, marker of fibrosis); red)co-staining with CD31 (marker of endothelial cells; green) followingseven or 14 days of treatment. (A) Vehicle treatment (B) 20uM MCT(C) 200uM MCT (D) 2mM MCT. Monocrotaline treatment resulted inan overall increase in CD31+ staining. In addition, at higherconcentrations, increases in larger and brighter CD31+ structures wereobserved following 7 day treatment that co-stained for aSMA. By 14days we observed distinct staining of the CD31 and aSMA markers.These observations are indicative of endothelial remodeling.

FIGURE 5. Alpha smooth muscle actin (aSMA, marker of fibrosis); red) co-staining with either CD31 (marker of endothelial cells; green) or Desmin(marker of stellate cells; green) following 14 days of treatment. (A) Vehicletreatment (B) 20uM MCT (C) 200uM MCT (D) 2mM MCT. Monocrotalinetreatment resulted in an increase in aSMA staining in the tissue, withaSMA staining adjacent to the large CD31+ structures and overlappedwith the Desmin+ cells. These observations are suggestive of onset oftissue remodeling involving both non-parenchymal cells types in the tissueand initiation of stellate mediated pro-fibrotic events.

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FIGURE 2. Dose dependent increases in cytokines observed followingmonocrotaline treatment. Cytokines were analyzed followingtreatment day 7 using an MSD pro-inflammatory 10-spot assay

SUMMARY: The mechanism of monocrotaline induced liver toxicity is complex and involves multiple pathways and cell types. In this study we used a 3D-bioprinted human liver tissue model (comprising of primary hepatocytes, hepatic stellate cells, and endothelial cells) to demonstrate the spectrum of changes induced by monocrotaline. Monocrotaline treatment led to:• Time- and dose-dependent decreases in tissue health as measured by albumin production• Increases in production of pro-inflammatory cytokines IL-1b, IL-4, IL-8 and IL-10 • Histologically, signs of tissue damage, including dissociation of the network of hepatocytes and reduced cellularity• Dose-dependent increase in CD31+ cells and a marked increase in the appearance of large and bright CD31+ staining cells, often forming clusters or

complex multi-cellular structures• Dose-dependent changes in organization of CD31 expressing endothelial cells and appearance of desmin and aSMA co-expressing stellate cells

indicative of tissue remodeling and initiation of early fibrotic events

These observations indicate the utility of the 3D tissue model to capture complex multi-cellular events that are not typically captured by traditional 2D in vitro systems.

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