Background Nonalcoholic fatty liver disease (NAFLD) is a chronic condition that originates as lipid accumulation within hepatocytes (steatosis) and progresses into nonalcoholic steatohepatitis (NASH), characterized by lipid accumulation, inflammation, oxidative stress, and fibrosis. NAFLD is now recognized as the most common cause of chronic liver disease in the western world, with an estimated prevalence of 25% worldwide, and is projected to become the leading indication for liver transplant by 2025. Despite decades of research, the mechanisms of NAFLD progression, therapeutic approaches and non-invasive diagnostics are still resoundingly absent. The study of steatosis and NASH has traditionally utilized rodent models, which are time consuming to generate and do not fully recapitulate the complex phenotypes associated with the human disease. Furthermore, current 2D cell culture models lack relevant liver cell types, do not accurately display diseased phenotypes, and have limited utility due to rapid loss of cell viability and function. To date, there are no current models exploring the role of cell donor heterogeneity and its impact on disease phenotype and the progression of disease. Thus, there is a significant need for a more predictive human multicellular 3D in vitro model to study the progression of steatosis into NASH. Methods ExVive™ Human Liver Tissue, a human in vitro 3D bioprinted liver model comprising primary human hepatocytes, hepatic stellate cells, and endothelial cells exhibits a complex, multicellular architecture similar to that of native liver and retains metabolic competence and liver-specific functions for at least four weeks in culture. To mimic the proposed pathogenesis of NASH via a “Two-Hit Hypothesis”, immune competent tissues containing Kupffer cells were exposed to steatogenic cues via a nutrient overload approach of simple sugars and fatty acids, followed by inflammatory stimulation using prototypical inducers. Safe Harbor Statement Any statements contained in this presentation that do not describe historical facts constitute forward-looking statements as that term is defined in the Private Securities Litigation Reform Act of 1995. Any forward-looking statements contained herein are based on current expectations, but are subject to a number of risks and uncertainties. The factors that could cause the Company's actual future results to differ materially from current expectations include, but are not limited to, risks and uncertainties relating to the Company's ability to develop, market and sell products and services based on its technology; the expected benefits and efficacy of the Company's products, services and technology; the Company’s ability to successfully complete studies and provide the technical information required to support market acceptance of its products, services and technology, on a timely basis or at all; the Company's business, research, product development, regulatory approval, marketing and distribution plans and strategies, including its use of third party distributors; the Company's ability to successfully complete the contracts and recognize the revenue represented by the contracts included in its previously reported total contract bookings and secure additional contracted collaborative relationships; the final results of the Company's preclinical studies may be different from the Company's studies or interim preclinical data results and may not support further clinical development of its therapeutic tissues; the Company may not successfully complete the required preclinical and clinical trials required to obtain regulatory approval for its therapeutic tissues on a timely basis or at all; and the Company’s ability to meet its fiscal year 2017 outlook and/or its long-range outlook. These and other factors are identified and described in more detail in the Company's filings with the SEC, including its Annual Report on Form 10-K filed with the SEC on June 9, 2016 and its Quarterly Report on Form 10-Q filed with the SEC on February 9, 2017. You should not place undue reliance on these forward-looking statements, which speak only as of the date that they were made. These cautionary statements should be considered with any written or oral forward-looking statements that the Company may issue in the future. Except as required by applicable law, including the securities laws of the United States, the Company does not intend to update any of the forward-looking statements to conform these statements to reflect actual results, later events or circumstances or to reflect the occurrence of unanticipated events. Steatosis Induction in 3D Bioprinted Liver Tissues Technology Overview Figure 1: 3D human tissue development using the NovoGen Bioprinter® Platform. Cells reside in heterogeneous and