TISSUE ENGINEERING: Part A

Volume 24, Numbers 7 and 8, 2018

ª Mary Ann Liebert, Inc.
DOI: 10.1089/ten.tea.2017.0161

ORIGINAL ARTICLE

Three-Dimensional Bioprinting of Hepatic Structures

with Directly Converted Hepatocyte-Like Cells
Kyojin Kang, MS,1–3 Yohan Kim, MS,1–3 Hyeryeon Jeon, MS,2,3 Seung Bum Lee, PhD,4 Ji Sook Kim, PhD,5
Su A Park, PhD,6 Wan Doo Kim, PhD,6 Heung Mo Yang, PhD,7 Sung Joo Kim, MD, PhD,7
Jaemin Jeong, PhD,2,3 and Dongho Choi, MD, PhD1–3

Three-dimensional (3D) bioprinting technology is a promising new technology in the field of bioartificial organ
generation with regard to overcoming the limitations of organ supply. The cell source for bioprinting is very
important. Here, we generated 3D hepatic scaffold with mouse-induced hepatocyte-like cells (miHeps), and
investigated whether their function was improved after transplantation in vivo. To generate miHeps, mouse
embryonic fibroblasts (MEFs) were transformed with pMX retroviruses individually expressing hepatic tran-
scription factors Hnf4a and Foxa3. After 8–10 days, MEFs formed rapidly growing hepatocyte-like colonies.
For 3D bioprinting, miHeps were mixed with a 3% alginate hydrogel and extruded by nozzle pressure. After
7 days, they were transplanted into the omentum of Jo2-treated NOD Scid gamma (NSG) mice as a liver
damage model. Real-time polymerase chain reaction and immunofluorescence analyses were conducted to
evaluate hepatic function. The 3D bioprinted hepatic scaffold (25 · 25 mm) expressed Albumin, and ASGR1 and
HNF4a expression gradually increased for 28 days in vitro. When transplanted in vivo, the cells in the hepatic
scaffold grew more and exhibited higher Albumin expression than in vitro scaffold. Therefore, combining 3D
bioprinting with direct conversion technology appears to be an effective option for liver therapy.

Keywords: miHep cells, 3D bioprinting, cell transplantation, direct conversion

Introduction Direct conversion technique has several advantages more

than pluripotent stem cells, such as lower tumori-
genic risk,13 faster conversion rate, and the ability to re-
C ell therapy using stem cells is being explored as
a novel approach that could potentially replace organ
transplantation. Cell therapies for liver diseases can be
pair injured tissues.12
Hepatocytes derived from induced pluripotent stem cells
categorized based on cell source (cell lines, primary hepa- (iPSCs) or embryonic stem cells (ESCs) cannot be main-
tocytes, stem cells, progenitor cells, or nonparenchymal cell tained because they do not multiply.14 Several researchers
types).1,2 Pluripotent stem cells have been used to gener- have suggested methods of long-term culture of hepatocytes,
ate hepatocyte-like cells.3–6 However, the use of pluripotent such as combining them with mesenchymal stem cells
stem cells has led to controversy regarding the danger (MSCs),15 use of collagen sandwiches,16 and the addition of
of tumor formation,7,8 failure of long-term differentiation,9 special media components.17 Although ‘‘sandwich culture,’’
and low differentiation efficiency.10 Therefore, developing an in vitro approach of mimicking three-dimensional (3D)
functional cells that are as effective as primary cells is culture, has been proposed as a solution to long-term culture
crucial. In this article, we introduce directly converted and maintenance of hepatocyte function, it has some limi-
hepatocyte-like cells. Direct conversion technique is con- tations because it cannot be used to construct an actual 3D
sidered to be one of the most effective methods of altering model18; 3D structures are particularly important for gen-
somatic cells without generating pluripotent stem cells.11,12 erating functional hepatocytes in an in vivo-like state.
1
Department of Translational Medicine, Graduate School of Biomedical Science and Engineering, Seongdong-gu, Korea.
2
Department of Surgery, Hanyang University College of Medicine, Seoul, Korea.
3
HY Indang Center of Regenerative Medicine and Stem Cell Research, Hanyang University, Seoul, Korea.
4
Laboratory of Radiation Exposure and Therapeutics, National Radiation Emergency Medical Center, Korea Institute of Radiological
and Medical Science (KIRAMS), Seoul, Korea.
5
Department of Pathology, Hanyang University College of Medicine, Seoul, Korea.
6
Department of Nature-Inspired Nanoconvergence Systems, Korea Institute of Machinery and Materials, Daejeon, Korea.
7
Department of Surgery, Sungkunkwan University College of Medicine, Seoul, Korea.

