Three-Dimensional Dynamic Cell Models for Metabolic Dysfunction-Associated Steatotic Liver Disease Progression

Zhengxiang Huang; Lili Li; Kevin Dudley; Lan Xiao; Gary Huang; V. Nathan Subramaniam; Chen Chen; Ross Crawford; Yin Xiao

doi:10.34133/bmef.0181

journal article Jan 01, 2025

Three-Dimensional Dynamic Cell Models for Metabolic Dysfunction-Associated Steatotic Liver Disease Progression

Zhengxiang Huang Lili Li

Kevin Dudley Lan Xiao

Gary Huang

V. Nathan Subramaniam Chen Chen

Ross Crawford Yin Xiao

BME Frontiers Vol. 6 · American Association for the Advancement of Science (AAAS)

View at Publisher Save 10.34133/bmef.0181

Abstract

Objective:
Metabolic dysfunction-associated steatotic liver disease (MASLD) is a complex, progressive disorder involving multiple cell types, ranging from simple steatosis to metabolic dysfunction-associated steatohepatitis (MASH), characterized by pro-inflammatory macrophage activation, and can eventually advance to fibrosis, initiated by hepatic stellate cells (HSCs). In vitro multi-cell coculture models are vital tools for elucidating the mechanisms underlying MASLD.
Impact Statement:
Existing in vitro models for MASLD, including traditional 2-dimensional (2D) cultures and advanced organ-on-a-chip and organoid systems, face challenges in representing multiple cell types and analyzing them individually. Here, utilizing a cell carrier developed in our laboratory, we introduce a series of 3D dynamic coculture models that simulate different stages of MASLD progression and enable individual cell type analysis.
Introduction:
Currently, no single system provides an optimal balance of control, reproducibility, and analytical convenience. Most in vitro models lack the ability to isolate and analyze individual cell types post-culture, making it difficult to study cell-specific responses in MASLD progression.
Methods:
The 3D hollow porous sphere cell carrier allows cells to grow on its surface, while the culture device (mini-bioreactor) creates a dynamic environment. The 3 distinct MASLD models were established based on cocultured cell types: steatosis (hepatocytes only), MASH (hepatocytes and macrophages in a 4:1 ratio), and fibrosis (hepatocytes, macrophages, and HSCs in an 8:2:1 ratio). Well-established MASLD mouse models were employed to validate our in vitro 3D dynamic MASLD models, using 7-week-old male C57BL/6J mice fed a high-fat diet.
Results:
Our models demonstrate a progressive decline in hepatocyte viability and increased lipid accumulation, mirroring in vivo pathology. Additionally, gene expression profiles of our models align with those observed in MASLD-affected mouse livers. Notably, comparative analysis highlights the role of pro-inflammatory macrophages in disrupting hepatocyte lipid metabolism.
Conclusion:
These models offer a robust platform for investigating MASLD mechanisms and show potential for screening anti-MASLD therapeutics.

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References

49

[1]

The global epidemiology of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH): a systematic review

Zobair M. Younossi, Pegah Golabi, James M. Paik et al.

Hepatology 2023 10.1097/hep.0000000000000004

[2]

Marjot T, Moolla A, Cobbold JF, Hodson L, Tomlinson JW. Nonalcoholic fatty liver disease in adults: Current concepts in etiology, outcomes, and management. Endocr Rev. 2019;41(1):66–117. 10.1210/endrev/bnz009

[3]

Donnelly KL, Smith CI, Schwarzenberg SJ, Jessurun J, Boldt MD, Parks EJ. Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease. J Clin Invest. 2005;115(5):1343–1351. 10.1172/jci23621

[4]

Chavez-Tapia NC, Rosso N, Tiribelli C. Effect of intracellular lipid accumulation in a new model of non-alcoholic fatty liver disease. BMC Gastroenterol. 2012;12(1):20. 10.1186/1471-230x-12-20

[5]

Ramos MJ, Bandiera L, Menolascina F, Fallowfield JA. In vitro models for non-alcoholic fatty liver disease: Emerging platforms and their applications. iScience. 2022;25(1): Article 103549. 10.1016/j.isci.2021.103549

[6]

Kostrzewski T, Maraver P, Ouro-Gnao L, Levi A, Snow S, Miedzik A, Rombouts K, Hughes D. A microphysiological system for studying nonalcoholic steatohepatitis. Hepatol Commun. 2020;4(1):77–91. 10.1002/hep4.1450

[7]