architecturally structured 3D environments in vivo. Using the proprietary NovoGen Bioprinter® Platform, Organovo builds 3D tissues through automated, spatially- controlled cellular deposition to better recapitulate native tissue structure and function. Figure 2: [A] Schematic of ExVive™ Human Liver Tissue, comprised of primary human hepatocytes (HCs), hepatic stellate cells (HSCs), and endothelial cells (ECs) bioprinted in a compartmentalized geometry onto the membranes of 24-well transwell culture inserts. [B] Representative immunofluorescence image of 3D human liver tissue showing distinct zones of non-parenchymal cells (NPC) in green and parenchymal (HC) cells in red. (Norona, et al. 2016) [C] ExVive™ Liver tissue exhibits sustained hepatocyte function as indicated by albumin levels versus standard 2D hepatocyte culture, as well as sustained CYP3A4 activity [D] (2D = matched hepatocytes, grown on collagen 1 coated plates). A. B. One-way ANOVA * p<0.05 ** p<0.001 *** p<0.001 **** p<0.0001 Day 3 Day 7 Day 14 Day 28 0 1 2 3 4 5 6 Fold Change Albumin vs Day 3 2D Bioprinted **** **** **** *** **** C. D. One-way ANOVA * p<0.05 ** p<0.001 *** p<0.001 **** p<0.0001 Figure 3: Characterization of bioprinted liver tissue with incorporation of Kupffer cells (KC). [A] Kupffer cells in bioprinted liver express prototypical markers such as CD68 and CD168, and a staining pattern similar to native liver. [B] ExVive™ Liver Tissue with Kupffer cells exhibited greater cytokine induction after lipopolysaccharide (LPS) treatment. Media samples from tissue treated with LPS (100 μg/mL for 24hr) were analyzed via electrochemiluminesce. A. B. CD68 DAPI CD163 DAPI 3D Bioprinted Tissue Native Tissue ExVive Liver Tissue ExVive Liver Tissue + KCs IL-1β TNF-α ExVive Liver Tissue ExVive Liver Tissue + KCs Figure 4: Chronic exposure (21 days) of 3D bioprinted liver tissues to palmitic acid (PA) induces lipid droplet formation. H&E staining of PA-treated tissue shows both macro- (arrows) and micro- (arrowheads) vesicular phenotypes. Perilipin (PLIN5) staining of lipid vesicles further confirms the steatotic phenotype. Control (glucose) + PA [low] + PA [high] Native Liver H&E H&E PLIN Perilipin Native tissues from Samsara Sciences, Inc. Figure 5: Chronic exposure of 3D bioprinted liver tissues to palmitic acid (PA) induces increased lipid accumulation (steatosis). Oil Red O (ORO) staining of PA-treated tissues shows a dose dependent increase in steatosis. Control Initial Conditions (21 Days) Optimized Conditions (7 Days) H&E + PA (High) + PA (High) +PA only +FFA [A] +FFA [B] +FFA [C] +FFA [D] Control H&E H&E Figure 6: [A] Earlier onset of steatosis under optimized conditions. [B] Optimized regimen of free fatty acids (FFA) increases degree of steatotic phenotype. H&E H&E A. B. ExVive ™ + KC - LPS - PA - + + + + Native NASH αSMA DESMIN DAPI F F F B. A. Figure 9: Chronic dosing of Immune competent 3D bioprinted liver tissues with inflammatory inducer (LPS) and palmitic acid (PA) on a high sugar background led to [A] Increased activation of hepatic stellate cells (αSMA staining), and subsequent increased fibrosis (F; regions of increased collagen staining in blue) and steatosis (green arrows), similar to native tissue samples and [B] significantly increased inflammatory cytokine levels. TCM Control (glucose only) Trichrome + Sugars + Sugars + FFA [C] Figure 7: Titration of sugars and fatty acids alone can induce fibrosis (yellow arrows). Addition of sugars and FFAs appear to induce microvesicular (green arrowheads) and macrovesicular steatosis (green arrows). TCM Normal Steatosis NASH H&E H&E Native tissues from Samsara Sciences, Inc. NASH Induction in 3D Bioprinted Liver Tissues H&E Figure 8: Triglyceride (TG) Quantification in 3D Bioprinted Liver Tissues after FFA Treatment. [A] Native human hepatic steatosis and NASH display a 1.7 and 4.5-fold increase in TG quantification when compared to normal livers respectively, n=1 [B] 7 days treatment with sugars and low or high doses of FFAs, in immune competent (+ KCs) 3D bioprinted liver tissues results in dose-dependent increases in TG levels and [C] significantly increased TG levels over time when compared to untreated glucose only controls. X denotes fold change normalized to controls at timepoint. n = 3 - 4 per group. Trichrome Steatosis Induction in 3D Bioprinted Liver Tissues Continued Native Liver Summary and Conclusion Key features of NASH such as steatosis, increased inflammatory cytokine release, hepatic stellate cell activation, and subsequent fibrogenesis, which are to date, largely lacking in other NAFLD in vitro models are attainable in the fully human ExVive™ Liver Tissue via a nutrient overload approach, analogous to a western style diet and inflammatory stimuli. The longevity of the ExVive™ Liver Tissue