TABLE 1.

Benefits, limitations, and validated utility of engineered primary human liver platforms for drug metabolism studies

Model	Benefits	Potential Limitations	Drug Metabolism Applications (Primary Human Liver Cells)
Cellular microarrays	Can be used to evaluate the metabolism of many drugs simultaneously in a small footprint	Primarily dependent on imaging-based readouts when using cells	These arrays have so far been adapted to cancerous hepatic cell lines and application to primary liver cells is pending (Lee et al., 2005, 2008; Yu et al., 2018)
	Low novel drug/chemical usage	Current array configurations are limited in their ability to interrogate responses of adhered cells to molecular gradients
	Dual advantage with the ability to evaluate the effects of combinatorial biochemical and biophysical signals on enhancement of liver cell functions.
Conventional cocultures (random distribution)	No specialized systems/equipment is needed to create randomly distributed cocultures in standard multiwell culture plates	Function of hepatocytes is highly dependent on the choice of the nonparenchymal support cell type	Measured activities of NAT2, UGT1A1, SULT, AKR, AO, FMO, CYP1A2, CYP2B6, CYP2C9, CYP2D6, and CYP3A4 after 8 days of culture (Kratochwil et al., 2017)
		Part of the randomly distributed cocultures can display morphologic and functional instability due to suboptimal (random) cell-cell contact/interactions	Measured activities of CYP2D6 and CYP3A4 for 14 days (Novik et al., 2017)
		Typically, have lower activities of some drug metabolism enzymes than micropatterned cocultures
Micropatterned cocultures (MPCCs)	Controlled homotypic hepatocyte interactions on micropatterned protein domains allow for proper cell polarity and higher/stable liver functions in coculture with fibroblasts for 4–6 weeks	Specialized masks created using lithographic techniques are needed to pattern ECM proteins (e.g., collagen) to enable subsequent clustering of the hepatocytes	Measured activities of NAT2, UGT1A1, SULT, AKR, AO, FMO, CYP1A2, CYP2B6, CYP2C9, CYP2D6, and CYP3A4 after 8 days of culture (Kratochwil et al., 2017)
	Available in industry standard multiwell formats (up to 384-well plates)	A single configuration containing all major liver cell types is currently lacking	Measured activities of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2E1, CYP3A4, UGT, and SULT for 30 days (Khetani and Bhatia, 2008; Lin et al., 2016)
	Diverse types of liver NPCs can be cultured in MPCCs without significantly affecting the homotypic interactions and polarity of hepatocytes on the micropatterned colonies	Use nonliver fibroblasts for inducing optimal functions in hepatocytes	Drug metabolite detection (Wang et al., 2010; Ballard et al., 2016)
			Drug clearance prediction (Chan et al., 2013; Lin et al., 2016)
			DDIs including P450 induction and inhibition (Khetani and Bhatia, 2008; Lin et al., 2016; Kratochwil et al., 2018)
			Transporter, metabolism, and/or P450 induction interplay on drug disposition (Ramsden et al., 2014a; Moore et al., 2016)
Self-assembled Spheroids	Many off-the-shelf plate formats are available for the creation of spheroids	Cellular necrosis can occur in the center of spheroids if their diameter exceeds 250–300 µm	Measured activities of CYP1A2, CYP2C8, CYP2C9, CYP2D6, and CYP3A4 for 35 days (Bell et al., 2016, 2018)
		Some plate formats can cause large variations in the sizes of the spheroids, which leads to nonuniform viability and function
		Single spheroids may not provide enough material for sensitive drug metabolite identification
		Heterogeneous cell distribution without any defined architecture
Bioprinted spheroids/organoids	Printing head allows control over the placement of cells in different locations/compartments (e.g., hepatic, vascular, and other nonparenchymal cell compartments)	Microscale printing resolution for control of single cell placement is currently lacking	Measured activity of CYP3A4 for 28 days (Norona et al., 2016)
	Software programming allows on-demand 3D architectures to the created with same instrumentation configuration (i.e., expensive masks are not needed as for lithographic techniques)	Low-throughput creation of the cultures
		The method requires complex and often expensive instrumentation with the need for well-trained technologists
		The method requires a larger number of cells than microscale patterning methods using lithography
		Printed tissues on the millimeter to centimeter scale can potentially display necrosis in the core of the tissues in the absence of a connected/perfused vasculature
Perfused liver culture/coculture platforms (some are also known as liver-on-a-chip)	Enable fluid flow to allow automated nutrient and waste exchange as well as the generation of in vivo-like molecular gradients (e.g., oxygen), which can lead to zonated hepatic phenotypes	Perfusion requires specialized fluid pumps and control systems	Measured activities of CYP2D6 and CYP3A4 for 6 days of culture (Novik et al., 2010)
	Many microfluidic perfusion devices are now commercially available for the creation of perfused liver cultures	Drugs can potentially bind and be sequestered by the tubing and materials used in the perfusion devices	Measured activities of CYP2C9, CYP3A4, and UGT for 14 days (Vernetti et al., 2016)
		Due to the need for tubing, perfusion devices have a large dead volume, which can necessitate higher quantities of the novel (and often very limited) compounds for treating cell cultures	Drug clearance prediction (Dash et al., 2009; Novik et al., 2010)
		Most devices are currently low-throughput, allowing up to 12 devices to be perfused at a single time	Drug metabolite identification (Sarkar et al., 2017)

AKR, aldo-keto reductase; FMO, flavin monooxygenase; NAT2, N-acetyltransferase 2; SULT, sulfotransferase; UGT1A1, UDP glucuronosyltransferase 1A1.