Current Advances on 3D-Bioprinted Liver Tissue Models

Abstract

The liver, the largest gland in the human body, plays a key role in metabolism, bile production, detoxification, and water and electrolyte regulation. The toxins or drugs that the gastrointestinal system absorbs reach the liver first before entering the bloodstream. Liver disease is one of the leading causes of death worldwide. Therefore, an in vitro liver tissue model that reproduces the main functions of the liver can be a reliable platform for investigating liver diseases and developing new drugs. In addition, the limitations in traditional, planar monolayer cell cultures and animal tests for evaluating the toxicity and efficacy of drug candidates can be overcome. Currently, the newly emerging 3D bioprinting technologies have the ability to construct in vitro liver tissue models both in static scaffolds and dynamic liver-on-chip manners. This review mainly focuses on the construction and applications of liver tissue models based on 3D bioprinting. Special attention is given to 3D bioprinting strategies and bioinks for constructing liver tissue models including the cell sources and hydrogel selection. In addition, the main advantages and limitations and the major challenges and future perspectives are discussed,
paving the way for the next generation of in vitro liver tissue models.

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Current Advances on 3D-Bioprinted Liver Tissue Models
Liang Ma,* Yutong Wu, Yuting Li, Abdellah Aazmi, Hongzhao Zhou,* Bin Zhang,*
and Huayong Yang
The liver, the largest gland in the human body, plays a key role in metabolism,
bile production, detoxification, and water and electrolyte regulation. The
toxins or drugs that the gastrointestinal system absorbs reach the liver first
before entering the bloodstream. Liver disease is one of the leading causes of
death worldwide. Therefore, an in vitro liver tissue model that reproduces the
main functions of the liver can be a reliable platform for investigating liver
diseases and developing new drugs. In addition, the limitations in traditional,
planar monolayer cell cultures and animal tests for evaluating the toxicity and
efficacy of drug candidates can be overcome. Currently, the newly emerging
3D bioprinting technologies have the ability to construct in vitro liver tissue
models both in static scaffolds and dynamic liver-on-chip manners. This
review mainly focuses on the construction and applications of liver tissue
models based on 3D bioprinting. Special attention is given to 3D bioprinting
strategies and bioinks for constructing liver tissue models including the cell
sources and hydrogel selection. In addition, the main advantages and
limitations and the major challenges and future perspectives are discussed,
paving the way for the next generation of in vitro liver tissue models.
1. Introduction
D bioprinting is an emerging technology that has great poten-
tial in tissue and organ construction because of its ability to pre-
cisely control the spatial distribution of cells and the surrounding
microenvironment.[] It can spatially arrange cells, biomaterials,
and growth factors in order to form tissue-like D architectures
that facilitate cells in performing their in vivo functions.[b] D
bioprinting can be applied to the in vitro construction of almost
every tissue and organ, which might help address the worldwide
crisis of organ shortage.[] Complex heterogeneous organs, such
as the liver and kidney, contain several types of cells arranged in
a high degree of complexity. One advantage of D bioprinting is
its ability to construct these complex heterogeneous organs.
Dr. L. Ma, Y. Wu, Y. Li, A. Aazmi, Dr. H. Zhou, Dr. B. Zhang, Prof. H. Yang
State Key Laboratory of Fluid Power and Mechatronic Systems
Zhejiang University
Hangzhou 310027, P. R. China
E-mail: liangma@zju.edu.cn; hz_zhou@zju.edu.cn; zbzju@zju.edu.cn
Dr. L. Ma, Y. Wu, Y. Li, A. Aazmi, Dr. H. Zhou, Dr. B. Zhang, Prof. H. Yang
School of Mechanical Engineering
Zhejiang University
Hangzhou 310027 P. R. China
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/adhm.202001517
DOI: 10.1002/adhm.202001517
The liver, which is the largest gland in
the human body, plays a key role in more
than  types of biochemical reactions,
such as metabolism, bile production, detox-
ification, coagulation, immunity, heat gen-
eration, and water and electrolyte regula-
tion. The liver contains several types of
resident cells: hepatocytes (HCs), hepatic
stellate cells (HSCs), liver sinusoid endothe-
lial cells (LSECs), Kuper cells (KCs), and
biliary epithelial cells (BECs).[] All these
cells are tightly arranged in a specific or-
der in a hexagonal block called the hep-
atic lobule, which is the fundamental build-
ing block of the liver; each liver contains
half to one million hepatic lobules. An ac-
inus is the smallest functional unit of the
liver. It comprises two one-sixth hepatic lob-
ules and is supplied by the portal vein and
the hepatic artery. An acinus (Figure 1)can
be divided into three regions on the basis
of the concentration gradient of metabo-
lites. With a decrease in nutrient and oxygen
concentrations from the outside (Figure , region I) to the cen-
ter (Figure , region III), the liver’s regeneration ability and
metabolic rate also decrease.[] In addition, the liver has two blood
supply systems: i) venous blood from the gastrointestinal (GI)
tract via the portal vein and ii) arterial blood from the systemic
circulation via the hepatic artery. Toxins and drugs that are ab-
sorbed by the GI tract reach the liver first before entering the
bloodstream.
Over the past few decades, liver disease has become one of
the leading causes of death worldwide. According to the Global
Health Observatory metadata, two liver-related diseases, hepati-
tis B and hepatitis C are listed among the top ten global diseases.
About one-fifth of the people in China suer from liver diseases.
Hepatitis B virus (HBV), hepatitis C virus (HCV), cirrhosis, hep-
atoma, alcoholic liver disease (ALD), nonalcoholic fatty liver dis-
ease (NAFLD), and drug-induced liver injury are the main dis-
eases that aect liver health.[]
To eectively reduce the morbidity and mortality associated
with liver diseases, it is necessary to develop drugs with better
ecacy and fewer side eects. New drugs are usually developed
via testing in D monolayer cell cultures and animals. However,
these methods are expensive and time-consuming. Besides, D
monolayer cell cultures do not truly reflect the actual metabolic
microenvironment of drugs in the human body, while there are
ethical issues in using animals for experiments, and metabolic
dierences between experimental animal models and human be-
ings. Clark and Steger-Hartmann investigated drug development
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Figure 1. Schematic of the construction of in vitro liver tissue models with 3D bioprinting.
in the past  years and found that developing a Food and Drug
Administration (FDA)-approved new drug takes ≈ years and
costs $. billion. Also, the failure rate is as high as %. Many
new drugs do not show any side eects on the human body in the
early stages of development, and about % of drug failures are
due to a lack of ecacy and toxicity to the human liver in clinical
trials.[] The pharmaceutical industry has an urgent need for liver
tissue models that can realize the liver’s complex physiological
microenvironment in vitro and reproduce the liver’s metabolic
functions. Therefore, it is necessary to develop a new technology
to mimic the human liver in vitro in order to enable stable drug
development, screening, and testing, ensuring high ecacy and
low toxicity of drugs, and advance liver disease research.[]
In vitro HC culture is an important tool for liver investiga-
tion and the first step in in vitro liver tissue model construction.