Freag MS, Namgung B, Reyna Fernandez ME, Gherardi E, Sengupta S, Jang HL. Human nonalcoholic steatohepatitis on a chip. Hepatol Commun. 2021;5(2):217–233. 10.1002/hep4.1647

[8]

Wang J, Wu X, Zhao J, Ren H, Zhao Y. Developing liver microphysiological systems for biomedical applications. Adv Healthc Mater. 2024;13(21): Article e2302217. 10.1002/adhm.202302217

[9]

Cho HJ, Kim HJ, Lee K, Lasli S, Ung A, Hoffman T, Nasiri R, Bandaru P, Ahadian S, Dokmeci MR, et al. Bioengineered multicellular liver microtissues for modeling advanced hepatic fibrosis driven through non-alcoholic fatty liver disease. Small. 2021;17(14): Article e2007425. 10.1002/smll.202007425

[10]

Ströbel S, Kostadinova R, Fiaschetti-Egli K, Rupp J, Bieri M, Pawlowska A, Busler D, Hofstetter T, Sanchez K, Grepper S, et al. A 3D primary human cell-based in vitro model of non-alcoholic steatohepatitis for efficacy testing of clinical drug candidates. Sci Rep. 2021;11(1):22765. 10.1038/s41598-021-01951-7

[11]

van Os EA, Cools L, Eysackers N, Szafranska K, Smout A, Verhulst S, Reynaert H, McCourt P, Mannaerts I, van Grunsven LA. Modelling fatty liver disease with mouse liver-derived multicellular spheroids. Biomaterials. 2022;290: Article 121817. 10.1016/j.biomaterials.2022.121817

[12]

Aasadollahei N, Rezaei N, Golroo R, Agarwal T, Vosough M, Piryaei A. Bioengineering liver microtissues for modeling non-alcoholic fatty liver disease. EXCLI J. 2023;22:367–391.

[13]

Gao W, Xiao L, Wang J, Mu Y, Mendhi J, Gao W, Li Z, Yarlagadda P, Wu C, Xiao Y. The hollow porous sphere cell carrier for the dynamic 3D cell culture. Tissue Eng Part C Methods. 2022;28(11):610–622. 10.1089/ten.tec.2022.0137

[14]

Coelho I, Duarte N, Macedo MP, Penha-Gonçalves C. Insights into macrophage/monocyte-endothelial cell crosstalk in the liver: A role for Trem-2. J Clin Med. 2021;10(6): Article 1248. 10.3390/jcm10061248

[15]

10.3389/fimmu.2020.01169

[16]

Animal models of non‐alcoholic fatty liver disease: current perspectives and recent advances

Jennie Ka Ching Lau, Xiang Zhang, Jun Yu

The Journal of Pathology 2017 10.1002/path.4829

[17]

Ipsen DH, Lykkesfeldt J, Tveden-Nyborg P. Animal models of fibrosis in nonalcoholic steatohepatitis: Do they reflect human disease? Adv Nutr. 2020;11(6):1696–1711. 10.1093/advances/nmaa081

[18]

10.1002/j.2040-4603.2013.tb00503.x

[19]

Mechanisms of NAFLD development and therapeutic strategies

Scott L. Friedman, Brent A. Neuschwander-Tetri, Mary Rinella et al.

Nature Medicine 10.1038/s41591-018-0104-9

[20]

10.1111/febs.15877

[21]

Cao Y, Mai W, Li R, Deng S, Li L, Zhou Y, Qin Q, Zhang Y, Zhou X, Han M, et al. Macrophages evoke autophagy of hepatic stellate cells to promote liver fibrosis in NAFLD mice via the PGE2/EP4 pathway. Cell Mol Life Sci. 2022;79(6):303. 10.1007/s00018-022-04319-w

[22]

Soret PA, Magusto J, Housset C, Gautheron J. In vitro and in vivo models of non-alcoholic fatty liver disease: A critical appraisal. J Clin Med. 2020;10(1): Article 36. 10.3390/jcm10010036

[23]

10.1101/gad.348340.121

[24]

10.1007/s00424-020-02417-x

[25]

Cordeiro S, Oliveira BB, Valente R, Ferreira D, Luz A, Baptista PV, Fernandes AR. Breaking the mold: 3D cell cultures reshaping the future of cancer research. Front Cell Dev Biol. 2024;12:1507388. 10.3389/fcell.2024.1507388

[26]

Sean G, Banes AJ, Gangaraju R. Organoids and tissue/organ chips. Stem Cell Res Ther. 2024;15(1):241. 10.1186/s13287-024-03859-1

[27]

Brandauer K, Schweinitzer S, Lorenz A, Krauß J, Schobesberger S, Frauenlob M, Ertl P. Advances of dual-organ and multi-organ systems for gut, lung, skin and liver models in absorption and metabolism studies. Lab Chip. 2025. 10.1039/d4lc01011f

[28]

A model of hepatic steatosis with declined viability and function in a liver-organ-on-a-chip

Natsupa Wiriyakulsit, Ploychanok Keawsomnuk, Saowarose Thongin et al.