576
CONSTRUCTION OF MULTILAYERED 3D-PRINTED HEPATIC STRUCTURES
3D bioprinting is one of the most promising 3D culture
methods using stem cells, which overcomes many limita-
tions of earlier cell transplantation methods.19 Nevertheless,
bioprinting faces some of the same challenges that confront all
tissue engineering and regenerative medicine technologies.20
We describe the use of mouse-induced hepatocytes (miHeps)
in conjunction with 3D bioprinting as a technique that could be
used in cell therapies and drug discovery to eventually facili-
tate transplantation of bioartificial organs.21,22
Materials and Methods
Generation of miHeps
The production of miHeps has been described previous-
ly.23 In brief, to produce miHeps, 5 · 104 mouse embryonic
fibroblasts (MEFs) were transduced with pMX retroviruses
expressing hepatic transcription factors Hnf4a and Foxa3
(a gift from Prof. Dong Wook Han of Konkuk University).
After 48 hrs, the cells were further cultured in miHep medium
on a Type I collagen-coated dish to induce lineage transition
toward miHeps. The cells were cultured in a 100 mm di-
ameter dish (Corning) with DMEM/F-12 (11965; Gibco,
CA) supplemented with 10% fetal bovine serum (Gibco),
10 mM nicotinamide (Sigma-Aldrich, MO), 0.1 mM dexa-
methasone (Sigma-Aldrich), 1% insulin-transferrin-selenium
(ITS) (Gibco), 1% penicillin/streptomycin (Gibco), 20 ng/mL
hepatocyte growth factor (HGF) (Peprotech, NJ), and 20 ng/
mL epidermal growth factor (EGF) (Peprotech) at 37C in a
CO2 incubator. The medium was changed daily.
Production of mCherry lentivirus
Viruses were produced as previously described.22 mCherry
was packaged by cotransfection with psPAX2 lentiviral
packaging plasmid and pCMV-VSV-G plasmid in human
embryonic kidney 293T cells. Culture supernatants were
harvested after 48 and 72 hrs and stored at -80C. Lentiviral
transduction of mCherry was carried out in culture medium
supplemented with 8 mg/mL polybrene (Sigma-Aldrich).
3D bioprinting
3D-printed strands of packaged cells were produced with
a 3D bioprinting system made by the Korea Institute of Ma-
chinery and Materials. The system can control the pressure
and velocity needed to fabricate the 3D hydrogel, which was
plotted through the pneumatic dispenser. The material dis-
pensed consisted of 3% alginate to form the hydrogel scaffold
and a calcium chloride (CaCl2) solution to solidify the hy-
drogel. Before 3D printing, the miHep cells were suspended
in 3% alginate solution. The 3D-printed scaffold measured 25
by 25 mm. For cell printing, constant air pressure was applied
to the dispenser, and the cell-encapsulating alginate was
squeezed out onto a 100 mm diameter culture dish. After
printing, the 3D alginate hydrogel was soaked in 1% CaCl2
solution to strengthen crosslinking, washed with phosphate-
buffered saline (PBS), and placed in miHep culture medium.
RNA isolation and quantitative real-time
polymerase chain reaction
Total RNAs were isolated using Trizol Reagent (Gibco).
Two-microgram RNA samples were reverse transcribed with
a Transcriptor First Strand cDNA Synthesis Kit (Roche, PA),
577
and real-time polymerase chain reaction (PCR) was per-
formed using 10 mL of quantitative polymerase chain reaction