However, because of the large dierence between in vivo and in
vitro normal cell culture conditions of HCs,[] primary HCs often
lose their phenotypic functions within  h after isolation.[] The
construction of a functional in vitro liver tissue model for drug
screening or transplantation must ensure that primary HCs still
maintain their original phenotype in vitro.[] The most common
method used to culture primary HCs is collagen coating, in which
a layer of collagen is coated on a Petri dish and newly isolated pri-
mary HCs are then seeded for cultivation which can prolong their
phenotypic functions.[a] However, this method has the limitation
in the creation of a heterogeneous liver structure with multiple
cell types and complex architecture.
D-bioprinted liver tissues and liver-on-chips can better sim-
ulate the in vivo microenvironment of the liver in static and dy-
namic manners separately.[] These emerging technologies can
manipulate cells with biomaterials and assemble them into a
specific structure with functions.[] D bioprinting overcomes
the limitations of D culture and better simulates the in vivo
microenvironment complexity. By forming scaolds, cell be-
havior can be determined and cell–cell or cell–matrix interac-
tions can be investigated.[] D-bioprinted microfluidic liver-on-
chips, devices that control and analyze fluids on the microme-
ter scale,[] can precisely manipulate HCs with the embedded
internal microchannels. By designing microchannel structures,
the physiological state of the liver can be simulated. Figure 
shows a schematic diagram of constructing in vitro liver tissue
models.
This review mainly focuses on D bioprinting-based in vitro
liver tissue models and liver-on-chips from fabrication ap-
proaches to their biomedical applications. Furthermore, a brief
introduction to D bioprinting strategies and bioinks is provided.
The main advantages and limitations and the major challenges
and future perspectives are discussed, paving the way for the next
generation of in vitro liver tissue models.
2. 3D Bioprinting
D bioprinting is a more precise way to construct in vitro liver
tissue models.[] In D bioprinting, HCs are encapsulated with
hydrogel, also known as bioink, and stacked layer by layer to form
a D liver tissue scaold. Next, we will briefly introduce the com-
monly used D bioprinting strategies and bioinks for liver tissue
model bioprinting.
2.1. Construction Strategies
The three most commonly used D bioprinting strategies for
constructing liver tissue scaolds are inkjet-based bioprinting,
extrusion-based bioprinting, and photocuring bioprinting (Fig-
ure 2).
2.1.1. Inkjet-Based Bioprinting
Inkjet-based bioprinting is an eective way to construct liver
tissue scaolds. Unlike the filaments generated by extrusion-
based bioprinting, inkjet-based bioprinting manipulates individ-
ual droplets as the basic unit.[] Thermal, piezoelectric, and elec-
trohydrodynamic methods are the three main approaches to re-
alizing droplet formation.[]
Inkjet-based bioprinting can control biological elements more
precisely compared to extrusion-based bioprinting. However,
the principle of inkjet-based bioprinting confines the choice of
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Figure 2. 3D bioprinting strategies for constructing scaffolds. a) Inkjet-based bioprinting, b) extrusion-based bioprinting, and c) photocuring-based
bioprinting.
bioink, and because of the nozzle configuration, only materials
with low viscosity can be printed.[] Because of its high resolution,
inkjet-based bioprinting can be applied in many fields, such as
tissue engineering, high-throughput drug screening, and cancer
investigation. Using inkjet-based bioprinting, HepG cells were
printed onto hydrogel with a biocompatible surfactant to facili-
tate droplet formation by a piezoelectric nozzle.[] Inkjet-based
printing can also be used to construct ministructures in liver-on-
a-chip, for example, Moya et al. printed an integrated minisensor
in a biochip.[]
2.1.2. Extrusion-Based Bioprinting
Extrusion-based bioprinting is the most widely used method for
constructing liver tissue scaolds. Unlike inkjet-based bioprint-
ing, extrusion-based bioprinting generates continuous filaments
to form D architectures of dierent sizes, shapes, and resolu-
tions, layer by layer, by the nozzle motion.[] On the basis of the
extrusion scheme, extrusion-based bioprinting is of three types:
pneumatic, piston, and screw. In addition, multimaterial extru-
sion bioprinting and coaxial extrusion bioprinting[] are com-
monly used to construct scaolds with multiple types of cells and
vascular structures.
Using extrusion-based bioprinting, a much larger variety of
materials with a wide range of viscosities can be constructed.[, ]
Printing parameters, such as temperature, pressure, velocity, and
cell density,[] are optimized to obtain a well-defined printing re-
sult with high shape fidelity, high cell viability, and high-activity
encapsulated cells. However, cells in the bioink might suer from
high shear stress when they pass through the nozzle, which could
damage the cells and decrease cell viability. Alginate scaolds for
the long-term culture of primary HCs have been constructed us-
ing extrusion-based bioprinting.[] To provide a better microenvi-
ronment for HCs, printing parameters[] and scaold structure,
such as the pore size and angle between two printed lines, are
carefully analyzed.[]
However, the resolution of scaolds constructed by extrusion-
based bioprinting is low because it largely depends on the noz-
zle scale, platform accuracy, and bioink characteristics. There-
fore, it is challenging to create heterogeneous tissues and organs
with high complexity. Currently, improvements to extrusion-
based bioprinting mainly focus on the resolution of scaolds[]
and multimaterial printing ability.[] One possible improvement
is preset extrusion bioprinting, in which dierent bioinks are
printed though a well-defined precursor cartridge, and after print-
ing with a micronozzle, multimaterials can be miniaturized to
form a multimaterial pattern according to the predefined config-
uration without significant deformation. HepG and endothelial
cells have been successfully constructed using preset extrusion
bioprinting in a liver tissue model with a well-organized hepatic
lobule structure.[] This method can create heterogeneous struc-
tures using multiple bioinks.
2.1.3. Photocuring-Based Bioprinting
Photocuring-based bioprinting cures the photosensitive hydro-
gel at the specified position using light irradiation. This method
has higher accuracy and can construct more sophisticated struc-
tures compared to inkjet-based bioprinting and extrusion-based
bioprinting.[] The printing speed and resolution are greatly im-
proved. In addition, unlike inkjet-based and extrusion-based bio-
printing, nozzle clogging is not an issue. Currently, photocuring-
based bioprinting is of two types, stereolithography (SLA) and
digital light processing (DLP).[] More complex microstructures
within hepatic lobules can be constructed using photocuring-
based bioprinting.[] However, this method can cause potential
phototoxicity to cells.
2.1.4. The Selection of Bioprinting Strategies
The principle to make the selection of bioprinting strategies is
mainly based on the purpose of the application where the in vitro
liver model is used. Take liver unit bioprinting for an example, to
achieve such a complex microstructure, the integration of multi-
ple bioprinting processes is really needed. Microextrusion-based
bioprinting have the advantage on the printing flexibility with the
materials, and it can be used to print the main part of the liver
unit containing hepatocytes; for the microvascular system em-
bedded, due to the high resolution, the digital light procession
(DLP) technology would be a better choice, for the ability to create
complex scaolds with much higher resolution. For bioprinting
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Tabl e 1 . Various hepatic cell types and their functions in 3D in vitro liver models.