Scientific Reports 2023 10.1038/s41598-023-44198-0

[29]

Karnawat K, Parthasarathy R, Sakhrie M, Karthik H, Krishna KV, Balachander GM. Building in vitro models for mechanistic understanding of liver regeneration in chronic liver diseases. J Mater Chem B. 2024;12(32):7669–7691. 10.1039/d4tb00738g

[30]

Trampuž SR, van Riet S, Nordling Å, Ingelman-Sundberg M. The role of CTGF in liver fibrosis induced in 3D human liver spheroids. Cells. 2023;12(2): Article 302. 10.3390/cells12020302

[31]

Goswami Y, Baghel A, Sharma G, Sharma PK, Biswas S, Yadav R, Garg PK, Shalimar, Tandon R. Liver organoids from hepatocytes of healthy humans and non-alcoholic fatty liver disease (NAFLD) patients display multilineage architecture and can be used to develop an in vitro model of steatohepatitis. J Clin Exp Hepatol. 2025;15(3): Article 102463. 10.1016/j.jceh.2024.102463

[32]

10.1016/j.cmet.2019.05.007

[33]

Duriez M, Jacquet A, Hoet L, Roche S, Bock MD, Rocher C, Haussy G, Vigé X, Bocskei Z, Slavnic T, et al. A 3D human liver model of nonalcoholic steatohepatitis. J Clin Transl Hepatol. 2020;8(4):359–370.

[34]

Deguchi S, Takayama K. State-of-the-art liver disease research using liver-on-a-chip. Inflamm Regen. 2022;42(1):62. 10.1186/s41232-022-00248-0

[35]

Investigating Nonalcoholic Fatty Liver Disease in a Liver-on-a-Chip Microfluidic Device

Manuele Gori, Maria Chiara Simonelli, Sara Maria Giannitelli et al.

PLoS ONE 2016 10.1371/journal.pone.0159729

[36]

Rada P, González-Rodríguez Á, García-Monzón C, Valverde ÁM. Understanding lipotoxicity in NAFLD pathogenesis: Is CD36 a key driver? Cell Death Dis. 2020;11(9):802. 10.1038/s41419-020-03003-w

[37]

Zhu H, Zhao T, Zhao S, Yang S, Jiang K, Li S, Kang Y, Yang Z, Shen J, Shen S, et al. O-GlcNAcylation promotes the progression of nonalcoholic fatty liver disease by upregulating the expression and function of CD36. Metabolism. 2024;156: Article 155914. 10.1016/j.metabol.2024.155914

[38]

Moreno-Vedia J, Girona J, Ibarretxe D, Masana L, Rodríguez-Calvo R. Unveiling the role of the fatty acid binding protein 4 in the metabolic-associated fatty liver disease. Biomedicine. 2022;10(1): Article 197.

[39]

Li Y, Xu K, Zhou A, Xu Z, Wu J, Peng X, Mei S, Chen H. Integrative transcriptomics and proteomics analysis reveals THRSP’s role in lipid metabolism. Genes. 2024;15(12): Article 1562. 10.3390/genes15121562

[40]

Rong J, Zhang Z, Peng X, Li P, Zhao T, Zhong Y. Mechanisms of hepatic and renal injury in lipid metabolism disorders in metabolic syndrome. Int J Biol Sci. 2024;20(12):4783–4798. 10.7150/ijbs.100394

[41]

Zhou C, Shen Z, Shen B, Dai W, Sun Z, Guo Y, Xu X, Wang J, Lu J, Zhang Q, et al. FABP4 in LSECs promotes CXCL10-mediated macrophage recruitment and M1 polarization during NAFLD progression. Biochim Biophys Acta Mol basis Dis. 2023;1869(7): Article 166810. 10.1016/j.bbadis.2023.166810

[42]

