Abstract
This study presents a simple and robust three-dimensional human hepatic tissue model to emulate steatotic and fibrotic conditions and provide an in vitro model for drug testing and mechanistic studies. Using a photolithographic biofabrication method with a photomask featuring hexagonal units, liver cells, including a human hepatic cell line (HepG2-C3A) and a human hepatic stellate cell line (LX-2) were embedded in gelatin methacryloyl hydrogel. Hepatic steatosis was induced by supraphysiological concentration of free fatty acids; hepatic fibrosis was induced by transforming growth factor-β1. Induction of steatosis was confirmed by Oil Red O and BODIPY staining and was inhibited with toyocamycin and obeticholic acid. Induction of fibrosis was confirmed by immunostaining for collagen type I and alpha smooth muscle actin and inhibited by rapamycin and curcumin treatment. This model was further preliminarily validated using primary human hepatocytes in a similar setup. These constructs provide a viable, biologically relevant, and higher throughput model of hepatic steatosis and fibrosis and may facilitate the study of the mechanisms of disease and testing of liver-directed drugs.
Similar content being viewed by others
Availability of data and materials
The datasets that support the findings of this study are available from the corresponding authors upon reasonable request. All requests for raw and analyzed data and materials will be promptly reviewed by the Brigham and Women’s Hospital and University of Florida to verify whether the request is subject to any intellectual property or confidentiality obligations. Any data and materials that can be shared will be released via a Material Transfer Agreement.
References
Wang XJ, Malhi H (2018) Nonalcoholic fatty liver disease. Ann Intern Med 169(9):itc65–itc80. https://doi.org/10.7326/AITC201811060
Friedman SL, Neuschwander-Tetri BA, Rinella M et al (2018) Mechanisms of NAFLD development and therapeutic strategies. Nat Med 24(7):908–922. https://doi.org/10.1038/s41591-018-0104-9
Chalasani N, Younossi Z, Lavine JE et al (2018) The diagnosis and management of nonalcoholic fatty liver disease: practice guidance from the American Association for the Study of Liver Diseases. Hepatology 67(1):328–357. https://doi.org/10.1002/hep.29367
Vuppalanchi R, Chalasani N (2009) Nonalcoholic fatty liver disease and nonalcoholic steatohepatitis: selected practical issues in their evaluation and management. Hepatology 49(1):306–317. https://doi.org/10.1002/hep.22603
Marchisello S, Di Pino A, Scicali R et al (2019) Pathophysiological, molecular and therapeutic issues of nonalcoholic fatty liver disease: an overview. Int J Mol Sci 20(8):1948. https://doi.org/10.3390/ijms20081948
Lonardo A, Sookoian S, Chonchol M et al (2013) Cardiovascular and systemic risk in nonalcoholic fatty liver disease—atherosclerosis as a major player in the natural course of NAFLD. Curr Pharm Des 19(29):5177–5192. https://doi.org/10.2174/1381612811319290003
Gastaldelli A, Cusi K (2019) From NASH to diabetes and from diabetes to NASH: mechanisms and treatment options. JHEP Rep Innov Hepatol 1(4):312–328. https://doi.org/10.1016/j.jhepr.2019.07.002
Gluchowski NL, Becuwe M, Walther TC et al (2017) Lipid droplets and liver disease: from basic biology to clinical implications. Nat Rev Gastroenterol Hepatol 14(6):343–355. https://doi.org/10.1038/nrgastro.2017.32
Koyama Y, Brenner DA (2017) Liver inflammation and fibrosis. J Clin Invest 127(1):55–64. https://doi.org/10.1172/JCI88881
Samuel VT, Shulman GI (2016) The pathogenesis of insulin resistance: integrating signaling pathways and substrate flux. J Clin Invest 126(1):12–22. https://doi.org/10.1172/JCI77812
Ekstedt M, Hagström H, Nasr P et al (2015) Fibrosis stage is the strongest predictor for disease-specific mortality in NAFLD after up to 33 years of follow-up. Hepatology 61(5):1547–1554. https://doi.org/10.1002/hep.27368
Baffy G, Brunt EM, Caldwell SH (2012) Hepatocellular carcinoma in non-alcoholic fatty liver disease: an emerging menace. J Hepatol 56(6):1384–1391. https://doi.org/10.1016/j.jhep.2011.10.027