Cell types Functions Ref.
Parenchymal cells Hepatocytes Major liver functions [13a,22–24,28a,29a,35–38 ]
Nonparenchymal cells Hepatic stellate cells Cocultivate to mimic hepatic microenvironment [28a,37b ]
Hepatic sinusoidal endothelial cells Promote liver vascularization in vitro [37a]
Kupffer cells To verify the regulatory mechanism of the cells on liver injury [40]
a bulk liver for liver regeneration, compare to the shape, how to
keep the printed bulk tissue alive and realize the hepatic func-
tion should be the main concern. To increase the mesoscale pore
networks inside the constructs to facilitate the mass transfer and
oxygen exchange, a sacrificial microgel-laden bioink-enabled bio-
printing was developed.[] To address the vascularization issue, a
coaxial extrusion bioprinting can be used to create a large volume
of hepatic tissue with sucient vascular systems.[] For drug
screening purpose, because of the cost eective requirement, the
core issue is to realize the metabolism eects of the hepatocytes.
Hence, microextrusion-based bioprinting is enough for most of
the application scenarios. More eorts need to be put on the de-
sign and integration of dierent measurement sensors into this
system to develop a microfluidic based high-throughput liver-on-
chip system.
2.2. Bioinks
Bioinks for D bioprinting have two main components, cells, and
the surrounding hydrogel; the two components have a “brick-
and-mortar” relationship.
2.2.1. Cell Sources
The liver is mainly composed of liver parenchymal cells and
liver nonparenchymal cells. Hepatic parenchymal cells perform
the major functions of the liver, while nonparenchymal cells
serve to connect and support the liver parenchymal cells. Hepatic
parenchymal cells are mainly hepatocytes. Liver nonparenchymal
cells can be divided into Kuper cells, liver endothelial cells, and
hepatic stellate cells. In this part, we will introduce the cells used
for D bioprinting according to these categories. The main cell
sources which were used in the D bioprinting are summarized
in Table 1.
Hepatocytes: Hepatocytes, responsible for major liver func-
tions, such as bile synthesis, metabolism of glucose, and toxic
substance are the main parenchymal cells in the liver which ac-
count for % of total cells and % of the total volume in the
liver.[] Primary hepatocytes, the cells directly isolated from the
liver, are the most desirable cell sources for bioprinting to con-
struct liver tissues in vitro[,, ] for their high metabolic activi-
ties. However, due to the lack of human-sourced primary hepato-
cytes, the cultivation of primary hepatocytes is still dicult and
these cells are easy to lose their phenotype;[] some hepatoma
sourced cell lines such as HepG[a,,a, ] and HUH[] which
can represent a large number of functions of the primary hepato-
cytes such as albumin secretion, urea synthesis, and cytochrome
 (CYP) related enzyme activities were widely used for bioprint-
ing of liver microenvironment instead.
Through detecting the cell morphology and characterization of
the primary hepatocytes used in D-bioprinted in vitro model, dif-
ferent bioink materials can be developed to simulate the in vivo
microenvironment as much as possible,[] and the physiologi-
cal changes of cells in vitro are also closer to the actual situation
compared with other liver cell lines.[] In some research, some
induced hepatocyte-like cells were also used in D bioprinting,
such as hepatocyte-like cells dierentiated from human adipose
stem cells,[] hepatic cells derived from human induced pluripo-
tent stem cells (hiPSCs)[a ] and embryonic stem cells (ESCs).[]
Among these cells, HepaRG cells, one hepatic progenitor cells
may be the most promising one in the bioprinting of liver con-
structs due to their ability to dierentiate into fully functional
hepatocytes in vitro including phase I and II metabolic activ-
ities and the ability to form bile canaliculi. In a recent study,
HepaRG cells were bioprinted as hepatorganoids, showed in
vivo hepatic functions and alleviated liver failure after trans-
plantation into mice which showed great potential in the liver
regeneration.[b ]
Hepatic Stellate Cells: Hepatic stellate cells account for %
of total liver cells and mainly exist in the Disse’s space. Physi-
ologically, hepatic stellate cells are in a resting state under nor-
mal conditions. When the microenvironment of hepatic stellate
cells alternated, hepatic stellate cells would be activated, which
makes collagen and some other related proteins rapidly increase,
playing a major role in the liver fibrosis. This process can be
caused by viruses, alcohol, and iron overload.[]In some research,
hepatic stellate cells were cocultivated with parenchymal cells to
mimic the hepatic microenvironment,[b ] helping parenchymal
cells maintain phenotype and functions.[a ]
Hepatic Sinusoidal Endothelial Cells: Hepatic sinusoidal en-
dothelial cells, a type of endothelial cells, which directly con-
tact with blood flow.[,] Dierent from ordinary vascular en-
dothelial cells, they have high permeability and can clear solu-
ble substances, which would be activated under inflammation.
In some of the research, human umbilical vein endothelial cells
(HUVECs) can be used instead of hepatic sinusoidal endothelial
cells for the study of the vascularized liver. It was showed that
HUVECs could generate into capillary-like sprouts in a printed
hydrogel environment which has the potential to support the
growth of HUVECs.[a ]
Kupffer Cells: Kuper cells are the macrophages scattered in
the liver sinusoids. The main functions of KCs are to process
and transmit antigens, regulate the body’s immune response, re-
move particulates and toxins in the portal vein. In Norona et al.’s
research, Kuper cells were added to an in vitro liver model
to explore their regulations responding to the injury/fibrogenic
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response following cytokine and drug stimuli in bioprinted liver
tissue.[]
2.2.2. Hydrogels
The hydrogel helps fix the cells in a suitable place to form a mi-
crostructure in vivo.[] Therefore, for D bioprinting, hydrogel
not only needs good mechanical properties to ensure that it can
be easily shaped in vitro but also needs to have good biocompat-
ibility for cells to stably proliferate and perform their physiologi-
cal functions.[d,e, ] Here, we will review several commonly used
hydrogels in the construction of in vitro liver models.
Alginate: The most commonly used hydrogel for liver tissue
bioprinting is alginate. Alginate is extracted from seaweed and,
because of its excellent formability, sucient biocompatibility,
and low cost, is often used to construct in vitro scaolds. Kim
and co-workers isolated mouse primary HCs and used % algi-
nate to D-bioprint a scaold for long-term D cell culture. Pri-
mary HCs cultured in alginate can survive for  days, which also
gradually increases the gene expression level of albumin (ALB),
hepatocyte nuclear factor  alpha (HNF-4𝛼), and Foxa3.[] The
in vitro HC microenvironment created using D bioprinting can
better maintain the morphological characteristics and functions
of HCs. Jeon et al. constructed a HepG cell scaold using %
alginate. Compared to D-cultured cells, D-cultured cells in the
scaold grew better and showed increased expression of hep-
atic markers such as ALB, asialoglycoprotein receptor  (ASGR1),
and alpha-fetoprotein (AFP)[a ] (Figure 3a). However, since pure
alginate does not provide a cell activation site to promote cell ad-
hesion and migration,[a ] it is necessary to explore materials that
are more prone to cell proliferation in vitro.