allows for the testing of several induction strategies such as various dosing and durations of insults (nutrients, inflammatory inducers, xenobiotics), and also has the potential to overlay a range of modulatory approaches to profile prophylactic and treatment oriented drug strategies. Together, these features suggest that the ExVive ™ Human Liver Tissue hold promise for the study of complex, chronic conditions such as NASH, which will enable a better understanding of disease processes, discovery of novel therapeutics, potential biomarkers, and the safety assessment of drugs in a disease-relevant background. Future Directions • Refinement of induction protocol to achieve optimal steatosis and controlled stimulation of chronic inflammation representative of native disease onset. • Correlation of in vitro model to clinical phenotype. • Modulation of disease progression in vitro: testing reference compounds and client proprietary drug candidates. References • Norona, et al. (2016) Toxicological Sciences. 154(2):354-367. • Nguyen, et al. (2016) PLoS One. 7;11(7):e0158674. Native tissues from Samsara Sciences, Inc. Poster No. THU-503 Modeling NAFLD Using 3D Bioprinted Human Liver Tissue Dwayne Carter 1 , Sharon Presnell 1 , David Brenner 2 , Alice Chen 1 | 1 Organovo, 6275 Nancy Ridge Drive, San Diego, CA 92121, United States | 2 University of California, San Diego, Department of Medicine, San Diego, CA, United States CHANGING THE SHAPE OF RESEARCH AND MEDICINE www.organovo.com Oil Red O Control +PA [low] +PA [high] Control PA [low] PA [high] Lipid Accumulation [Fold x] One-way ANOVA * p<0.05 ** p<0.001 **** p<0.0001 ORO Figure 10: NASH phenotype is achievable across multiple donors. Chronic dosing with NASH induction conditions in immune competent 3D bioprinted liver tissues comprised of three different hepatocyte donors induces key NASH phenotypes such as steatosis (macrovesicular, green arrows and microvesicular , green arrowheads), and fibrosis (yellow arrows). Funding: Research reported in this publication was supported by the National Institute Of Diabetes And Digestive And Kidney Diseases of the National Institutes of Health under Award Number R44DK115242. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. NASH-Induced 3D Bioprinted Liver Tissues A. B. C. 7 Days Treatment 0.00 0.25 0.50 0.75 1.00 1.25 1.50 Triglycerides (mM) Control PA 1:1 OA:PA 2:1 OA:PA ** **** *** ** **** * * 2.9X 1.7X 2.1X 2.2X 2.8X 2.4X 2.5X 3.2X 2.9X 1.1X 1.6X TxDay 7 TxDay 14 TxDay 21 Significance determined via two-way ANOVA with a Tukey’s multiple comparisons: Control vs. treatment, within day comparisons Time point comparison within PA Time point comparison within FFA [C] Time point comparison within FFA [B] * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001
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BackgroundNonalcoholic fatty liver disease (NAFLD) is a chronic condition that originates as lipid accumulation within hepatocytes (steatosis) and progresses into nonalcoholic steatohepatitis (NASH), characterized by lipid accumulation, inflammation, oxidative stress, and fibrosis. NAFLD is now recognized as the most common cause of chronic liver disease in the western world, with an estimated prevalence of 25% worldwide, and is projected to become the leading indication for liver transplant by 2025. Despite decades of research, the mechanisms of NAFLD progression, therapeutic approaches and non-invasive diagnostics are still resoundingly absent. The study of steatosis and NASH has traditionally utilized rodent models, which are time consuming to generate and do not fully recapitulate the complex phenotypes associated with the human disease. Furthermore, current 2D cell culture models lack relevant liver cell types, do not accurately display diseased phenotypes, and have limited utility due to rapid loss of cell viability and function. To date, there are no current models exploring the role of cell donor heterogeneity and its impact on disease phenotype and the progression of disease. Thus, there is a significant need for a more predictive human multicellular 3D in vitro model to study the progression of steatosis into NASH.
MethodsExVive™ Human Liver Tissue, a human in vitro 3D bioprinted liver model comprising primary human hepatocytes, hepatic stellate cells, and endothelial cells exhibits a complex, multicellular architecture similar to that of native liver and retainsmetabolic competence and liver-specific functions for at least four weeks in culture. To mimic the proposed pathogenesis of NASH via a “Two-Hit Hypothesis”, immune competent tissues containing Kupffer cells were exposed to steatogenic cues via a nutrient overload approach of simple sugars and fatty acids, followed by inflammatory stimulation using prototypical inducers.
