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Copyright 2015 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Decellularization and Recellularization of Rat Livers With

Hepatocytes and Endothelial Progenitor Cells
*Pengcheng Zhou, *Yan Huang, Yibing Guo, *Lei Wang, *Changchun Ling,
*Qingsong Guo, *Yao Wang, *Shajun Zhu, *Xiangjun Fan, *Mingyan Zhu, Hua Huang,
*Yuhua Lu, and *Zhiwei Wang
*Department of General Surgery; Department of Emergency Surgery; Surgical Comprehensive Laboratory; and
Department of Pathology, Affiliated Hospital of Nantong University, Nantong, China

Abstract: Whole-organ decellularization has been identified as a promising choice for tissue engineering. The aim
of the present study was to engineer intact whole rat liver
scaffolds and repopulate them with hepatocytes and
endothelial progenitor cells (EPCs) in a bioreactor.
Decellularized liver scaffolds were obtained by perfusing
Triton X-100 with ammonium hydroxide. The architecture
and composition of the original extracellular matrix were
preserved, as confirmed by morphologic, histological, and
immunolabeling methods. To determine biocompatibility,
the scaffold was embedded in the subcutaneous adipose
layer of the back of a heterologous animal to observe the

infiltration of inflammatory cells. Hepatocytes were

reseeded using a parenchymal injection method and cultured by continuous perfusion. EPCs were reseeded using
a portal vein infusion method. Morphologic and functional
examination showed that the hepatocytes and EPCs grew
well in the scaffold. The present study describes an effective method of decellularization and recellularization of
rat livers, providing the foundation for liver engineering
and the development of bioartificial livers. Key
Words: DecellularizationExtracellular matrixLiver
Tissue EngineeringEndothelial progenitor cells
Recellularization.

Liver transplantation is currently regarded as the

only definitive and curative therapy for end-stage liver
disease. However, the increasing demand for transplantable livers far exceeds the availability of donor
livers (1). In addition, surgical complications, chronic
rejection, and high expenses limit the wide application
of liver transplantation. Hepatocyte transplantation
offers an alternative method to treat patients with
liver diseases (2,3). Although clinical studies have
shown the efficacy of hepatocyte transplantation
(36), it is associated with problems predominantly
related to the limited cell supply and low engraftment
efficiency (79). Bioartificial livers (BALs) are a temporary alternative to liver transplantation; they can be
used to sustain the patient until a suitable donor organ

becomes available (10). However, BAL cannot substitute liver transplantation permanently. Using the
concept of tissue engineering, artificial threedimensional scaffolds have been generated and shown
to successfully enhance the attachment and survival of
hepatocytes (1114). However, the liver is a complex
organ that requires a constant delivery of nutrients
and oxygen, and the removal of metabolic products.
In addition, the artificial scaffolds are not tissuespecific because of the lack of specific cell binding
factors for cell functions. In recent years, with the
development of regenerative medicine, a promising
approach for organ replacement has emerged.
Bioscaffolds derived from decellularized organs have
been used to create materials for tissue engineering
applications. Using this technology, organs such as the
heart (1519), lung (2028), liver (2937), and kidney
(3845) have been decellularized and recellularized
successfully. The decellularized scaffolds, consisting
of extracellular matrix (ECM), show good biocompatibility, provide tissue microarchitecture and intact
vascular systems, and maintain biological factors that
promote cell attachment, migration, and proliferation
(46). With these advantages, decellularized scaffolds

doi:10.1111/aor.12645
Received February 2015; revised August 2015.
Address correspondence and reprint requests to Dr. Zhiwei
Wang, Department of General Surgery, Affiliated Hospital of
Nantong University, No. 20, XISI Road, Nantong, Jiangsu Province 226001, China. E-mail: [email protected] or Dr. Yuhua Lu,
Departments of General Surgery, Affiliated Hospital of Nantong
University, No. 20, XISI Road, Nantong, Jiangsu Province 226001,
China. E-mail: [email protected]

Artificial Organs 2015, ():