Schattenberg JM, Galle PR (2010) Animal models of non-alcoholic steatohepatitis: of mice and man. Digestive diseases (Basel, Switzerland) 28(1):247–254. https://doi.org/10.1159/000282097
Adams LA, Angulo P (2006) Treatment of non-alcoholic fatty liver disease. Postgrad Med J 82(967):315–322. https://doi.org/10.1136/pgmj.2005.042200
Machado MV, Michelotti GA, **e G et al (2015) Mouse models of diet-induced nonalcoholic steatohepatitis reproduce the heterogeneity of the human disease. PLoS ONE 10(5):e0127991. https://doi.org/10.1371/journal.pone.0127991
Kanuri G, Bergheim I (2013) In vitro and in vivo models of non-alcoholic fatty liver disease (NAFLD). Int J Mol Sci 14(6):11963–11980. https://doi.org/10.3390/ijms140611963
Kozyra M, Johansson I, Nordling Å et al (2018) Human hepatic 3D spheroids as a model for steatosis and insulin resistance. Sci Rep 8(1):14297. https://doi.org/10.1038/s41598-018-32722-6
Cole BK, Feaver RE, Wamhoff BR et al (2018) Non-alcoholic fatty liver disease (NAFLD) models in drug discovery. Expert Opin Drug Discov 13(2):193–205. https://doi.org/10.1080/17460441.2018.1410135
Hassan S, Sebastian S, Maharjan S et al (2020) Liver-on-a-chip models of fatty liver disease. Hepatology 71(2):733–740. https://doi.org/10.1002/hep.31106
Gori M, Simonelli MC, Giannitelli SM et al (2016) Investigating nonalcoholic fatty liver disease in a liver-on-a-chip microfluidic device. PLoS ONE 11(7):e0159729. https://doi.org/10.1371/journal.pone.0159729
Ma L, Wu Y, Li Y et al (2020) Current advances on 3D-bioprinted liver tissue models. Adv Healthc Mater 9(24):2001517. https://doi.org/10.1002/adhm.202001517
Sacchi M, Bansal R, Rouwkema J (2020) Bioengineered 3D models to recapitulate tissue fibrosis. Trends Biotechnol 38(6):623–636. https://doi.org/10.1016/j.tibtech.2019.12.010
Leite SB, Roosens T, El Taghdouini A et al (2016) Novel human hepatic organoid model enables testing of drug-induced liver fibrosis in vitro. Biomaterials 78:1–10. https://doi.org/10.1016/j.biomaterials.2015.11.026
Norona LM, Nguyen DG, Gerber DA et al (2016) Editor’s highlight: modeling compound-induced fibrogenesis in vitro using three-dimensional bioprinted human liver tissues. Toxicol Sci 154(2):354–367. https://doi.org/10.1093/TOXSCI/KFW169
Norona LM, Nguyen DG, Gerber DA et al (2019) Bioprinted liver provides early insight into the role of Kupffer cells in TGF-β1 and methotrexate-induced fibrogenesis. PLoS ONE 14(1):e0208958. https://doi.org/10.1371/journal.pone.0208958
Kang K, Kim Y, Jeon H et al (2018) Three-dimensional bioprinting of hepatic structures with directly converted hepatocyte-like cells. Tissue Eng A 24(7–8):576–583. https://doi.org/10.1089/ten.tea.2017.0161
Moroni L, Burdick JA, Highley C et al (2018) Biofabrication strategies for 3D in vitro models and regenerative medicine. Nat Rev Mater 3(5):21–37. https://doi.org/10.1038/s41578-018-0006-y
Ma X, Qu X, Zhu W et al (2016) Deterministically patterned biomimetic human iPSC-derived hepatic model via rapid 3D bioprinting. Proc Natl Acad Sci USA 113(8):2206–2211. https://doi.org/10.1073/pnas.1524510113
Mao Q, Wang Y, Li Y et al (2020) Fabrication of liver microtissue with liver decellularized extracellular matrix (dECM) bioink by digital light processing (DLP) bioprinting. Mater Sci Eng C 109:110625. https://doi.org/10.1016/j.msec.2020.110625
Ivanov VV, Decker C (2001) Kinetic study of photoinitiated frontal polymerization. Polym Int 50(1):113–118. https://doi.org/10.1002/1097-0126(200101)50:1<113::AID-PI594>3.0.CO;2-X
Knight E, Przyborski S (2015) Advances in 3D cell culture technologies enabling tissue-like structures to be created in vitro. J Anat 227(6):746–756. https://doi.org/10.1111/joa.12257
Tsang VL, Chen AA, Cho LM et al (2007) Fabrication of 3D hepatic tissues by additive photopatterning of cellular hydrogels. Faseb J 21(3):790–801. https://doi.org/10.1096/fj.06-7117com
Aubin H, Nichol JW, Hutson CB et al (2010) Directed 3D cell alignment and elongation in microengineered hydrogels. Biomaterials 31(27):6941–6951. https://doi.org/10.1016/j.biomaterials.2010.05.056