(qPCR) PreMix (Dyne bio, Korea), 1 mL of cDNA, and oli-
gonucleotide primers on a CFX Connect Real-Time PCR
Detection system (Bio-rad, CA). Reactions were analyzed in
triplicate for each gene; the primers used for mouse Albumin,
ASGPR1, AFP, HNF4a, and 18s rRNA are listed in Supple-
mentary Table S1 (Supplementary Data are available online
at www.liebertpub.com/tea). PCR cycles consisted of 40 cycles
of 95C for 20 s and 60C for 40 s. Melting curves and melting
peak data were obtained to characterize the PCR products.
Hematoxylin and eosin staining
and immunohistochemistry
Four-micrometer sections were deparaffinized by washing
three times with xylene for 5 min each time. The printed mi-
Heps were rehydrated in a series of 100%, 90%, and 70%
ethanol for 5 min each, and finally rinsed in tap water. The
sections were stained with hematoxylin (Sigma) for 5 min and
counterstained with eosin for 1 min after washing with running
water for 5 min. Finally, they were washed in running water
and dehydrated in 95% ethanol for 5 min. The 3D-printed
mCherry-miHep alginate scaffold was fixed in 4% buffered
formalin and embedded in paraffin. Four-micrometer sections
were cut, mounted on silane-coated glass slides, depar-
affinized, and rehydrated in graded alcohols. After washing,
endogenous peroxidase was blocked with 3% H2O2 in meth-
anol for 15 min at room temperature. Antigen was retrieved by
autoclaving the slides in pH 6.0 citric acid buffer.
For immunohistochemistry, the slides were incubated in a
moist chamber with rabbit anti-mCherry antibody (1:200;
Abcam, Cambridge, United Kingdom), goat antimouse se-
rum Albumin antibody (1:200; Abcam), mouse antihuman
cytokeratin 18 (1:200; DAKO, CA), cytokeratin 19 (1:100;
Santa Cruz Biotechnology, TX), or Ki67 (1:200; Leica, NCL,
United Kingdom) overnight at 4C. The next day, the cells
were washed twice with staining solution in PBS supple-
mented with 1% fetal bovine serum for 5 min, then incubated
with secondary antibody, goat antimouse IgG Alexa Fluor
488 (1:250; Molecular probes, OR), goat antirabbit IgG
TRITC (1:250; Molecular Probes), or donkey antigoat IgG
Alexa Fluor 488 (1:250; Molecular Probes) for 1 hr 30 min at
room temperature. Nuclei were counterstained with Hoechst
33342 (1:10000; Molecular Probes) in PBS. After incubation
with secondary antibodies, the slides were washed in running
tap water, dehydrated, cleared, and mounted.
Experimental animal model
NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice were purchased
from the Jackson Laboratory and housed under specific
pathogen-free conditions in accordance with the Principles of
Laboratory Animal Care and the Guide for the Use of La-
boratory Animals of Samsung Biomedical Research Institute
(20170116003). Twenty-four hours after intraperitoneal injec-
tion of Jo2 antibody/PBS (BD Pharmingen, CA) at 0.1–0.2 mg/
kg body weight, a 3D alginate hydrogel containing miHeps
(*0.5 · 0.5 cm) was implanted into the peritoneal cavity of
mice. After implantation, the mice received 100 mg/L cipro-
floxacin (CJ Pharma, Korea) in their drinking water to prevent
infection. At termination, mouse tissue and the 3D alginate
hydrogel scaffold were immediately fixed in 10% formalin.
578 KANG ET AL.

