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He, et al.
and assembled into complex heterogeneous architectures , co-printing with human umbilical vein endothelial
[2]
and abundant cell-cell and cell-extracellular matrix cells and adipose-derived stem cells, the phenotype and
(ECM) interactions have been established in bioprinted biofunction of hiPSC-HPCs were found to be enhanced in
cell-laden structures. These characteristics could meet long-term culture. In addition, spheroid-based bioprinting
the demands of liver tissue engineering. Hepatocytes, as has been shown to possess unique advantages in terms
parenchymal cells of the liver, are attachment-dependent of biological characteristics of the spheroids . Higher
[16]
and require close interactions with other liver cells and cell viability and better maintenance of hepatic function
within specific hepatic microenvironments . The rapid have been shown in bioprinted hepatoblast spheroids
[3]
loss of the normal phenotype and biological function compared to bioprinted hepatoblasts using single-cell
of hepatocytes occurs in conventional two-dimensional dispersion. However, most current studies use midterm-
(2D) cultures; however, hepatic tissue models created differentiated hepatoblasts or HPCs in bioprinting, which
by 3D bioprinting have been shown to better facilitate are involved in long-term, post-printing differentiation.
the phenotype restoration and function preservation of Methods for in vitro differentiation into hepatocytes
hepatocytes, showing great superiority over 2D cultures . through bioprinting remain relatively underdeveloped.
[4]
To date, bioprinted hepatic tissue models have been The bioprinting process and bioink components could
widely used for disease modeling [5,6] , drug screening , potentially affect the differentiation efficiency of the
[7]
hepatotoxicity evaluation [8-10] , and tissue regeneration [11,12] . cells, which needs to be optimized and standardized for
Hepatocytes used in bioprinting mainly include successful bioprinting, and fully matured hepatocytes
hepatocarcinoma cell lines, primary human hepatocytes are required to construct hepatic tissue models for drug
(PHHs) [11,13] , and hepatocytes derived from human- screening and toxicological studies . In our previous
[25]
induced pluripotent stem cells (hiPSCs) [14-16] . Among study, we developed an effective differentiation system
hepatocarcinoma cell lines, HepG2 [8,17-19] , Huh-7 , to generate large quantities of mature hepatocytes from
[20]
and HepaRG [5,9,12] are commonly used in hepatic tissue hiPSCs . Therefore, we considered maturating hiPSC-
[26]
models; however, these hepatoma cells cannot adequately HPCs based on our well-developed differentiation
represent typical hepatocytes because of a deficiency in protocol and then applied functional hepatocytes in
liver-specific functions . PHHs isolated from native the bioprinting of hepatic tissue models. hiPSC-Heps
[21]
liver tissue express excellent biofunctions but are obtained using our optimized protocol showed excellent
difficult to culture and expand in vitro. hiPSC lines are expression of liver-specific functions similar to PHHs,
capable of self-renewal. They can be expanded in vitro and they were successfully applied in bioartificial liver
on a large scale and converted into hepatocyte-like cells systems for acute liver failure treatment [26,27] .
through certain differentiation processes [22,23] . At present, In this study, we bioprinted hiPSC-Heps using
most of the hepatocyte-like cells derived from hiPSCs an alginate-gelatin bioink to construct a hepatic tissue
retain a relatively immature phenotype and express model. We evaluated the success of cell growth, liver-
limited metabolic functions compared to PHHs . specific function, and drug-induced hepatotoxicity of the
[24]
Nonetheless, hiPSCs could be a promising renewable 3D-printed (3DP) model compared with the conventional
and easily accessible cell source for the generation of 2D-cultured (2D) and the non-printed sandwich-cultured
functional hepatocytes which may obtain full maturity (SW) models. The results of this study demonstrated the
with the application of future biotechnologies. When feasibility of hiPSC-Heps bioprinting using an alginate-
applied in the bioprinting of hepatic tissue models using gelatin bioink and confirmed that using the 3DP model
proper printing techniques and bioinks, hiPSC-derived showed biofunctional superiority, thus providing potential
hepatocytes (hiPSC-Heps) have demonstrated well- applications in the prediction of drug-induced liver injury.
maintained cellular phenotypes and biofunctions .
[25]
Bioprinting of hiPSCs and human embryonic stem cells 2. Materials and methods
was first reported in 2015 by Faulkner-Jones et al. The 2.1. Cell culture and bioink preparation
[14]
cells were bioprinted using RGD-coupled alginate bioink
and then were further differentiated into hepatocyte-like The cell differentiation timeline from hiPSCs to hiPSC-
cells. During the differentiation process after printing, Heps is shown in Figure 1A. hiPSC-Heps were obtained
the cells retained pluripotency with positive hepatocyte according to the manufacturer’s protocol , in which,
[27]
nuclear factor 4 alpha expressions, and they displayed hiPSCs were cultured in RPMI1640 medium with a
biological functions and cellular morphology similar to combination of Activin A, BMP4, bFGF, B27, and
functional hepatocytes. In another study by Ma et al. , Wnt3a for 1 day (24 h), and transferred to RPMI1640
[15]
hiPSC-derived hepatic progenitor cells (hiPSC-HPCs) medium with a combination of Activin A, BMP4, and
were bioprinted into biomimetic hepatic lobule patterns bFGF for 3 days to induce differentiation into definitive
using digital light processing-based 3D printing. In endoderm cells. Subsequently, the endoderm cells were

International Journal of Bioprinting (2022)–Volume 8, Issue 3 177