Gelatin: Gelatin and its derivatives are another kind of widely
used hydrogel for liver D bioprinting. Until recently, gelatin
methacrylamide (GelMA), a photosensitive gelatin, was a pop-
ular bioink for bioprinting because of its high formability and
excellent biocompatibility.[] Billiet et al. analyzed the printabil-
ity of GelMA and printed an interconnected pore network us-
ing –% (w/v) GelMA. They also investigated factors that
aect GelMA formability, such as rheological properties of the
material, printing pressure, printing needle model, tempera-
ture, and cell density. HepG cell scaolds constructed with
GelMA have the required stiness, high shape reliability, and
good biocompatibility.[] However, because of the potential cy-
totoxicity of the photoinitiator to cells, GelMA-based bioinks are
not approved for clinical trials yet.
Collagen: Collagen, an extracellular matrix (ECM), is an ideal
natural biomaterial for encapsulating HCs. However, it has poor
mechanical strength, so collagen scaolds easily collapse. Hence,
various methods have been developed to improve the mechanical
properties of collagen, for example, compositing collagen with
other supporting materials. Polycaprolactone (PCL) is widely
used in bone tissue engineering because of its excellent mechan-
ical properties. Lee et al. used PCL to build a framework and then
mixed collagen gel and cells in the canals of the framework. The
PCL framework protected the collagen gel from collapsing and
maintained its shape and structure[a ] (Figure b). Lee et al. also
used neutralized collagen as a bioink for printing liver mass. To
retain the scaold structure and strength, they used genipin solu-
tion, an excellent natural crosslinker, as a crosslinking agent.[a]
Mazzocchi et al. investigated the performance of a bioink com-
prising collagen and hyaluronic acid in dierent ratios and found
that HCs and HSCs can maintain ALB secretion and urea synthe-
sis and that they respond appropriately to acetaminophen (APAP)
for  weeks in a : collagen:hyaluronic acid scaold.[]
dECM: The natural cell matrix has better biological proper-
ties and can be used as a bioink. Hiller et al. prepared a new
bioink with the human ECM added to construct a D-bioprinted
liver tissue model, which showed better HC activities compared
to the bioink without human ECM; the optimal human ECM con-
centration was .– mg mL−.[c ] Lee et al. developed a liver
decellularized extracellular matrix (dECM) bioink. Compared to
collagen, the liver dECM induced better stem cell dierentiation
and enhanced HepG cell function.[a ] Yu et al. also used the
dECM bioink to print hepatic lobes by DLP.[a ]
The liver dECM bioink can be applied to D cell-printing tech-
nology, providing suitable biomechanics and a biochemical mi-
croenvironment, which may support future applications in the
field of liver tissue engineering. Compared to the commonly used
hydrogel, the liver dECM bioink has several advantages, such as
excellent biocompatibility and induction of stem cell dierentia-
tion. However, its main drawback, which limits its applications,
is that like Matrigel, the liver dECM is extracted from real tissue,
so the batch-to-batch composition varies, and the liver dECM is
not approved for clinical trials.
In addition to material optimization, coculturing other cells
with HCs can also increase their survivability and functionality.
Lee et al. cocultured human umbilical vein endothelial cells (HU-
VECs) and human lung fibroblasts (HLFs) with HCs in collagen
hydrogel and found that the heterotypic interaction between lung
parenchymal cells and HCs improves the viability and function
of HCs.[a ]
3. 3D-Bioprinted In Vitro Liver Tissue Models
Using D bioprinting, numerous in vitro liver tissue models
have been constructed to achieve liver functions for dierent re-
search purposes. In this section we mainly focus on the static
D cell cultures, these static liver tissue models can be classified
into two main categories, scaold-based models and scaold-free
spheroids.
3.1. Scaffold-Based Models
The in vitro liver tissue models mainly include static D cul-
ture of HCs using D bioprinting. We will present scaold-based
liver tissue models classified into physiological and disease mod-
els. Table 2 summarizes scaold-based D-bioprinted liver tis-
sue models based on the cell source, biomaterial, printing shape,
manufacturing strategy, and main research application.
3.1.1. Liver Physiological Models and Liver Regeneration
HCs grow in a D microenvironment. However, after isola-
tion and culture in a Petri dish, which provides a D microen-
vironment, primary HCs gradually lose their phenotype and
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Tabl e 2 . Summary of 3D-bioprinted scaffold-based liver tissue models.
Cell sources Biomaterials Shape/structure Manufacturing
strategy
Research applications Ref.
Mouse primary hepatocytes Alginate Grid Extrusion-based
bioprinting
Hepatocyte culture in vitro scaffold [23]
HepG2 cells Alginate Grid Extrusion-based
bioprinting
HepG2 cells culture in vitro scaffold [13a ]
HepG2 cells GelMA (two different
photoinitiators, I2959
and VA-086)
Grid Extrusion-based
bioprinting
Exploring the material properties of GelMA [22]
Primary rat hepatocytes; human
endothelial cells; human lung
fibroblasts (HLFs)
Collagen, PCL Grid Extrusion-based
bioprinting
Construction of collagen scaffold by PCL;
coculture
[37a ]
Hepatocyte-like cells (AHLCs)
differentiated from human adipose
stem cells
Neutralized collagen with
genipinsolution(an
excellent natural
crosslinker)
Grid Extrusion-based
bioprinting
Modification of collagen [ 35a]
Lx2 and an HSC stellate
cell/hepatocyte cell
Collagen and hyaluronic
acid
Four-spoke wheel
structure
Extrusion-based
bioprinting
Exploring the optimal ratio of collagen and
hyaluronic acid for culturing liver cells
[36]
HepG2 cell Liver decellularized
extracellular matrix
(dECM) bioink
Grid Extrusion-based
bioprinting
Developing a new biomaterial for 3D bioprinting [35a ]
Human-induced pluripotent stem cell
(hiPSC)-derived cardiomyocytes
and hepatocytes
Liver decellularized
extracellular matrix
(dECM) bioink
Lobular liver structures Digital light
processing
(DLP)-based
3D
bioprinting
Construction of hepatic lobular structure
scaffold
[33a ]
HUH7 Gelatin Grids with different
angles
Extrusion-based
bioprinting
Investigating the effect of the hole size and the
angle between the lines on cells
[24]
HepaRG and human HSCs Gelatin and PEG Lobule shape DLP-based 3D
bioprinting
A hepatic lobule equivalence consisting a
hollow-channel system for perfusion
[28a ]
Human-induced pluripotent stem
cells (hiPSCs) and human
umbilical vein endothelial cells
GelMA and GMHA Lobule structure and
vascular structure
DLP-based 3D
bioprinting
Printing a hepatic lobular structure scaffold
containing two liver-related cells
[29a ]
Human mature hepatocytes (hHeps),
human umbilical vein endothelial
cells (HUVECs), and human
mesenchymal stem cells (hMSCs)
Liver-like tissue 3D bioprinter
with needle
array system
Combining 3D bioprinting technique with a
needle array system
[33b ]
HepaRG cells Alginate and gelatin Cuboid structure Extrusion-based
bioprinting
3D-bioprinted hepatorganoids making mice
have human-specific drug metabolism
function
[35b ]
Hepatocyte and endothelial cells Alginate, gelatin, and collagen Hepatic lobule
structure
Extrusion-based
bioprinting
A preset extrusion bioprinting technique to
create sophisticated liver lobule models
[32]
Mouse-induced hepatocyte-like cells Alginate Grid Extrusion-based
bioprinting
The scaffold with miHeps transplanted into
mice as a liver damage model
[38]
HepaRG cells Alginate/gelatin bioink with
hECM
Grid Extrusion-based
bioprinting
Exploring the effect of hECM on the mechanical
properties and biocompatibility of the
materials
[35c ]
Human hepatic stellate cells (HSCs),
HUVECs, and cryopreserved
primary human hepatocytes
NovoGel 2.0 Hydrogel A mini liver tissue Extrusion-based
bioprinting
A 3D liver tissue containing various calls for
assessment of organ-level response to clinical
drug-induced toxicity In vitro
[37b ]
Hepatocytes, HSCs, and endothelial
cells
NovoGel 2.0 Hydrogel A miniliver tissue Extrusion-based
bioprinting
A bioprinted liver model to model liver injury
leading to fibrosis
[37c ]
Primary human hepatocytes, HSCs,
human umbilical vein endothelial
cells, and Kupffer cells
NovoGel 2.0 Hydrogel A mini liver tissue Extrusion-based
bioprinting
To explore the impact of KCs on the liver injury
and fibrosis
[40]
Hepg2 Alginate, gelatin, and
fibrinogen
Grid Extrusion-based
bioprinting
To compare drug sensitivities of several
anticancer drugs to the HepG2 cells in both
3D models and 2D monolayers
[35d ]
HC, hepatocyte.