Statistical analysis reprogramming pMX vectors encoding two transcription

Quantitative data are presented as mean – standard devi-
ation with inferential statistics ( p-values). Statistical sig-
nificance was evaluated using two-tailed t-tests with
significance set at *p < 0.05, **p < 0.01, and ***p < 0.001.
Results
Induction of hepatic lineage cells from MEFs by direct
conversion methods
To generate the miHeps, MEFs were prepared from an
E16.5 embryo. The fibroblasts were virally transduced with
factors, Foxa3 and HNF4a (Fig. 1A). After 10–12 days,
hepatocyte-like cells appeared. To evaluate the hepatic
characteristics of these miHeps, hepatic marker gene ex-
pression levels were measured by quantitative real-time
polymerase chain reaction (qRT-PCR). Expression levels of
Albumin, AFP, ASGR1, and HNF4a in miHeps were signifi-
cantly higher than in MEFs (Fig. 1B). Moreover, miHeps
were positive for hepatic proteins, Albumin, E-cadherin,
ASGR1, HNF4a, CYP1a2, and CK18 on immunofluores-
cence staining as compared with MEFs (Fig. 1C). Also, Al-
bumin secretion and urea synthesis were increased on miHeps
compared with MEFs (Supplementary Fig. S1). These results

FIG. 1. Generation and

characterization of miHeps.
(A) Scheme of miHep pro-
duction. Epithelial
hepatocyte-like cells appear
10–12 days after infection
with retroviruses carrying
two hepatic transcription
factors. (B) Hepatocyte
marker gene expression
evaluated by qRT-PCR. Ex-
pression of Albumin, AFP,
ASGR1, and HNF4a is up-
regulated during the genera-
tion of miHeps. *p < 0.05,
**p < 0.01, and ***p < 0.001.
MEFs, mouse embryonic fi-
broblasts; miHeps, mouse-
induced hepatocytes; mPHs,
mouseprimary hepatocytes.
(C) Hepatic marker protein
expression evaluated by im-
munofluorescence assays be-
tween MEFs and miHeps.
The miHeps stain for
Albumin, CK18, HNF4a,
E-cadherin, CYP1a2, and
ASGR1. Scale bar, 25 mm.
qRT-PCR, quantitative real-
time polymerase chain
reaction.
CONSTRUCTION OF MULTILAYERED 3D-PRINTED HEPATIC STRUCTURES 579

suggest that direct conversion techniques are effective for
generating hepatocyte-like cells from fibroblasts.
3D bioprinting of miHeps with alginate,
and improvement of hepatic function
minished on day 14 after printing (Fig. 2C). AFP, which is
not expressed in normal hepatocytes, gradually decreased.
Overall, these hepatic function data suggest that directly
converted miHeps mature during incubation upon 3D-
printed scaffold.

Before 3D printing, we labeled miHeps with mCherry

lentivirus to trace miHeps proliferation and migration in
3D scaffold (Fig. 2A). To produce 3D hepatic scaffold,
mCherry-miHeps and alginate mixtures were ejected from
a 3D printer (Fig. 2B). Under fluorescence microscopy, we
confirmed mCherry-miHeps distribution on day 1 (Fig. 2B).
They were cultured in vitro with miHeps medium for 28 days
to form miHeps colonies. To evaluate gene expression in the
scaffold, we performed qRT-PCR at several time points.
Expression of Albumin gradually increased, for up to 28 days,
but ASGR1 and HNF4a increased only on day 28 and di-
Proliferation of functional miHeps
in 3D-printed scaffold
We investigated whether 3D-printed hepatic scaffold
facilitates proliferation of miHeps. As we expected, the mass
of mCherry expressing miHeps increased gradually day by
day (Fig. 3A–E), and proliferation of the cells was confirmed
by fluorescence microscopy (Fig. 3F–J). miHeps appeared to
be highly compact by days 14 and 28 after printing, as revealed
by hematoxylin and eosin (H&E) staining (Fig. 3K–O). Sur-
prisingly, the cells strongly expressed Albumin and CK18,

FIG. 2. Generation of 3D hepatic structures using a 3D bioprinter. (A) mCherry lentivirus infection into miHeps for
tracing proliferation and migration. Scale bar, 100 mm. (B) The 3D bioprinter and the shape of the printed 3D hepatic
structures. Five layers of alginate scaffold each measuring 25 · 25 mm were constructed. And confirming distribution of
mCherry-expressing miHeps under fluorescence microscopy on day 1. Scale bar, 500 mm. (C) Hepatocyte marker gene
expression in the 3D structures evaluated by qRT-PCR after 28 days. Expression of Albumin, ASGR1, and HNF4a increased
over time, whereas AFP expression gradually decreased. **p < 0.01, ***p < 0.001. 3D, three-dimensional.
580 KANG ET AL.

