In Vivo and In Vitro Models to Study Liver Fibrosis: Mechanisms and Limitations

Abstract

Liver fibrosis is a common result of liver injury owing to various kinds of chronic liver diseases. A deeper understanding of the pathophysiology of liver fibrosis and identifying potential therapeutic targets of liver fibrosis is important because liver fibrosis may progress to advanced liver diseases, such as cirrhosis and hepatocellular carcinoma. Despite numerous studies, the underlying mechanisms of liver fibrosis remain unclear. Mechanisms of the development and progression of liver fibrosis differ according to etiologies. Therefore, appropriate liver fibrosis models should be selected according to the purpose of the study and the type of underlying disease. Many in vivo animal and in vitro models have been developed to study liver fibrosis. However, there are no perfect preclinical models for liver fibrosis. In this review, we summarize the current in vivo and in vitro models for studying liver fibrosis and highlight emerging in vitro models, including organoids and liver-on-a-chip models. In addition, we discuss the mechanisms and limitations of each model. Liver fibrosis is a common result of liver injury owing to various kinds of chronic liver diseases. A deeper understanding of the pathophysiology of liver fibrosis and identifying potential therapeutic targets of liver fibrosis is important because liver fibrosis may progress to advanced liver diseases, such as cirrhosis and hepatocellular carcinoma. Despite numerous studies, the underlying mechanisms of liver fibrosis remain unclear. Mechanisms of the development and progression of liver fibrosis differ according to etiologies. Therefore, appropriate liver fibrosis models should be selected according to the purpose of the study and the type of underlying disease. Many in vivo animal and in vitro models have been developed to study liver fibrosis. However, there are no perfect preclinical models for liver fibrosis. In this review, we summarize the current in vivo and in vitro models for studying liver fibrosis and highlight emerging in vitro models, including organoids and liver-on-a-chip models. In addition, we discuss the mechanisms and limitations of each model. SummaryBecause the development and progression of liver fibrosis differ based on the etiology, it is important to select an appropriate liver fibrosis model according to the purpose of study and type of disease. To study liver fibrosis, many in vivo animal and in vitro models have been developed. This review summarizes and analyzes the various in vivo and in vitro liver fibrosis models and their implications and limitations. Because the development and progression of liver fibrosis differ based on the etiology, it is important to select an appropriate liver fibrosis model according to the purpose of study and type of disease. To study liver fibrosis, many in vivo animal and in vitro models have been developed. This review summarizes and analyzes the various in vivo and in vitro liver fibrosis models and their implications and limitations. Liver fibrosis is fibrous scar formation by extracellular matrix (ECM) accumulation resulting from chronic liver inflammation caused by conditions including chronic viral hepatitis B and C, autoimmune hepatitis, alcoholic liver disease (ALD), primary biliary cholangitis, primary sclerosing cholangitis (PSC), and nonalcoholic fatty liver disease (NAFLD).1Friedman S.L. Liver fibrosis–from bench to bedside.J Hepatol. 2003; 38: S38-S53Abstract Full Text Full Text PDF PubMed Google Scholar,2Bataller R. Brenner D.A. Liver fibrosis.J Clin Invest. 2005; 115: 209-218Crossref PubMed Google Scholar Liver fibrosis may progress to cirrhosis and further to hepatocellular carcinoma (HCC).3Lim Y.S. Kim W.R. The global impact of hepatic fibrosis and end-stage liver disease.Clin Liver Dis. 2008; 12 (vii): 733-746Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar Treating underlying liver disease may ameliorate liver fibrosis, even at the cirrhosis stage.4Arthur M.J. Reversibility of liver fibrosis and cirrhosis following treatment for hepatitis C.Gastroenterology. 2002; 122: 1525-1528Abstract Full Text Full Text PDF PubMed Google Scholar,5Chang T.T. Liaw Y.F. Wu S.S. et al.Long-term entecavir therapy results in the reversal of fibrosis/cirrhosis and continued histological improvement in patients with chronic hepatitis B.Hepatology. 