Table 1.

In vitro cell lines that have been used to investigate NAFLD, highlighting their advantages and disadvantages

Cell line	Description	Advantages	Disadvantages	References
HepG2	Human hepatocellular carcinoma cell line	–Cheap –Easy to maintain –Uptake and storage of exogenous FA –Uptake of lipoprotein remnants	–Low drug-metabolizing capacity –Rapid dedifferentiation –Transcript differences with human hepatocytes –Fetal phenotype –Cancer origin/phenotype –Low FA oxidation –PNPLA3 I148M mutation –Mainly LDL secretion	Green et al. (2015, 2020); Gunn et al. (2017); Jennen et al. (2010); Lőrincz et al. (2021); Srivastava and Chan (2008)
HepG2/C3A	Clonal derivative of HepG2	–Closer phenotype to human hepatocytes –Growth on glucose deficient medium –In liver spheroids, high sensitivity to drug-induced liver injury	–Strong contact inhibition of growth –Capable of growing on free-glucose medium –Cancer origin/phenotype –Lower metabolism than HepG2 –Low basal expression of CYP2E1	Baquerizo et al. (2015); Flynn and Ferguson (2008); Garcia et al. (2011); Gaskell et al. (2016)
HepaRG	Human hepatocellular carcinoma cell line	–Highly differentiation into hepatocytes –Expression of several CYP and phase II enzymes –Uptake and storage of exogenous FA –Secretion of lipoproteins –No PNPLA3 mutation	–Low levels of CYP2D6 and CYP2E1 –Cancer origin/phenotype –Overexpression of CYP3A4 –No bile collection	German and Madihally (2019); Green et al. (2020); Jennen et al. (2010); Lőrincz et al. (2021)
L02	Human fetal liver cell line	–Easy culture –Functional similarities with hepatocytes	–Fetal phenotype	Hu et al. (2013)
Huh7	Human hepatocellular carcinoma cell line	–Lipolytic enzyme expression –Effective CYP3A4 activity when confluent	–Fetal phenotype –Cancer origin/phenotype –Low FA oxidation on glucose –Steatosis overestimation	Green et al. (2020); Gunn et al. (2017)
Hepa1c1c7	Mouse hepatocellular carcinoma cell line	–High induction of CYP1A1 –Suitable for study of chemoprotective enzymes by organic selenium-containing compounds	–Cancer origin/phenotype	El-Sayed et al. (2007)
AML-12	Mouse liver cell line	–Contains human transgenic TGF-α –High expression of gap junction proteins	–Expression of liver-specific proteins decreases in long-term	Wu et al. (1994)
Hepa1-6	Mouse carcinoma cell line	–Suitable for drug evaluation –Favourable immune profile	–Fetal phenotype –Cancer origin/phenotype	Kachlishvili et al. (2020); Lacoste et al. (2017); Urs (2018)
RAW264.7	Mouse monocyte/macrophage-like cells	–Easy expansion in culture –High efficiency for DNA transfection –Sensitivity to RNA interference	–Genotypic and phenotypic drifts due to repeated passaging (until passage no. 30)	Hartley et al. (2008); Taciak et al. (2018)
THP-1	Human monocytic cell line	–High growth rate –High reproducibility	–Lack of surface and cytoplasmic immunoglobulin –Dependent on M-CSF or similar for differentiation into macrophages –Genotypic and phenotypic drifts due to repeated passaging (until passage no. 25) –Differences with human phenotype	Chanput et al. (2014)
Bone marrow-derived macrophages	Mouse primary macrophage cells	–Homogeneous distribution –High proliferative capacity –Transfectable	–Dependent on M-CSF or similar for differentiation into macrophages –Shorter life-span	Weischenfeldt and Porse (2008)
LX-2	Hepatic stellate cells (HSCs) from normal liver	–Activated phenotype with fibroblast-like appearance –Inducible retinoid metabolism –High transfectability –Growth in low-serum conditions	–Low-serum/basement matrix required for quiescent phenotype –Genotypic and phenotypic drifts due to repeated passaging (until passage no. 50)	Vande Bovenkamp et al. (2007); Weiskirchen et al. (2013); Xu et al. (2005)
T6 stellate cells	Rat primary HSCs from liver cell line	–Inducible retinoid metabolism –Morphologic and proliferative characteristics from activated HSCs –Form cytosolic lipid droplets	–Retinoid protein expression and processing similar to quiescent cells –Low-serum/basement matrix required for quiescent phenotype	Van de Bovenkamp et al. (2007); Vogel et al. (2000); Xu et al. (2005)
Primary cell lines	–Primary hepatocytes –Primary KC –Primary HSC	–Isolation from NAFLD/NASH patients is possible –Interindividual variation studies are possible –FA oxidation –High functionality –Human metabolism is reproduced	–Difficult isolation –Low availability –Lose differentiation in long-term –Lose proliferation in long-term –Difficult reproducibility	Brandon et al. (2003); Green et al. (2020); Wilkening et al. (2003)
3T3-L1 MBX	Mouse fibroblast	–Adipocyte differentiation –Robust response to insulin –Present lipid droplets	–Low proliferation rate	ATCC; Berger and Géloën (2019)
3T3-J2	Embryonic mouse fibroblast	–Expression of genes present in liver –Use in multicellular aggregates to enhance liver functions –Stabilisation of liver phenotype without NPC is possible	–Non-liver source –Fibroblast density modulates hepatic function in dose-dependent manner	Bhatia et al. (1998); Ware et al. (2021)
Liver sinusoidal endothelial cells (LSEC)	Primary human cell line	–High endocytosis capacity –High permeability –Long life-span	–Low availability –Difficult isolation –Low proliferation rate –Rapid loss of differentiation	German and Madihally (2019); Poisson et al. (2017)
Human umbilical vein endothelial cells (HUVEC)	Human non-pathological tissue	–Generation of vascular-like endothelial network when cultured with hepatocytes in spheroids –Easy access –Established protocols for tissue culture in a serum-free medium	–Non-liver source –Less migration of lymphocytes than LSEC –Reduced viability when co-cultured with HepaRG than LSEC	German and Madihally (2019); Medina-Leyte et al. (2020)
Caco-2	Human epithelial cells from colorectal adenocarcinoma	–Spontaneous change in enterocytic characteristics at confluence –Tight junctions between cells	–Genotypic and phenotypic drifts due to repeated passaging –Presence of multilayer zones	Briske-Anderson et al. (1997); Poncede León-Rodríguez et al. (2019)
NCTC-1469	Mouse NPC from liver cell line	–Expression of estrogen receptors		Asahi et al. (2010)
Pluripotent stem cells (PSC)	Stem cells that can differentiate into endoderm, mesoderm or ectoderm	–Differentiation into desired cell type –Similar phenotype to primary cell lines –Unlimited culture time	–Fetal phenotype –No standardized differentiation protocol –Epigenetic memory may prevent differentiation –Differentiation is time consuming –Cost	Soret et al. (2021); Zeilinger et al. (2016)