FIG. 3. Proliferation of functional miHeps in 3D hepatic scaffolds in vitro. (A–E) Bright field images of miHeps grown in
3D hepatic structures. Scale bar, 100 mm. (F–J) Fluorescence images of miHeps labeled with mCherry. Scale bar, 100 mm.
(K–O) Hematoxylin and eosin staining. Scale bar, 50 mm. (P–Y) Immunohistochemical analysis of the expression of
Albumin and CK18 using confocal microscopy. Scale bar, 50 mm.

demonstrating that rapidly proliferating miHep cells retained
typical hepatic characteristics in the scaffold (Fig. 3P–Y). In
addition, no cell death was apparent in these structures (Sup-
plementary Fig. S2). These data demonstrate that the 3D-
printed framework facilitates miHep cell proliferation without
loss of hepatic function.
Transplantation of a 3D-printed scaffold
with miHeps into mice
To evaluate miHeps proliferation and hepatic function
in vivo, 3D hepatic scaffold was transplanted into omentum
of Jo2-treated NOD Scid gamma (NSG) mice. 3D-printed
hepatic scaffold was stabilized for 7 days in vitro and
transplanted (Fig. 4A). Twenty-eight days after transplan-
tation, miHep scaffold was found in the omentum, and some
pieces were observed to be also surrounding the liver (Fig. 4B).
We harvested the scaffold to check the status of the miHeps
and confirmed hepatic marker gene expression. As shown in
Figure 3, in vivo, proliferating miHeps in the scaffold could
be detected after 28 days (Fig. 4C). Surprisingly, the mi-
Heps grew more rapidly and expressed higher levels of
Albumin in vivo than in vitro (Fig. 4C, D). Also, we per-
formed immunocytochemistry to evaluate whether miHeps
differentiated into cholangiocytes in vivo. Interestingly,
some miHeps were stained with CK19 antibody, and they
were colocalized with mCherry expression around the bile
duct on day 28 (Supplementary Fig. S3). These proliferating
cells were detected (Supplementary Fig. S4). These results
confirm that miHeps proliferate and differentiate faster when
transplanted on 3D scaffold than in vitro.
We further examined the hepatic function of 3D hepatic
scaffold around the liver. We were able to detect mCherry
fluorescence around mouse liver tissue on days 14 and 28
FIG. 4. 3D hepatic scaffolds transplanted into mouse omenta. (A) The experimental schedule. Seven days after 3D
printing, 3D hepatic structures were transplanted in jo2-treated NSG mouse omenta, and harvested on days 14 and 28. (B)
The transplanted 3D hepatic scaffold is well-engrafted around liver tissue at 28 days (red arrow). (C) Proliferation of
miHeps in 3D hepatic scaffolds seen by fluorescence microscopy. The miHeps formed larger colonies in vivo than in vitro
by days 14 and 28. Scale bar, 100 mm. (D) Hepatocyte marker genes (Albumin and AFP) evaluated by qRT-PCR in vitro and
in vivo cultured miHeps. **p < 0.01, ***p < 0.001. NSG, NOD Scid gamma.

FIG. 5. Histological analysis of 3D hepatic scaffolds with mouse liver. (A–D) Fluorescence images of miHeps labeled
with mCherry on days 14 and 28. Scale bar, 250 mm. (E–H) Immunohistochemical analysis of levels of Albumin and CK18
by confocal microscopy on days 14 and 28. Scale bar, 250 mm. White asterisk is mouse liver tissue. (I–L) Hoechst (blue)
labels all nuclei. Red (mCherry) and green (Albumin and CK18) fluorescence colocalize (M–T, enlarged in Q–T).