P. ZHOU ET AL.

are regarded as a promising choice for liver tissue

engineering.
The ultimate goal of the decellularization process
is to remove cellular material while minimizing the
damage to the ECM. Commonly used methods of
decellularization include a combination of physical,
chemical, and enzymatic approaches (47). Because of
the thickness and complex intrinsic structures of the
liver, perfusion decellularization is the best choice for
the construction of liver scaffolds. In the present
study, we used Triton X-100 (Amresco, Solon, OH,
USA), which is a non-ionic detergent, as the main
detergent to remove cellular components. After the
decellularization process, we examined the
decellularization efficiency, ultrastructure and ECM
components, and the biocompatibility of the scaffold.
Revascularization remains the major challenge for
creating an artificial organ. All organs require a vascular
network to supply oxygen and nutrients, and the intact
vascular structure of the artificial organ can be directly
connected to the circulation of the recipient. The advantage of decellularized scaffolds over polymer scaffolds is
the presence of an intact vascular network. Researchers
have used microvascular endothelial cells and human
umbilical vein endothelial cells for endothelialization of
decellularized liver scaffolds (29,34,48,49). Endothelial
progenitor cells (EPCs) are a cell population that is
released mainly from the bone marrow into the peripheral blood circulation and have been confirmed to participate in vasculogenesis (5052). EPCs have been used
in the endothelialization of blood vessels (53,54) and
heart valves (55,56); however, they have not been used
in the endothelialization of liver decellularized scaffolds. In the present study, we cultured EPCs from rat
bone marrow and repopulated them into decellularized
liver scaffolds.
The methods used for reseeding of the whole organ
include direct parenchymal injection and infusion. In
our study, we compared these two methods for
hepatocyte reseeding. After the reseeding process,
the scaffold was transferred to the chamber and cultured under continuous perfusion. During the culture
process, the specific function of the cells in the scaffold
was monitored. At the end of the culture, the scaffold
was harvested and histological examination was performed to observe the repopulation status.
MATERIALS AND METHODS
Animals
C57BL/6 mice weighing 2025 g, Sprague Dawley
(SD) rats weighing 300350 g, and 4-week-old SD
rats were purchased from the Experimental Animal
Center of Nantong University, Nantong, China. All
Artif Organs, Vol. , No. , 2015

the animals were kept under constant environmental

conditions with a 12-h light/dark cycle and free access
to water and food. All animal procedures were
performed according to institutional and national
guidelines and approved by the Animal Care Ethics
Committee of Nantong University.
Harvesting of livers from SD rats
SD rats were anesthetized by intraperitoneal injection of chloral hydrate (5%, 0.5 mL/100 g). Under
deep anesthesia, rats were treated with topical skin
disinfectant, and a laparotomy extending from the
pubis to the xyphoid was performed in the abdomen.
The distal end of the portal vein (PV) was ligated, and
then the vein was cannulated with a 22-G cannula and
fixed with 3-0 silk sutures. A total of 2 mL heparin
sodium (100 U/mL) was injected through the vein for
anticoagulation. Then, the infrahepatic inferior vena
cava was transected to allow the outflow of the
perfusate. A total of 50 mL phosphate-buffered saline
(PBS) was perfused slowly through the PV clear blood
from the liver. Then, the suprahepatic inferior vena
cava, the hepatic artery, and the common bile duct
were freed. Finally, the whole liver was isolated and
transferred to a cell culture dish.
Decellularization of the liver
The cannula in the PV was connected to the peristaltic pump (Masterflex Technical Hoses Ltd.,
Oldham, UK), and the perfusion rate for each step
was set at 4 mL/min. The decellularization process
was initiated by PV perfusion with PBS for 1 h, followed by distilled water for 30 min. Then, the liver
was perfused with 0.02% ethylenediaminetetraacetic
acid (EDTA) (Sinopharm Chemical Reagent Co.
Ltd., Shanghai, China) in PBS for 30 min. Then, as
the most important step of the decellularization, 1%
(w/v) Triton X-100/0.1% ammonium hydroxide
(Xilong Chemical Reagent Co. Ltd.) in distilled water
was perfused for 20 h. Subsequently, distilled water
was used to rinse cellular lysis and circuit debris for
2 h. Finally, the liver was perfused with PBS for 4 h to
maintain isotonicity. The decellularized liver scaffold
was preserved in PBS at 4C.
Analysis of ECM components
To examine the ECM components of the scaffolds,
tissues were randomly cut from fresh livers (n = 3) and
decellularized liver scaffolds (n = 3) and fixed with
4% formaldehyde, dehydrated, and embedded in paraffin. Tissue sections were deparaffinized and stained
with hematoxylin and eosin (H&E), Massons
trichrome, and Sirius red stain. Slides were visualized
and recorded under an Olympus microscope