2010; 52: 886-893Crossref PubMed Scopus (791) Google Scholar Although many targets for liver fibrosis were investigated, effective medications have not been developed.6Zhang J. Liu Q. He J. Li Y. Novel therapeutic targets in liver fibrosis.Front Mol Biosci. 2021; 8: 766855Crossref PubMed Scopus (10) Google Scholar Therefore, exploring the precise pathophysiologies of liver fibrosis is crucial for better understanding and for discovering new therapeutic targets. Various animal models are used in preclinical studies on liver fibrosis. Because no model is perfect, the selection of relevant animal models to study target fibrotic diseases is crucial. A better understanding of the pathogenesis of each animal model and its implications and limitations is essential. In this review, we summarized the currently available in vivo and in vitro models for studying liver fibrosis and discussed emerging in vitro models. In vivo animal models are the gold standard in studying liver fibrosis. The main effectors that produce ECM in liver fibrosis are activated hepatic stellate cells (HSCs). Various types of liver cells are involved in HSC activation in liver fibrosis. These cells include immune cells (monocyte-derived macrophages, Kupffer cells, B cells, and T cells), cholangiocytes, liver sinusoidal endothelial cells (LSECs), and hepatocytes. These cells produce inflammatory and fibrotic cytokines (transforming growth factor-β [TGF-β], platelet-derived growth factor [PDGF], CTGF, interleukin 1β, and C-C motif chemokine ligand 2) and mediators (reactive oxygen species and nitric oxide) to affect HSC activation (Figure 1A). Because various cell types are involved in HSC activation and fibrosis, simple in vitro cell culture models cannot entirely recapitulate the disease course of liver fibrosis. To study different etiologies of liver fibrosis, hepatotoxin, cholestasis, and nonalcoholic steatohepatitis (NASH)-induced liver fibrosis models are commonly used (Table 1), and each model shows different patterns of fibrosis (Figure 1B).Table 1In Vivo Animal Models of Liver FibrosisModelsMethodsDurationAdvantagesLimitationsReferencesCCl4IP injectionInhalation4–12 wkSimplicity (IP)High reproducibility, induction of portal hypertension (inhalation)High toxicityRisk of peritonitis (IP injection)Requirement of special equipment (inhalation)11–13TAAIP injectionOral administration6–8 wk (IP injection)2–4 mo (oral)SimplicityHigh reproducibilityLong time to induce liver fibrosisHighly toxic15, 16DMNIP injection4–8 wkSimplicityInducing significant fibrosisMore suitable to study HCCRisk of carcinogenesis in researcher17, 18HFDFeeding2–6 mo (for fatty liver)50 wk (for fibrosis)Inducing obesity and insulin resistanceLong time to induce liver fibrosis27HFD with glucose/fructose water ± cholesterolFeeding16 wk to 12 moSignificant feature of metabolic syndromeLong time to induce liver fibrosis29, 30, 32, 33MCD dietFeeding5–8 wkShort time to induce fibrosisReduces body weightNo feature of metabolic syndrome38, 39CDAA dietFeedingMore than 20 wkBody weight gain with insulin resistanceUseful to study NASH-induced HCCHindrance of studying liver fibrosis by HCC development40CD-HFDFeeding6–24 wkHuman-relevant NASH fibrosisLong time to induce liver fibrosis41, 43, 46, 47CDA-HFDFeeding12 wkNASH with severe fibrosis with short durationNo feature of obesity or insulin resistance49BDLSurgery3 wkRelevant to cholestasisUseful to study relationship between gut microbiome and fibrosisHigh surgery skill level requiredHigh mortality rate52–56DDC dietFeeding4–8 chronic chronic at in the of and development of liver of studying liver fibrosis by HCC of to with water or wk wk to mo for studying alcoholic liver to induce liver for to induce hepatocellular on and primary biliary growth in a new Models of Liver from and such as in vivo from to of the in activated of from primary by with of to or with of activated of from culture with various or from or liver or cell of of cells in with of vitro culture with liver for to culture cells on a of a liver liver and relevant of in a new hepatocellular on and primary biliary growth is the used for liver fibrosis models. is to and by and endothelial J. et models of liver PubMed Scopus Google and of of as a 2003; PubMed Google of by Biosci. 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