Hahn MS, Miller JS, West JL (2006) Three-dimensional biochemical and biomechanical patterning of hydrogels for guiding cell behavior. Adv Mater 18(20):2679–2684. https://doi.org/10.1002/adma.200600647
Bajaj P, Marchwiany D, Duarte C et al (2013) Patterned three-dimensional encapsulation of embryonic stem cells using dielectrophoresis and stereolithography. Adv Healthc Mater 2(3):450–458. https://doi.org/10.1002/adhm.201200318
Pi Q, Maharjan S, Yan X et al (2018) Digitally tunable microfluidic bioprinting of multilayered cannular tissues. Adv Mater 30(43):e1706913. https://doi.org/10.1002/adma.201706913
Ying GL, Jiang N, Maharjan S et al (2018) Aqueous two-phase emulsion bioink-enabled 3D bioprinting of porous hydrogels. Adv Mater 30(50):e1805460. https://doi.org/10.1002/adma.201805460
Gong J, Schuurmans CC, van Genderen AM et al (2020) Complexation-induced resolution enhancement of 3D-printed hydrogel constructs. Nat Commun 11(1):1267. https://doi.org/10.1038/s41467-020-14997-4
Yue K, Li X, Schrobback K et al (2017) Structural analysis of photocrosslinkable methacryloyl-modified protein derivatives. Biomaterials 139:163–171. https://doi.org/10.1016/j.biomaterials.2017.04.050
Xu L, Hui AY, Albanis E et al (2005) Human hepatic stellate cell lines, LX-1 and LX-2: new tools for analysis of hepatic fibrosis. Gut 54(1):142–151. https://doi.org/10.1136/gut.2004.042127
Štampar M, Frandsen HS, Rogowska-Wrzesinska A et al (2020) Hepatocellular carcinoma (HepG2/C3A) cell-based 3D model for genotoxicity testing of chemicals. Sci Total Environ 755(2):143255. https://doi.org/10.1016/j.scitotenv.2020.143255
Ramaiahgari SC, Den Braver MW, Herpers B et al (2014) A 3D in vitro model of differentiated HepG2 cell spheroids with improved liver-like properties for repeated dose high-throughput toxicity studies. Arch Toxicol 88(5):1083–1095. https://doi.org/10.1007/s00204-014-1215-9
Seo W, Jeong W-I (2016) Hepatic non-parenchymal cells: master regulators of alcoholic liver disease? World J Gastroenterol 22(4):1348–1356. https://doi.org/10.3748/wjg.v22.i4.1348
Friedman SL (2008) Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. Physiol Rev 88(1):125–172. https://doi.org/10.1152/physrev.00013.2007
Prestigiacomo V, Weston A, Messner S et al (2017) Pro-fibrotic compounds induce stellate cell activation, ECM-remodelling and Nrf2 activation in a human 3D-multicellular model of liver fibrosis. PLoS ONE 12(6):e0179995. https://doi.org/10.1371/journal.pone.0179995
Baze A, Parmentier C, Hendriks DFG et al (2018) Three-dimensional spheroid primary human hepatocytes in monoculture and coculture with nonparenchymal cells. Tissue Eng C Methods 24(9):534–545. https://doi.org/10.1089/ten.tec.2018.0134
Loessner D, Meinert C, Kaemmerer E et al (2016) Functionalization, preparation and use of cell-laden gelatin methacryloyl-based hydrogels as modular tissue culture platforms. Nat Protoc 11(4):727–746. https://doi.org/10.1038/nprot.2016.037
Klotz BJ, Gawlitta D, Rosenberg A et al (2016) Gelatin-methacryloyl hydrogels: towards biofabrication-based tissue repair. Trends Biotechnol 34(5):394–407. https://doi.org/10.1016/j.tibtech.2016.01.002
Ying G, Jiang N, Yu C et al (2018) Three-dimensional bioprinting of gelatin methacryloyl (GelMA). Bio-des Manuf 1(4):215–224. https://doi.org/10.1007/s42242-018-0028-82
Hoch E, Schuh C, Hirth T et al (2012) Stiff gelatin hydrogels can be photo-chemically synthesized from low viscous gelatin solutions using molecularly functionalized gelatin with a high degree of methacrylation. J Mater Sci Mater Med 23(11):2607–2617. https://doi.org/10.1007/s10856-012-4731-2
Senoo H (2004) Structure and function of hepatic stellate cells. Med Electron Microsc 37(1):3–15. https://doi.org/10.1007/s00795-003-0230-3
Fausto N, Campbell JS (2003) The role of hepatocytes and oval cells in liver regeneration and repopulation. Mech Dev 120(1):117–130. https://doi.org/10.1016/S0925-4773(02)00338-6
Tsuchida T, Friedman SL (2017) Mechanisms of hepatic stellate cell activation. Nat Rev Gastroenterol Hepatol 14(7):397–411. https://doi.org/10.1038/nrgastro.2017.38