RECELLULARIZATION OF HEPATOCYTES AND EPCs

(Olympus Microscope Corp., Tokyo, Japan) for H&E
and Massons trichrome stain and a Nikon E200
(Nikon, Nanjing, China) for detection of the Sirius red
stain. To determine whether collagen I, collagen IV,
laminin, and fibronectin were retained in the
decellularized scaffolds, the slides were blocked
against nonspecific binding with a solution consisting
of 1% bovine serum albumin (BSA) (Biosharp, Hefei,
China) and 0.1% Triton X-100 in PBS for 30 min at
37C. Primary antibodies were diluted with blocking
buffer, anti-collagen I (Bioss Ltd., Beijing, China),
anti-collagen IV (Bioss), anti-laminin (Santa Cruz
Biotechnology [Shanghai] Co. Ltd., Shanghai, China),
and anti-fibronectin (Abcam, Cambridge, UK), and
incubated with the slides overnight at 4C. After that,
slides were incubated with secondary biotinylated
goat anti-rabbit antibodies (Zsbio, Beijing, China).
Slides were visualized and recorded under an
Olympus microscope for H&E, Massons trichrome
stain, and immunohistochemical analysis, and a Nikon
E200 for detection of the Sirius red stain.
Scanning electron microscopy (SEM) observation
Samples were randomly cut from the decellularized
liver scaffolds (n = 3) and sectioned into small blocks.
First, samples were fixed in 2.5% glutaraldehyde overnight followed by three washes in PBS for 10 min
each. Then, samples were fixed in 1% osmic acid in the
dark. This was followed by another three PBS washing
steps of 10 min each. After that, samples were dehydrated in a gradient series of alcohol for 10 min each
(30, 50, 70, 90, 95, and 100%). Next, isoamyl acetate
was added to exchange the alcohol twice for 10 min
each. Subsequently, samples were critical point dried
and sputter-coated with gold. Finally, images were
observed and recorded under a scanning electron
microscope (HITACHI, Tokyo, Japan).
DNA content assay
Fresh livers and the decellularized liver scaffolds
(n = 3) were lyophilized. Three small samples from
each scaffold weighing 25 mg were cut and crushed
into small pieces, and then digested with proteinase
K for 24 h at 60C. DNA was isolated using the
Dneasy Tissue kit (Tiangen, Beijing, China) according to the manufacturers instructions. The concentration of DNA (ng/mL) was measured using an
ultraviolet spectrophotometer. Finally, the DNA
content in the tissues was determined.
GAG content assay
Glycosaminoglycan (GAG) content was measured
using the GAG assay kit (Hermes Criterion Biotechnology, Vancouver, Canada). Fresh livers and the

decellularized liver scaffolds (n = 3) were used.

