Table 1.

Human primary and immortalized hepatic cells used for modelling MAFLD.

Cell Type	Cell Lines	Major Features
Immortalized Cell lines	HepG2	One of the earliest in vitro cell models for recapitulating MAFLD development in hepatocytes [43]. Treatment with unsaturated free fatty acids (FFA) such as oleic acid induces hallmarks of early MAFLD development, including elevation of intracellular triglyceride levels, lipid micro-vesicle and macro-vesicle formation, increased lipid peroxidation, and reduced cell viability [43,44,69,72,73,74]. Treatment with saturated FFAs such as palmitic acid further enhances lipid accumulation, changes in oxidative phosphorylation, and increased cell apoptosis and ER stress responses [44,73,74]. The ease of manipulating immortalized cell lines facilitates loss and gain of function studies to unravel mechanisms of drug response and MAFLD development [75]. Cancer origin of cells and molecular changes introduced by the immortalization process are concerns for physiological relevance of such cell line models.
	Huh7	Huh7 accumulates a much higher level of triacylglycerols (TAGs) compared to HepG2 when exposed to bovine or human Serum, highlighting the diverse fatty acid metabolic activity across different cell lines [69,76].
	WRL68	Cells treated with FFAs develop similar steatosis phenotypes in comparison with HepG2 [77].
	HepaRG	Cells exhibit greater sensitivity to drug-induced steatosis compared to HepG2 [78]. Cells treated with FFAs develop similar steatosis phenotypes compared to HepG2 [79].
Tissue-derived primary cells	Primary Hepatocyte (PHH)	PHH derived from human liver tissue remains the most physiologically relevant hepatocyte cell model. However, usage in the modeling of human liver diseases has been limited due to donor availability. Immortalized PHH could potentially provide a renewable source of human hepatocytes for MAFLD studies [80]. MAFLD phenotypes could be induced with treatment using FFA (oleic acid, palmitic acid, and stearic acid) and fructose [41,44,45,70,80,81,82]. Conditioned media from hepatocytes treated with FFA with and without fructose can induce fibrogenic responses in hepatic stellate cells [45,70]. 3D spheroid culture of PHH improves hepatocyte function and maintenance compared to 2D culture and is favorable for chronic MAFLD modeling [41,82]. Hepatocytes from donors harboring previously reported TM6SF2 E167K genetic variant exhibit increased lipid accumulation under FFA treatment compared to other donors [82]. This study highlights the value of patient-derived hepatocytes in evaluating genetic risk variants identified in genome-wide association studies.
	Bipotent ductal stem cells	Derivation of bipotent ductal stem cells from NASH patient tissue biopsies using organoid culture platform [46]. Upregulation of pro-inflammatory pathway genes, cytochrome p450-related pathways genes, and genes associated with fibrogenesis and tumorigenesis in specific NASH patient-derived ductal organoids [46]. Differentiated NASH patient-derived organoids exhibit enhanced NASH phenotypes compared to healthy controls [46]. Patient-specific idiopathic response, similar to MAFLD studies using PHH models, was observed [46].
Pluripotent stem cells (PSC)-derived primary cells	Hepatocyte-like cells (HLC)	Induced PSC (iPSC) technology enables the establishment of patient-specific MAFLD models for precision therapeutic studies [19,49]. HLCs generated from NASH patient-derived iPSC express disease signatures observed in patient tissues [49]. The ease of genetic manipulations of iPSC and capturing of patient-specific genotypes facilitate the generation of human genetic MAFLD models [19,49]. Self-renewing PSCs enable the generation of large numbers of primary HLC cells for downstream molecular profiling [19,48,49,83,84], and drug screening experiments [83]. Fetal nature of PSC-derived cells remains a concern for physiological relevance.