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Figure 3. Different constructions of 3D-bioprinted liver tissue models. a) PCL as a framework material to construct a collagen-containing grid struc-
ture. Reproduced with permission.[37a ] Copyright 2016, IOP. b) 3D-bioprinted hepatic grid structures using alginate. Reproduced with permission.[13a ]
Copyright 2017, Joe Bok Chung. c) Hepatic lobule structure bioprinted by DLP. Reproduced with permission.[ 29a] Copyright 2016, National Academy
of Sciences. d) Preset extrusion bioprinting to create a hepatic lobule structure. Reproduced with permission.[32] Copyright 2020, Wiley-VCH GmbH.
e) Liver-like tissue constructed with 3D bioprinting and needle array technology. Reproduced with permission.[33b] Copyright 2017, Nature Publishing
Group. PCL, polycaprolactone; DLP, digital light processing.
function.[] In contrast, cells in scaolds have a D microenvi-
ronment and attach to the hydrogel instead of sticking to a flat
surface. Therefore, D-bioprinted liver tissue models can repre-
sent the main structure and functions of the liver.
Researchers tried to encapsulate HCs in a hydrogel to create
a D microenvironment with D bioprinting. Lewis et al. used
gelatin to construct scaolds containing an undierentiated HC
line (HUH) with dierent shapes and pore sizes and tested HC-
specific functions to determine the optimal scaold structure.[]
They found that the grid scaold shape and pore size significantly
aect HC growth in vitro. Later, researchers changed their focus
from shapes that mimic the liver to the reproduction of hepatic
functions in vitro.
Various kinds of scaolds with complex structures have been
designed to satisfy multiple liver functions. Grix et al. followed
a stereolithographic bioprinting approach to produce a hepatic
lobule equivalent containing a hollow-channel system to mimic
blood vessels for perfusion culture.[a ] Ma et al. constructed a
hexagonal hepatic lobule containing human induced pluripo-
tent stem cells (hiPSCs) and supporting cells from endodermal
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and mesodermal origins by DLP, forming a complex microstruc-
ture and liver microenvironment with multiple cells to achieve
a complex liver tissue model structure with sucient HC
functions[a ] (Figure c). Mao et al. successfully constructed liver
microtissue with the liver dECM bioink mixed with GelMA via
DLP and encapsulated human-induced hepatocytes (hiHeps) in
the liver microtissue for  week that expressed excellent HC
functions.[b ] These scaold-based in vitro liver tissue models
can reproduce several liver key functions, such as ALB secretion,
urea production, and cytochrome P (CYP)-related enzyme
activities.
D-bioprinted in vitro liver tissue models also can be used
to investigate liver regeneration. A recent study used well-
established hepatic progenitor (HepaRG) cells to create a D-
bioprinted hepatorganoids model. These hepatorganoids when
transplanted into mice showed in vivo hepatic functions and the
ability to alleviate liver failure, indicating that D-bioprinted hep-
atorganoids can be used for liver regeneration.[b ]
Vascularization is a major issue in D-bioprinted liver tissue
models. Traditional D-bioprinted liver tissue models lack a vas-
cular system. However, with the development of coaxial printing,
sacrificial printing, and preset extrusion, it is now possible to em-
bed the vascular system into D-bioprinted tissues and organs.
A recent study adopted preset extrusion and D-bioprinted hep-
atic lobules with HCs and endothelial cells surrounding the HCs,
with a lumen in the center of the hepatic lobules, which repre-
sented the portal vein structure in vivo[] (Figure d). Preset ex-
trusion can construct heterogeneous, multicellar, and multima-
terial structures simultaneously.
3.1.2. Liver Disease Models and Drug Screening
In addition to physiological liver tissue models, scaold-based
liver tissue models can be applied as liver disease models. Liver
diseases, such as HBV, HCV, cirrhosis, and hepatoma, are some
of the leading causes of death and pose a serious threat to human
health worldwide. A more accurate prediction of drug-induced
hepatotoxicity can be expected using D-bioprinted HC con-
structs with primary hepatocytes. In the pharmaceutical indus-
try, these liver disease models can be applied in the new drug
development to decrease the failure rate of drug development.
Here, we take drug-induced liver injury, hepatoma, and liver fi-
brosis models for examples to show how D bioprinting is used
to create liver disease models.
Drug-induced liver injury (DILI) is one of the major causes
of drug development failure. D-bioprinted liver tissue models
are a powerful tool for testing the hepatotoxicity of drug candi-
dates during drug screening.[] Nguyen et al. D-bioprinted de-
fined liver tissue constructs with patient-derived hepatocytes and
nonparenchymal cells and assessed the organ-level response to
clinical drug-induced hepatotoxicity.[b] Dose responses of hep-
atotoxic drug Trovafloxacin and nontoxic drug Levofloxacin were
tested to evaluate the ability of this D model in the assessment
of tissue-level DILI. The results showed that Trovafloxacin has
the ability to induce significant dose-dependent DILI at a clin-
ically relevant dose (≤×−). This kind of D-bioprinted
model showed a clinically relevant injury response to hepatotoxic
drugs.