Washington K, Wright K, Shyr Y et al (2000) Hepatic stellate cell activation in nonalcoholic steatohepatitis and fatty liver. Hum Pathol 31(7):822–828. https://doi.org/10.1053/hupa.2000.8440
Takahara I, Akazawa Y, Tabuchi M et al (2017) Toyocamycin attenuates free fatty acid-induced hepatic steatosis and apoptosis in cultured hepatocytes and ameliorates nonalcoholic fatty liver disease in mice. PLoS ONE 12(3):e0170591. https://doi.org/10.1371/journal.pone.0170591
Jung IR, Choi SE, Hong SA et al (2017) Sodium fluorocitrate having protective effect on palmitate-induced beta cell death improves hyperglycemia in diabetic db/db mice. Sci Rep 7(1):12916. https://doi.org/10.1038/s41598-017-13365-5
Friedman SL (2010) Evolving challenges in hepatic fibrosis. Nat Rev Gastroenterol Hepatol 7(8):425–436. https://doi.org/10.1038/nrgastro.2010.97
Mederacke I, Hsu CC, Troeger JS et al (2013) Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology. Nat Commun 4:2823. https://doi.org/10.1038/ncomms3823
Gressner AM, Weiskirchen R, Breitkopf K et al (2002) Roles of TGF-beta in hepatic fibrosis. Front Biosci 7:d793–d807. PMID: 11897555. https://pubmed.ncbi.nlm.nih.gov/11897555/
Yang L, Roh YS, Song J et al (2014) Transforming growth factor beta signaling in hepatocytes participates in steatohepatitis through regulation of cell death and lipid metabolism in mice. Hepatology 59(2):483–495. https://doi.org/10.1002/hep.26698
Kim YJ, Lee ES, Kim SH et al (2014) Inhibitory effects of rapamycin on the different stages of hepatic fibrosis. World J Gastroenterol 20(23):7452–7460. https://doi.org/10.3748/wjg.v20.i23.7452
Saadati S, Sadeghi A, Mansour A et al (2019) Curcumin and inflammation in non-alcoholic fatty liver disease: a randomized, placebo controlled clinical trial. BMC Gastroenterol 19(1):133. https://doi.org/10.1186/s12876-019-1055-4
Farzaei MH, Zobeiri M, Parvizi F et al (2018) Curcumin in liver diseases: a systematic review of the cellular mechanisms of oxidative stress and clinical perspective. Nutrients 10(7):855. https://doi.org/10.3390/nu10070855
Arab JP, Arrese M, Trauner M (2018) Recent insights into the pathogenesis of nonalcoholic fatty liver disease. Annu Rev Pathol 13:321–350. https://doi.org/10.1146/annurev-pathol-020117-043617
Funding
YSZ received funding from National Institutes of Health (K99CA201603, R00CA201603, R21EB025270, R21EB026175, R01EB028143, R03EB027984), National Science Foundation (1935105), Brigham Research Institute New England Anti-Vivisection Foundation, and American Fund for Alternatives to Animal Research (AFAAR). AZ received funding from National Institutes of Health (K08DK113244, R01MD012579). SD received funding from National Institutes of Health (R01MD012579-UT20664DS).
Author information
Authors and Affiliations
Contributions
SM, SD, AZ, and YSZ were involved in conceptualization. SM, DB, and PS were involved in data curation. SM, DB, and YSZ performed formal analysis. SM, DB, PS, HL, WL, AZ, and YSZ conducted investigation. SM, DB, SD, and YSZ contributed to methodology. AZ and YSZ contributed to project administration. AZ and YSZ contributed resources. AZ and YSZ conducted supervision. SM, AZ, and YSZ were involved in validation. SM and YSZ contributed to visualization. SM, AZ, and YSZ were involved in writing original draft. SM, DB, PS, HL, WL, SD, AZ, and YSZ were involved in writing review and editing.
Corresponding authors
Ethics declarations
Ethical approval
This article does not contain any studies with human or animal subjects performed by any of the authors.
Conflict of interest
The authors have no relevant conflicts of interest to declare.
Consent to participate
Consent to participate is not applicable in this study.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Maharjan, S., Bonilla, D., Sindurakar, P. et al. 3D human nonalcoholic hepatic steatosis and fibrosis models. Bio-des. Manuf. 4, 157–170 (2021). https://doi.org/10.1007/s42242-020-00121-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s42242-020-00121-4