581
582
(Fig. 5A–D). Albumin (Fig. 5E, G) and CK18 (Fig. 5F, H)
were also expressed, and expression of both proteins colo-
calized with mCherry expression (Fig. 5 M–P, enlarged
panel in Q–T). These observations indicate that bioprinted
3D hepatic scaffold can engraft around liver tissue, and may
offer an alternative to liver transplantation.
Discussion
Liver transplantation is a vital procedure for patients with
end-stage chronic liver disease.24,25 However, the number of
potential liver transplantation recipients are increasing faster
than the number of potential donors.21 To overcome this
donor shortage, many researchers have attempted cell ther-
apies using stem cells,26 and are now focusing on generating
3D scaffold made up of stem cells within a matrix.27 In this
study, we used a direct conversion technique to generate
hepatocyte-like cells as an alternative cell source for cell
therapy, together with 3D printing to form 3D scaffold of
liver organoids. Although hepatocyte-like cells can be de-
rived from human pluripotent stem cells,3–6 there are dis-
advantages to this process. The major issues are that their
differentiation into functional mature hepatocytes is chal-
lenging to achieve, and sometimes these cells can form
tumors in vivo.27 In contrast, directly converted hepatocyte-
like cells do not need to follow liver development mecha-
nism during differentiation, and they do not form teratomas
after transplantation.27 Therefore, we transduced HNF4a
and Foxa3 into MEFs, and by 3 or 4 days after infection,
the fibroblasts had transformed into polygonal epithelial
cells referred to as miHeps.28 This process activates the
mesenchymal–epithelial transition and hepatocyte markers.29
Lim et al. have reported that treatment with A83-01, which
is a transforming growth factor beta receptor inhibitor,
boosts miHeps generation with just one gene. miHeps of
generation was first reported with small molecules.29 For
clinical trials of directly converted hepatocyte-like cells to
be approved, they must be generated without exogenous
genes or induced pluripotent stem cells.30 Therefore, we
will attempt miHeps generation without exogenous genes
such as mRNA or other small molecules in the future.
The geometry of the extracellular matrix that was formed
using collagen sandwiches can maintain hepatocytes long
term and to expand their function.18 Therefore, we hy-
pothesized that miHeps would also maintain their hepatic
function and proliferation on 3D scaffold or 3D structure.
Although collagen is theoretically the optimal extracellular
matrix material, we used alginate hydrogels as bioink. This
is because collagen requires certain temperatures and pH
levels to harden,31 and 3D bioprinters are incapable of
handling collagen as an extracellular matrix material.
Therefore, further development of bioinks and techniques
for seeding cells under the desired conditions is needed.
In summary, we have shown that miHeps can be used as a
liver cell source in 3D hepatic scaffold and that they in-
crease in number and function when seeded upon scaffold
and incubated. Thus, the transplantation of 3D-printed mi-
Heps suggest that 3D bioprinting techniques will improve
cell therapy techniques and survival rates. Although chal-
lenges related to 3D bioprinters, bioink, and cell sources
remain, bioprinting is poised to become one of the most
promising methods of creating bioartificial organs.
KANG ET AL.
Acknowledgment
This work was carried out with the support of the ‘‘Co-
operative Research Program for Agriculture Science and
Technology Development (Project No. PJ01100202),’’
Rural Development Administration, Republic of Korea.
Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Dongho Choi, MD, PhD
Department of Surgery
Hanyang University College of Medicine
Seoul 04763
Korea
E-mail: crane87@hanyang.ac.kr
Jaemin Jeong, PhD
Department of Surgery
Hanyang University College of Medicine
Seoul 04763
Korea
E-mail: jmj1103@gmail.com
Received: April 10, 2017
Accepted: July 10, 2017
Online Publication Date: January 9, 2018
Reproduced with permission of copyright owner. Further reproduction
prohibited without permission.