D-bioprinted hepatoma models are the most common liver
disease models used to determine the molecular mechanism un-
derlying hepatoma origins, hepatoma progress, metastasis, and
anticancer drug screening. HepG cells are the most commonly
used hepatoma cell source because of their cost-eectiveness.
Researchers D-bioprinted HepG cells into scaolds and cul-
tured hepatoma constructs for long-time culture. Xinwei Zhou
et al. constructed a D-bioprinted hepatoma model with HepG
cells encapsulated in sodium alginate/gelatin/fibrinogen hydro-
gel. Anticancer drugs -fluorouracil, mitomycin, and their com-
bination were tested using this model, and significant dierences
were found between D and D cell culture conditions.[d ] Re-
cent studies have pushed D-bioprinted in vitro hepatoma mod-
els to a more biological relevant level. Sun et al. tested a D-
bioprinted hepatoma model with HepG cells and found signif-
icant upregulation of tumor-related genes, including ALB,AFP,
CD133, interleukin  (IL-8), epithelial cell adhesion molecule (Ep-
CAM), CD24, and transforming growth factor beta (TGF-𝛽), com-
pared to their D cell culture counterpart.[]
Liver fibrosis is a pathophysiological process caused by vari-
ous insults such as chemical exposure, drugs, metabolic disease,
alcoholism, viral infection, and the resulting complex HC–NPC
interactions. The cumulation of such interactions causes liver
stiening, leading to liver cirrhosis and failure. Because of the
complex factors involved, it is dicult to reproduce a liver fibro-
sis model in vitro following standard approaches. D bioprinting
is an eective way to construct liver fibrosis models for drug eval-
uation and therapy because of its multimaterial spatial distribu-
tion ability. Norona et al. D-bioprinted human liver tissue with
primary HCs, HSCs, and endothelial cells to test methotrexate
(MTX)- and thioacetamide (TAA)-induced fibrogenesis.[c] They
applied this liver tissue model to analyze the eects of exposure
to TGF-𝛽 and MTX for  days and determine the role of KCs
in fibrogenesis.[]
These D-bioprinted hepatic disease models have the impor-
tance in the following aspects: ) modeling various human hep-
atic diseases in vitro to explore the underlying molecular mecha-
nisms; ) elucidating the microenvironmental cues of cell–cell
and cell–matrix interactions on liver cellular functions; ) en-
abling accurate prediction of clinically relevant results and de-
creasing the risk of drug development failure owing to DILI or
lack of ecacy, and ) helping to develop cell-based treatments
in the clinic.
3.1.3. Limitations
Although scaolds constructed by D bioprinting can simulate
the in vivo D microenvironment of the liver, scaold-based in
vitro liver tissue models have many limitations. The static model
lack of the reflection of the dynamic response of drug under per-
fusion HCs culture condition. Compared to the cell size, the scale
of the printed hydrogel structure is large, making it dicult to
precisely manipulate cells. The cells are randomly distributed in
the scaold and cannot reproduce the subtle anisotropy of the
liver.[] Additionally, cells in scaolds usually obtain nutrients by
soaking in the medium, and it is dicult to realize subtle changes
in the oxygen and nutrient concentrations.[, ]
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3.2. Scaffold-Free Spheroids
In addition to cells encapsulated in biomaterial scaolds, another
kind of D culture model free of hydrogel is based on cellular
spheroids. Spheroids are self-organizing, spherical cellular ag-
gregates without scaolds that make up for some of the deficien-
cies of D culture. Primary HCs can maintain their phenotype
and function by forming spheroids.[a, ]
Yanagi et al. proposed a strategy for forming liver buds with-
out biomaterials such as collagen, gelatin, and Matrigel. D bio-
printing was combined with needle array technology to assemble
liver bud-like spheroids[b ] (Figure e). Hundreds of these liver
bud-like spheroids were fused with D bioprinting and trans-
planted into nude rats for investigating liver regeneration. Re-
sults showed that transplantable liver organoids could be eec-
tively generated in vitro.
Conventional approaches to constructing HC spheroids
include hanging drop,[] microwell array,[] and magnetic
assembly.[] Each method has advantages and drawbacks. More-
over, the applicative cell types for each method dier because of
diverse phenotypes and in vitro cell culture characteristics, which
also aect the eectiveness of cell aggregate formation, including
spheroid diameter and stability. We identified HC spheroids as
cell-laden scaold-free hydrogel constructs (the HCs were usu-
ally encapsulated within hydrogel).
The design and construction of such models vary with dif-
ferent applications. Compared to conventional tissue engineer-
ing approaches, D-bioprinted cell-laden hydrogel spheroids pre-
cisely control the spatial distribution and composition of cell
types and ECM factors, resulting in a better recapitulation of the
in vivo microenvironment of the liver. The distribution of cells en-
capsulated in the hydrogel is dierent in dierent studies. Several
types of cells could even spontaneously aggregate and form into
spheroids in the hydrogel. The hydrogel can be removed later,
and the cell aggregates used for subsequent analysis show simi-
lar behaviors as their in vivo counterparts.
Compared to scaold-based models, scaold-free models have
a low cost and do not need hydrogel. However, they cannot be
used to construct complex heterogeneous microstructures.
4. Liver-on-Chips with 3D Bioprinting
Liver-on-chips are a kind of dynamic microfluidic based biochip
models for in vitro HC culture. In contrast to the previous static
platforms, dynamic microfluidic-based in vitro liver systems can
allow the precise control over the HC culture microenvironment
such as temperature, pH, cell shear stress, oxygen, nutrient sup-
ply, and waste removal. D bioprinting can be applied in every
stage of the fabrication of liver-on-chips including the chip frame-
work, the liver tissue scaolds embedded as well as the whole
chip printing.
4.1. Liver-on-Chips
Introducing the flow using microfluidic technology into HC cul-
ture can better mimic the liver’s vascular system. Compared to
the static models, the microfluidic biochip decreases shear stress
on cells during medium perfusion by using microcolumn or mi-
croporous membranes (Figure 4a). Therefore, HCs cultured in-
side a microfluidic biochip have a better chance to grow with
their in vivo functions. Because of the small scale of microfluidic
biochips, they can implement more complex structural designs to
better simulate the microstructures and related functions of the
liver, and these designs can be used as bioreactors or to model
liver diseases for drug screening.
The hepatic lobule is the basic building block of the liver. Liver
sinusoids are arranged from the center of the hepatic lobule to
the surroundings to form a hexagonal vascular network struc-
ture. Blood is inputted from the surrounding hepatic artery and
portal vein and then outputted to the central hepatic vein.[] Di-
verse biochips have been constructed by imitating the liver on the
basis of the microstructures and functions.
Banaeiyan et al. designed and constructed a high-throughput
microfluidic biochip consisting of integrated hepatic-lobule-like
hexagonal regions and diusion microchannels, which mim-
icked the blood circulation with convection diusion in the liver.