Safe Harbor StatementAny statements contained in this presentation that do not describe historical facts constitute forward-looking statements as that term is defined in the Private Securities Litigation Reform Act of 1995. Any forward-looking statements contained herein are based on current expectations, but are subject to a number of risks and uncertainties. The factors that could cause the Company's actual future results to differ materially from current expectations include, but are not limited to, risks and uncertaintiesrelating to the Company's ability to develop, market and sell products and services based on its technology; the expected benefits and efficacy of the Company's products, services and technology; the Company’s ability to successfully complete studies and provide the technical information required to support market acceptance of its products, services and technology, on a timely basis or at all; the Company's business, research, product development, regulatory approval, marketing and distribution plansand strategies, including its use of third party distributors; the Company's ability to successfully complete the contracts and recognize the revenue represented by the contracts included in its previously reported total contract bookings and secure additional contracted collaborative relationships; the final results of the Company's preclinical studies may be different from the Company's studies or interim preclinical data results and may not support further clinical development of its therapeutic tissues; theCompany may not successfully complete the required preclinical and clinical trials required to obtain regulatory approval for its therapeutic tissues on a timely basis or at all; and the Company’s ability to meet its fiscal year 2017 outlook and/or its long-range outlook. These and other factors are identified and described in more detail in the Company's filings with the SEC, including its Annual Report on Form 10-K filed with the SEC on June 9, 2016 and its Quarterly Report on Form 10-Q filed withthe SEC on February 9, 2017. You should not place undue reliance on these forward-looking statements, which speak only as of the date that they were made. These cautionary statements should be considered with any written or oral forward-looking statements that the Company may issue in the future. Except as required by applicable law, including the securities laws of the United States, the Company does not intend to update any of the forward-looking statements to conform these statements toreflect actual results, later events or circumstances or to reflect the occurrence of unanticipated events.
Steatosis Induction in 3D Bioprinted Liver Tissues
Technology Overview
Figure 1: 3D human tissue development using the NovoGen Bioprinter® Platform. Cells reside in heterogeneous and architecturally structured 3D environments in vivo. Using the proprietary NovoGen Bioprinter® Platform, Organovo builds 3D tissues through automated, spatially-controlled cellular deposition to better recapitulate native tissue structure and function.
Figure 2: [A] Schematic of ExVive™ Human Liver Tissue, comprised of primary human hepatocytes (HCs), hepatic stellate cells (HSCs), and endothelial cells (ECs) bioprinted in a compartmentalized geometry onto the membranes of 24-well transwell culture inserts. [B] Representative immunofluorescence image of 3D human liver tissue showing distinct zones of non-parenchymal cells (NPC) in green and parenchymal (HC) cells in red. (Norona, et al. 2016) [C] ExVive™ Liver tissue exhibits sustained hepatocyte function as indicated by albumin levels versus standard 2D hepatocyte culture, as well as sustained CYP3A4 activity [D] (2D = matched hepatocytes, grown on collagen 1 coated plates).
A. B.
One-way ANOVA* p<0.05** p<0.001*** p<0.001**** p<0.0001
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Figure 3: Characterization of bioprinted liver tissue with incorporation of Kupffer cells (KC). [A] Kupffer cells in bioprinted liver express prototypical markers such as CD68 and CD168, and a staining pattern similar to native liver. [B] ExVive™ Liver Tissue with Kupffer cells exhibited greater cytokine induction after lipopolysaccharide (LPS) treatment. Media samples from tissue treated with LPS (100 μg/mL for 24hr) were analyzed via electrochemiluminesce.
A. B.
CD68DAPI
CD163DAPI
3D Bioprinted Tissue Native Tissue
ExVive Liver Tissue ExVive Liver Tissue + KCs
IL-1β TNF-α
ExVive Liver Tissue ExVive Liver Tissue + KCs
Figure 4: Chronic exposure (21 days) of 3D bioprinted liver tissues to palmitic acid (PA) induces lipid droplet formation. H&Estaining of PA-treated tissue shows both macro- (arrows) and micro- (arrowheads) vesicular phenotypes. Perilipin (PLIN5)staining of lipid vesicles further confirms the steatotic phenotype.
Control (glucose) + PA [low] + PA [high] Native Liver
H&E
H&E
PLIN
Peril
ipin
Nat
ive
tissu
es fr
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cien
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Inc.
Figure 5: Chronic exposure of 3D bioprinted liver tissues to palmitic acid (PA) induces increased lipid accumulation (steatosis). Oil Red O (ORO) staining of PA-treated tissues shows a dose dependent increase in steatosis.
Control
Initial Conditions (21 Days)
Optimized Conditions (7 Days)
H&E
+ PA (High) + PA (High)
+PA only +FFA [A]
+FFA [B] +FFA [C] +FFA [D]
Control
H&E
H&E
Figure 6: [A] Earlier onset of steatosis under optimized conditions. [B] Optimized regimen of free fatty acids (FFA) increases degree of steatotic phenotype.
