Table 1.

Summary of HBV in vitro hepatocyte culture models

Classification	Cell line	Advantages	Shortcomings	HBV infection rate and application of the models
HBV replication cell lines	(1) HepG2.2.15 cells	cccDNA accumulation Stable and continuous HBV gene expression and replication	Low viral replication level Antigen expression instability Virions are produced from the integrated DNA	Screening and evaluation of antiviral drugs, etc. [90].
	(2) HepAD38 (EF9,EFS19) cells	Cells differentiate quickly Produce high titers of viral particles cccDNA accumulation Hepatoma cells stably expressing HBV from a Tet-on/Tet-off system	Incomplete viral life cycle Virions are produced from the integrated DNA	Screening and evaluation of antiviral drugs, etc. A potential source for tissue culture derived virions [91].
	(3) Ad-HBV1.3-systems	No species barrier Efficient expression of HBV HBV expression and mutation can be controlled Direct observation of transfection and infection efficiency (integrated green fluorescent protein gene)	Missing HBV natural infection stage	Used to establish animal models of acute hepatitis B infection [92].
	(4) HBV baculovirus system	Easy detection of riboprotein-bound HBV DNA High HBV replication level Formation of infectious viruses and a detectable intracellular cccDNA pool	Nonreceptor-mediated entry Gene transfer is restricted to certain species Missing HBV natural infection stage	Quantify the effect of antiviral agents on nuclear HBV DNA Used for studying the resistance of HBV to nucleoside analogs [93].
Cell lines that can be infected with HBV	(1) Human fetal hepatocytes	Phenotypically and biologically functionally close to primary adult human hepatocytes	Low infection efficiency Short infection time Limited availability Large donor-donor variations	HBV infection rate12%-90% [22, 94]. Coculturing with hepatic non-parenchymal cells and subsequent addition of 2% DMSO leads to the formation of hepatocyte islands with prolonged phenotypic maintenance [25]. The early events in viral entry into cells as well as viral replication [23].
	(2) Adult human hepatocytes	The gold standard host cell to HBV infection experiments Closest to the physiological characteristics of hepatocytes in vivo Close to the natural process of infection	Limited life cycle Unpassable culture Phenotypically unstable in vitro Rapidly lose permissiveness for HBV infection Large donor-donor variations	HBV infection rate 20%-100% [26, 28]. Used for studying the process of HBV infection [5, 28]. Studying on apoptosis [26]. Preparation of 3D primary hepatocyte culture system for analyses of liver diseases, drug metabolism, and toxicity [40, 41].
	(3) Co-culture system	Test the utility of various direct-acting antivirals (DAAs) and putative host-targeting antivirals (HTAs); Assessing preclinically the efficacy of other entry inhibitors and possibly (vaccine-induced) neutralizing antibodies;	Wide variability between donors in terms of HBV permissiveness	Inflammation and drug-Induced Hepatotoxicity [95].
	(4) Primary Tupaia hepatocytes	The only species susceptible for HBV infection besides humans and chimpanzees	Expensive	HBV infection rate >70% [52]. Used for in vitro as well asin vivo infection experiments [96]. HBV specific receptor identification [78].
	(5) HepaRG cells	Preserve the specific functional properties of hepatocytes Support the complete HBV life cycle Produce HBV cccDNA	Strict culture conditions Low infection efficiency	HBV infection rate <30% [56, 78]. HBV molecular mechanism and screening, evaluation of anti-HBV drugs; cccDNA spread etc. [57]. Drug metabolism and toxicity [58, 59].
	(6) In vitro systems based on induced pluripotent stem (iPS) cell-derived human hepatocytes	Biological characteristics similar to those of normal liver cells Support the complete life cycle of the virus Complete natural immune system	Complicated operation	HBV infection rate 25% [97]. Drug hepatotoxicity screening [98]. The life cycle of HBV virus and virus-induced hepatic dysfunction [66].
	(7) NTCP overexpressing hepatoma cell lines	Support the complete life cycle of the virus Flexibility and easy handling	Low susceptibility to serum-derived HBV The multiplicity of infection (MOI) needed to achieve infection is extremely high No substantial viral spreading following infection	HBV infection rate 50% [99]. Large-scale screening of antiviral drugs for targeting NTCP [91].