HepG cells and hiPSC-derived iCell HCs were cultured in the
microfluidic biochip. The ALB secretion and urea synthesis lev-
els indicated that the functions of both cell types were well main-
tained. Figure b shows a functional bile-canaliculi network in
hiPSC-derived iCell HCs cultured in the microfluidic biochip.[]
A hepatic lobule comprises several liver sinusoids with four types
of cells (LSECs, KCs, HSCs, and HCs). Du et al. cocultured these
four types of cells in a two-layered microfluidic biochip. LSECs,
KCs, and HSCs were attached to a porous PE membrane, and
HCs were cultured in the lower chamber to simulate the liver
sinusoid structure[] (Figure c).
As stated in the previous section, an acinus is the smallest
functional unit of the liver. With the concept to mimic the func-
tion of the liver acinus, many liver chips have been developed.
Shih et al. designed a system simulating the in vivo liver acinar
structure. They formed a concentration gradient in the triangu-
lar cell culture area through a polydimethylsiloxane (PDMS) col-
umn and fluid flow control[] (Figure d). Lee et al. designed and
manufactured a two-layered liver-on-a-chip to simulate the liver–
biliary duct system. The biochip was used for in vitro drug exper-
iments. Results showed a more sensitive drug response in D
compared to D culture conditions[] (Figure e).
With a sophisticated microstructure, microfluidic biochips can
control the flow of fluid or precisely manipulate cells to perform
drug screening and detection. However, in some biochips that
aim to achieve a complex liver structure, such as hepatic lobules
or liver sinusoids, multiple cells are separated by columns or just
stacked layer by layer, and it is dicult to reproduce the in vivo
microenvironment of the liver and form liver minitissue by mi-
crofluidic technology. Therefore, it is necessary to develop novel
microfluidic biochips that can provide hepatic cells with a D
growing condition to better simulate the in vivo microenviron-
ment of the liver.
4.2. 3D-Bioprinted Liver-on-Chips
The traditional methods of manufacturing the microfluidic liver-
on-chips are photolithography and replica molding, both are
expensive and time-consuming. D bioprinting is a rapidly
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Figure 4. Liver-on-chips that mimic the liver structures and functions. a) HCs cultured in the cell culture area with microfluidic endothelial-like barrier
mimicking hepatic sinusoids. Reproduced with permission[68] Copyright 2007, Wiley-VCH GmbH. b) Two-layer biochip simulation. Reproduced with
permission[54] Copyright 2017, IOP. c) Four types of cells used to simulate hepatic sinusoids. Reproduced with permission.[55] Copyright 2017, The Royal
Society of Chemistry. HC, hepatocyte; LSEC, liver sinusoid endothelial cell; KC, Kupffer cell; HSC, hepatic stellate cell; PE, polyester. d) The concentration
gradient of the culture medium changes gradually from top to bottom in the upper and lower channels. Reproduced with permission[56] Copyright 2013,
Springer Nature. e) Double-layer structure mimicking the liver–biliary duct system. Reproduced with permission[57] Copyright 2019, IOP.
developing manufacturing method that can be used to construct
the microfluidic liver-on-chips. Not only can D bioprinting con-
struct the microfluidic liver-on-chips in less time at low cost, but
it can also be used to construct complex cell-laden scaolds in the
chip for perfusion cultivation. Table 3 summarizes D-bioprinted
liver-on-chips based on the cell source, printing shape, manufac-
turing strategy, and main research application.
4.2.1. Printed Framework of Liver-on-Chips
D printing is a rapid and cost-eective approach to create com-
plex frameworks of the liver-on-chips. D vascular network in
the extracellular matrix containing HepG cells was printed by
Pimentel et al. using PVA/PCL as a sacrificial layer. The prolif-
eration and spheroid formation of HepG cells can be induced
with perfusion culture, which can simulate the gradient changes
of the tissue in the tumor necrosis area[] (Figure 5a). Moya et al.
applied inkjet-based bioprinting to print multielectrodes as sen-
sors; the scale to monitor oxygen concentration was ≈ m[]
(Figure b). Ong et al. compared two methods, PolyJet and SLA,
to construct microfluidic perfusion devices; the resolution of the
devices was ≈ and  m, respectively.[]
4.2.2. Liver-on-Chips Comprising Bioprinted Liver Tissue Scaffolds
The application of D bioprinting in microfluidic biochips can
also help simulate the in vivo microenvironment of the liver more
precisely with perfusion.
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Tabl e 3 . 3D-bioprinted liver-on-chips.
Functions Cell sources Shape/structure Manufacturing strategy Research applications Ref.
Bioprinted framework
of liver chips
HepG2 vascular structure Sacrificial method Simulate vascular flow [59]
Hepatocyte Rectangle with
multi-inkjet-printed sensors
Inkjet-based bioprinting Sensors to monitor oxygen
concentration in liver chip
[18]
Liver-on-chips
comprising
bioprinted liver
tissue scaffolds
Epithelial cells and HCs Scaffold in chip Photolithography and replica molding
techniques applied in bioreactor and
extrusion-based bioprinting applied in
scaffold
Perfusion culture [12]
HepG2/C3A cells Hexagonal structure Photolithography and replica molding
techniques applied in bioreactor and
extrusion-based bioprinting applied in
scaffold
A biochip allowed for direct access
with bioprinter
[61]
3D-bioprinted
liver-on-chips
Hepatocytes and mouse
embryonic fibroblasts
Multiwell plate inserts 3D printing and micromolding Developing biologist-friendly culture
plates
[63]
Human HepaRG cells and
HUVEC
Two layers containing
vascular/biliary fluidic
channels
Extrusion-based bioprinting Liver chips containing vascular and
biliary systems
[57]
HepaRG and Human hepatic
stellate cells (SteCs)
Lobule shape Digital light processing (DLP)-based 3D
bioprinting
A hepatic lobule equivalence
consisting a hollow-channel system
for perfusion
[28a ]
HepG2 and human umbilical
vein endothelial cells
Cuboid Extrusion-based bioprinting A novel 3D bioprinting method for
organ-on-a-chip applications
[64]
HC, hepatocyte.
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Figure 5. Liver-on-chips fabricated with 3D bioprinting. a) 3D vascular network constructed by sacrificial layer printing. Reproduced with permission.[59]
Copyright 2018, Elsevier. b) Inkjet printing method to print oxygen concentration detector. Reproduced with permission.[18] Copyright 2018, The Royal
Society of Chemistry. c) A liver-on-a-chip platform with bioprinted hepatic spheroids. Reproduced with permission.[61] Copyright 2016, IOP. d) A bio-
printed 3D micro-organ cultured in a microscale in vitro device. Reproduced with permission.[12] Copyright 2011. IOP. e) Hepatic lobule equivalent with
hollow channels. Reproduced with permission.[28a ] Copyright 2018, MDPI. f) 3D bioprinting technology for the chips. Reproduced with permission.[64]
Copyright 2016, The Royal Society of Chemistry. HC, hepatocyte.
