Table 2.

Human multi-cellular 3D MAFLD models.

Key Approach	Cell Culture Method	Major Features
Self-organizing liver organoid models	Step-wise differentiation from pluripotent stem cells (PSC)	PSC-derived organoids that are composed of multiple cell types, including hepatocyte-like cells, Stellate-like cells, and Kupffer-like cells [50]. Organoids exhibit steatosis, inflammation, and fibrosis response upon free fatty acid (FFA) treatment [50]. Increased organoid stiffness recapitulated in vivo liver fibrogenesis event and was employed for drug response study [50]. Inter and Intra batch variability observed. Further characterization of biochemical changes in cells during FFA treatment is required [50].
		PSC-derived liver epithelial organoids that are expandable and can differentiate into hepatocytes [126]. Epithelial organoid-derived hepatocytes readily take up FFA and accumulate lipid droplets, enabling the testing of various drugs for reducing steatosis [126].
		PSC-derived organoids that are primarily composed of hepatocytes at the core and cyst-forming cholangiocytes in the peripheral [47]. Structural features in liver organoid enabled modeling of tissue architecture changes in the liver during MAFLD progression, including bile canaliculi network disruption and ductular reaction [47]. Organoids lack non-parenchymal cell types, which limits modeling of inflammation and fibrogenesis [47].
	Co-culture of parenchymal and non- parenchymal liver cell types	Co-culture of different hepatic and non-hepatic cells to form 3D spheroid cultures in suspension [154,155,156] or matrices [157]. The majority of these co-cultured spheroids do not recapitulate liver tissue structure [154,155,156,157]. The inclusion of fibroblast and stellate cell lines enable the modeling of fibrogenesis event, and the inclusion of Kupffer cells allow the modeling of inflammatory events [154,155,156,157]. The direct co-culture of mature functional cell types enabled better control of cell type proportions to achieve higher homogeneity and reproducibility of organoids for quantitative applications, especially in drug testing [154,155,157]. This approach enables genetic manipulation of selected cell populations before co-culture to enable cell-type specific targeting.
Bio-engineered liver models	Microfluidics culture	Culture composed of largely immortalized hepatic cells [79,158] or primary hepatocytes [159]. Microfluidics enabled cell culture with circulation to mimic vascular flow [79,158,159]. The system facilitates the continuous exchange of molecules, including nutrients and metabolites, which mimics physiological conditions during MAFLD development. The introduction of vascular flow enhances cellular function compared to static cultures [79,158,159]. The media flow also improves cell viability in the core of hepatic spheroid [79].
		The control of system parameters achievable with the microfluidics platform enables the seeding of multiple cell types together [51,160,161,162], or in separate chambers [162] within a chip. The use of microfabrication techniques enables precision placement of cells [162]. Separation of cells in chambers with porous walls mimics the vascular system. It also enables the layering of cells to achieve a similar spatial arrangement of cells observed in the liver tissue [163]. Incorporation of PSC differentiation approach in microfluidics platform enables generation of organoids-on-a-chip [51]. Manipulation of chip configuration enables recapitulation of liver lobule distribution of cells to mimic in vivo tissue organization [160]. Limited throughput and the requirement of specialized equipment and techniques limit the wide adoption of microfluidics platforms.
	Precision Cut Tissue Slice (PCTS)	PCTS enables the direct use of patient tissue from biopsy for drug response studies [164]. PCTS maintains intact cellular interactions and organizations observed in MAFLD liver tissues, which may be favorable for drug response study [164]. The short-term culture may also enable the capture of host-pathogen interactions that may influence drug treatment response [164]. Limited application due to short culture period (only up to 5 days) and availability of human tissue [164].
	Organ scaffolds	Co-culture of iPSC-derived HLC, HUVEC, mesenchymal stromal cells, fibroblast, and blood-derived macrophages in decellularized liver tissue [165]. One of the largest centimeter-size liver organoids cultured. Authors employ a peristaltic pump system to deliver nutrients to the core of the tissue. This enhanced the viability of cells and penetration of MAFLD phenotype throughout the tissue [165]. The tissue-like culture exhibit MAFLD hallmarks which enabled steatosis, inflammation, and ballooning scoring comparable to patient tissue [165].