Ren Y, Zhao H, Yin C, Lan X, Wu L, du X, Griffiths HR, Gao D. Adipokines, hepatokines and myokines: Focus on their role and molecular mechanisms in adipose tissue inflammation. Front Endocrinol. 2022;13: Article 873699. 10.3389/fendo.2022.873699

[43]

Xing H, Wu Z, Jiang K, Yuan G, Guo Z, Yu S, He S, Zhong F. FABP4 deficiency ameliorates alcoholic steatohepatitis in mice via inhibition of p53 signaling pathway. Sci Rep. 2024;14(1):21135. 10.1038/s41598-024-71311-8

[44]

10.1111/jdi.13735

[45]

Järvinen E, Hammer HS, Pötz O, Ingelman-Sundberg M, Stage TB. 3D spheroid primary human hepatocytes for prediction of cytochrome P450 and drug transporter induction. Clin Pharmacol Ther. 2023;113(6):1284–1294. 10.1002/cpt.2887

[46]

Mickols E, Meyer A, Handin N, Stüwe M, Eriksson J, Rudfeldt J, Blom K, Fryknäs M, Sellin ME, Lauschke VM, et al. OCT1 (SLC22A1) transporter kinetics and regulation in primary human hepatocyte 3D spheroids. Sci Rep. 2024;14(1):17334. 10.1038/s41598-024-67192-6

[47]

Kermanizadeh A, Brown DM, Stone V. The variances in cytokine production profiles from non- or activated THP-1, Kupffer cell and human blood derived primary macrophages following exposure to either alcohol or a panel of engineered nanomaterials. PLOS ONE. 2019;14(8): Article e0220974. 10.1371/journal.pone.0220974

[48]

10.1016/j.stemcr.2021.11.002

[49]

Holmgren G, Ulfenborg B, Asplund A, Toet K, Andersson CX, Hammarstedt A, Hanemaaijer R, Küppers-Munther B, Synnergren J. Characterization of human induced pluripotent stem cell-derived hepatocytes with mature features and potential for modeling metabolic diseases. Int J Mol Sci. 2020;21(2): Article 469. 10.3390/ijms21020469

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References

Details

Published: Jan 01, 2025
Vol/Issue: 6

Authors

Z

Zhengxiang Huang

School of Medicine and Dentistry, Griffith University, Gold Coast, QLD 4222, Australia.; Institute for Biomedicine and Glycomics, Griffith University, Southport, QLD 4222, Australia.; School of Biomedical Sciences, University of Queensland, St Lucia, QLD 4072, Australia.

L

Lili Li

School of Medicine and Dentistry, Griffith University, Gold Coast, QLD 4222, Australia.; Nanjing Stomatological Hospital, Affiliated Hospital of Medical School, Research Institute of Stomatology, Nanjing University, Nanjing, China.

K

Kevin Dudley

Central Analytical Research Facility, Queensland University of Technology, Brisbane, QLD 4000, Australia.

L

Lan Xiao

School of Medicine and Dentistry, Griffith University, Gold Coast, QLD 4222, Australia.; Institute for Biomedicine and Glycomics, Griffith University, Southport, QLD 4222, Australia.

G

Gary Huang

Hepatogenomics Research Group, Centre for Genomics and Personalised Health, School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD 4059, Australia.

V

V. Nathan Subramaniam

Hepatogenomics Research Group, Centre for Genomics and Personalised Health, School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD 4059, Australia.

C

Chen Chen

School of Biomedical Sciences, University of Queensland, St Lucia, QLD 4072, Australia.

R

Ross Crawford

Orthopaedic Research Unit, School of Mechanical, Medical & Process Engineering, Faculty of Engineering, Queensland University of Technology, Brisbane, QLD 4000, Australia.; Centre for Biomedical Technologies, Queensland University of Technology, Brisbane, QLD 4000, Australia.

Y

Yin Xiao

School of Medicine and Dentistry, Griffith University, Gold Coast, QLD 4222, Australia.; Institute for Biomedicine and Glycomics, Griffith University, Southport, QLD 4222, Australia.

Funding

National Health and Medical Research Council Award: APP2000647

Cite This Article

Zhengxiang Huang, Lili Li, Kevin Dudley, et al. (2025). Three-Dimensional Dynamic Cell Models for Metabolic Dysfunction-Associated Steatotic Liver Disease Progression. BME Frontiers, 6. https://doi.org/10.34133/bmef.0181

Three-Dimensional Dynamic Cell Models for Metabolic Dysfunction-Associated Steatotic Liver Disease Progression

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