To test the eect of drugs on the liver, Bhise et al. D-
bioprinted a scaold containing hepatic spheroids in a biore-
actor for perfusion[] (Figure c). Snyder et al. D-bioprinted
a scaold comprising two layers of two types of cells, ep-
ithelial cells and HCs, using extrusion-based bioprinting in
a microfluidic biochip[] (Figure d). Chang et al. combined
D bioprinting with microfluidic biochip technology to print
a D heterogeneous cell-encapsulated hydrogel-based matrix
for discovering and developing new drugs and testing their
hepatotoxicity.[]
D bioprinting helps in better control of the cell–hydrogel mix-
ture in the biochip, which can also be better assembled into a
more ideal shape for further investigation.
4.2.3. 3D-Bioprinted Liver-on-Chips
After studying and summarizing most of the methods of reduc-
ing shear stress on HCs, Gheibi et al. combined D bioprinting
and micromolding to construct a polymer insert with micropat-
terns compatible with a standard culture plate. This device cre-
ates low-volume culture conditions under which primary HCs
can maintain dierentiated phenotypes.[] Lee et al. constructed
a D-bioprinted liver-on-a-chip using polyethylene vinyl acetate
(PEVA) and a HepaRG–liver dECM bioink mixture.[] Grix et al.
used stereolithographic bioprinting to produce a hollow-channel
system in a hepatic lobule equivalent, and the scale could main-
tain ≈ m for medium perfusion[a ] (Figure e). Lee and Cho
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et al. constructed an organ-on-a-chip containing HepG cells and
HUVECs in a one-step process. They used D bioprinting to con-
struct a PCL outer frame and an inner hydrogel–cell structure,
improving the protein absorption defects of ordinary PDMS-
based biochips[] (Figure f).
In conclusion, inkjet-based bioprinting, extrusion-based bio-
printing, and photocuring-based bioprinting can be used to man-
ufacture liver-on-a-chip, and the resolution increases in the or-
der extrusion-based bioprinting <inkjet-based bioprinting <
photocuring-based bioprinting. By using these D bioprinting
methods in the area of liver-on-chips, the time and cost of man-
ufacturing are significantly reduced.
4.2.4. Limitations
Although D bioprinting can provide a rapid and cost-eective
way to fabricate dynamic liver-on-chips. Still, there are many lim-
itations with the current technology. First of all, the liver is a com-
plex heterogeneous organ with multiple cell types and form dif-
ferent levels of microstructures, each microarchitecture requires
dierent fabrication technologies. To fully reproduce the liver
functions in vitro, a combination of dierent D bioprinting tech-
nics and other fabrication methods in a certain order is highly re-
quired. Secondly, the resolution of current D bioprinting tech-
nics are far to the satisfactory to reproduce the complex hepatic
microenvironment, the liver has abundant vascular systems and
vascularization is one of the great issues that needs to be ad-
dressed in the in vitro liver modeling to ensure a long-term ef-
fect of investigation. Last but not least, when it terms to these
perfusion-based devices for the drug screen, the throughput is of
great importance; currently, the D-bioprinted liver-on-chips lack
high-throughput ability which will hamper their applications in
the pharmaceutical industry for drug screening.
5. Challenges and Future Directions
Research on in vitro liver tissue models has developed rapidly
in recent years. D bioprinting can be an eective way to build
static liver tissue models and dynamic microfluidic-based liver-
on-chips for the culture of HCs in vitro but still has some limita-
tions.
D bioprinting and microfluidic liver-on-chips can simulate
the in vivo microenvironment of the liver to a certain extent, but
we are still a long way o from complete realization of in vitro
primary HC culture with full liver functions. The characteristics
of hydrogel used in D bioprinting still need to be investigated.
A hydrogel that not only has excellent mechanical properties and
biocompatibility but also induces HCs to maintain their origi-
nal functional and morphological characteristics for long term
investigation need to be developed. To more precisely construct
scaolds, the resolution of D-bioprinted devices also needs im-
provement.
The scale of microfluidic-based liver-on-chips is similar to the
cell size, which can better manipulate cellular movement and cul-
ture cells. These liver-on-chip models have the advantages of a
small sample-loading requirement and sensitive HC behavior de-
tection. By further designing and optimizing the structure, liver
sinusoids or microcapillaries in hepatic lobules that are closer to
the liver microenvironment can be reproduced in vitro by con-
trolling the culture medium.
Cost and eciency are two main concerns in drug develop-
ment, so the new generation of in vitro liver tissue models should
consider these two factors. Liver-on-a-chip processes with high-
throughput[] and multidrug screening ability along with precise
control of the flow and drug dosage are necessary. Additionally,
achieving multiorgan interaction is of significance for the future
in vitro liver tissue models. By integrating dierent organs on a
biochip to realize human-on-a-chip, the complete PBPK process
of drugs in the human body can be achieved and can provide us
with more authentic and informative data for estimating drug ef-
ficacy and hepatotoxicity.
6. Conclusions
Drug development based on monolayer cell cultures and animal
tests are expensive and lack ecacy. Therefore, it is necessary
to develop in vitro liver tissue models. D-bioprinted liver tissue
scaolds and liver-on-chips can help construct in vitro liver tissue
models. D bioprinting for constructing in vitro liver tissue mod-
els was first proposed to cultivate primary HCs under D culture
conditions. Next, diverse structures were designed to mimic the
in vivo microenvironment of the liver as closely as possible, such
as imitating the hepatic lobule shape or constructing a hollow-
channel system to mimic blood vessels.
Microfluidic liver-on-chips precisely manipulate liquids
through microchannels where HCs can be cultured without
shear stress and damage. Moreover, the liver microstructure can
be simulated by designing multiple flow channel structures.
D-bioprinted liver-on-chips are used to simulate dierent liver
structures and functions. D-bioprinted liver tissue scaolds
and liver-on-chips are important methods of constructing in
vitro liver tissue models. By applying D bioprinting technology
in liver-on-a-chip construction, the development of in vitro liver
tissue models will become more ecient and cost-eective,
besides providing a powerful platform for screening drugs
and determining the molecular mechanism underlying liver
diseases.
Acknowledgements
L.M. and Y.W. contributed equally to this work. The authors would like to
thank the support by the National Key Research and Development Pro-
gram of China (2018YFA0703000), National Natural Science Foundation
of China (Grant No. 51875518), Key Research and Development Projects
of Zhejiang Province (Grant No. 2017C01054) and the Fundamental Re-
search Funds for the Central Universities (Grant Nos. 2019XZZX003-02
and 2019FZA4002).
Conflict of Interest
The authors declare no conflict of interest.
Keywords
3D bioprinting, bioinks, hepatocytes, liver in vitro models, liver-on-chips
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Received: August 25, 2020
Revised: September 27, 2020
Published online:
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Liang Ma received his Ph.D. degree in mechanical engineering from University of Washingtonin 2012.
He was a postdoctoral researcher in University of Texas at Austin and then joined Zhejiang California
Nanosystems Institute, Zhejiang University as research associate. He is now an assistant professor
in the School of Mechanical Engineering, Zhejiang University.His research interests include high-
resolution 3D bioprinter development and 3D bioprinting of tissues and organs especially tumor in
vitro models and organs-on-chip.
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