H&E
H&E
A.
B.
ExVive™ +KC -LPS -PA -
++++
Native NASH
αSMADESMINDAPI
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Figure 9: Chronic dosing of Immune competent 3D bioprinted liver tissues with inflammatory inducer (LPS) and palmitic acid (PA) on a high sugar background led to [A] Increased activation of hepatic stellate cells (αSMA staining), and subsequent increased fibrosis (F; regions of increased collagen staining in blue) and steatosis (green arrows), similar to native tissue samples and [B] significantly increased inflammatory cytokine levels.
TCM
Control (glucose only)
Tric
hrom
e
+ Sugars + Sugars + FFA [C]
Figure 7: Titration of sugars and fatty acids alone can induce fibrosis (yellow arrows). Addition of sugars and FFAs appear toinduce microvesicular (green arrowheads) and macrovesicular steatosis (green arrows).
TCM
Normal Steatosis NASH
H&E
H&E
Native tissues from Samsara Sciences, Inc.
NASH Induction in 3D Bioprinted Liver Tissues
H&E
Figure 8: Triglyceride (TG) Quantification in 3D Bioprinted Liver Tissues after FFA Treatment. [A] Native human hepaticsteatosis and NASH display a 1.7 and 4.5-fold increase in TG quantification when compared to normal livers respectively, n=1[B] 7 days treatment with sugars and low or high doses of FFAs, in immune competent (+ KCs) 3D bioprinted liver tissuesresults in dose-dependent increases in TG levels and [C] significantly increased TG levels over time when compared tountreated glucose only controls. X denotes fold change normalized to controls at timepoint. n = 3 - 4 per group.
Tric
hrom
e
Steatosis Induction in 3D Bioprinted Liver Tissues Continued
Native Liver
Summary and ConclusionKey features of NASH such as steatosis, increased inflammatory cytokine release, hepatic stellate cell activation, and subsequent fibrogenesis, which are to date, largely lacking in other NAFLD in vitro models are attainable in the fully human ExVive™ Liver Tissue via a nutrient overload approach, analogous to a western style diet and inflammatory stimuli. The longevity of the ExVive™ Liver Tissue allows for the testing of several induction strategies such as various dosing and durations of insults (nutrients, inflammatory inducers, xenobiotics), and also has the potential to overlay a range of modulatory approaches to profile prophylactic and treatment oriented drug strategies. Together, these features suggest that the ExVive™ Human Liver Tissue hold promise for the study of complex, chronic conditions such as NASH, which will enable a better understanding of disease processes, discovery of novel therapeutics,potential biomarkers, and the safety assessment of drugs in a disease-relevant background.
Future Directions• Refinement of induction protocol to achieve optimal steatosis and controlled stimulation of chronic inflammation representative
of native disease onset.• Correlation of in vitro model to clinical phenotype.• Modulation of disease progression in vitro: testing reference compounds and client proprietary drug candidates.
References• Norona, et al. (2016) Toxicological Sciences. 154(2):354-367. • Nguyen, et al. (2016) PLoS One. 7;11(7):e0158674.
Nat
ive
tissu
es fr
om S
amsa
ra S
cien
ces,
Inc.
Poster No. THU-503
Modeling NAFLD Using 3D Bioprinted Human Liver Tissue Dwayne Carter1, Sharon Presnell1, David Brenner2, Alice Chen1 | 1Organovo, 6275 Nancy Ridge Drive, San Diego, CA 92121, United States
| 2University of California, San Diego, Department of Medicine, San Diego, CA, United States
CHANGING THE SHAPE OF RESEARCH AND MEDICINE www.organovo.com
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Accu
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atio
n [F
old
x]
One-way ANOVA* p<0.05** p<0.001**** p<0.0001
ORO
Figure 10: NASH phenotype is achievable across multiple donors. Chronic dosing with NASH induction conditions in immune competent 3D bioprinted liver tissues comprised of three different hepatocyte donors induces key NASH phenotypes such as steatosis (macrovesicular, green arrows and microvesicular, green arrowheads), and fibrosis (yellow arrows).
Funding: Research reported in this publication was supported by the National Institute Of Diabetes And Digestive And Kidney Diseases of the National Institutes of Health under Award Number R44DK115242. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
NASH-Induced 3D Bioprinted Liver Tissues
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TxDay 7 TxDay 14 TxDay 21Significance determined via two-way ANOVA with a Tukey’s multiple comparisons:Control vs. treatment, within day comparisonsTime point comparison within PATime point comparison within FFA [C]Time point comparison within FFA [B]* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001
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