Three small samples from each scaffold weighing
10 mg were incubated with papain extraction reagent
(Sigma-Aldrich Corp., St. Louis, MO, USA) for 3 h
at 65C. Assays were then performed according to
the manufacturers instructions. Absorbance was
recorded on a microplate spectrophotometer. GAG
content was calculated based on the standard curve
obtained from the standard GAG specimen.
Biocompatibility of the decellularized liver scaffolds
in heterologous animals
Tissues cut from the decellularized liver scaffolds
(n = 3) were sectioned into 5 mm in diameter and 3 mm
in thickness and immersed in 0.1% peracetic acid for
3 h on the shaker. Then, the blocks were washed twice
with PBS on the shaker for 30 min each. The operation
was performed under sterile conditions. A 5-mm incision was made on the back of C57BL/6 mice (n = 20),
and the tissue blocks were embedded in the subcutaneous adipose layer on the back. The incision was closed
with 5-0 sutures and disinfected with iodine after the
operation for 4 consecutive days. In addition, 20 000
units of penicillin were injected intraperitoneally for 3
consecutive days after the operation. The tissues were
harvested on postoperative days 5, 7, 10, 14, and 21 and
stained with H&E.
Hepatocyte culture
The rat normal liver cell line BRL was obtained
from the cell bank of the Chinese Academy of
Science, Shanghai. Cells were cultured in Dulbeccos
modified Eagles medium (DMEM) (Hylcone,
Shanghai, China) supplemented with 10% fetal
bovine serum (FBS) (Gibco, Grand Island, NY,
USA), penicillin (1 105 U/L), and streptomycin
(100 mg/L) (Gibco) on tissue culture flasks. The cells
were cultured in an incubator at 37C and 5% CO2.
The medium was changed every other day.
Toxicity assay of the decellularized liver scaffolds
Tissues were immersed in 0.1% peracetic acid for
3 h and then washed three times with PBS on the
shaker for 30 min each. DMEM was added to the
scaffolds at a concentration of 0.1 mL/g and incubated at 37C for 24 h to extract liquid. The extracted
liquid was filtered through 0.22-m filters before use.
Two types of medium were prepared: a, DMEM with
10% FBS; and b, the extracted liquid with 10% FBS.
BRL cells were seeded in DMEM with 10% FBS at a
density of 2000 cells/well on four 96-well plates and
divided into two groups, A and B. The medium was
removed overnight. A total of 100 L of a and b
media was added to groups A and B, respectively.
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P. ZHOU ET AL.

Then, 10 L of the CCK-8 (Dojindo, Laboratories,

Kumamoto, Japan) solution was added into each well
of one plate at 0, 12, 24, and 48 h and incubated at
37C for 2 h. Absorbance was measured at 450 nm on
a microplate reader.
Repopulation of BRL cells into the decellularized
liver scaffolds in vitro
A circulation perfusion device (EQUL Ltd.,
Houston, TX, USA) consisting of a peristaltic pump,
oxygenator, and chamber was set up (Fig. 7b,c). The
device was filled with 50 mL of DMEM supplemented
with 10% FBS, 10 ng/mL epidermal growth factor
(Peprotech, Rocky Hill, NJ, USA), penicillin
(1 105 U/L), and streptomycin (100 mg/L) and
placed in the incubator filled with 5% CO2 at 37C. To
recellularize the scaffold in vitro, a total of 2 107 cells
were prepared. We compared the two methods, the
multiposition parenchymal injection method and
infusion method. For the multiposition parenchymal
injection method, the prepared cells were dissolved in
2 mL of medium and injected at 10 sites (four sites in
the left lateral lobe, two in the left median lobe, two in
the right median lobe, and two in the right lateral
lobe) with a 29-G needle (BD, Suzhou, China)
(Fig. 7a). For the infusion method, the prepared cells
were dissolved in 2 mL of medium, and injected
through the PV in four steps, at 15 min intervals. Each
method was performed a total of four times. The
medium was collected and the engraftment rate was
calculated from the total number of cells minus the
number of cells floating in the medium. After 40 min
of stationary culture, the scaffold was transferred to
the chamber under aseptic conditions. The cannula in
the PV was connected with the circulation as the inlet,
and the other vessels worked as the outlet.
Dynamic three-dimensional culture of the
recellularized liver scaffold and the function
of hepatocytes
The medium was continuously perfused through
the PV at 1 mL/min for 2 days and changed to 2 mL/
min thereafter. The medium was changed daily and
detected using an automatic biochemical analyzer
(Siemens Healthcare Diagnostics Ltd., Tarrytown,
NY, USA) for assessment of liver function according
to albumin and total bile acid secretion. 2-D culture in
dishes was set as a control. After 7 days of culture in
vitro, the recellularized liver scaffold was fixed and
evaluated by H&E staining, SEM examination, and
albumin immunofluorescence. For the assessment of
albumin immunofluorescence, tissue sections were
blocked against nonspecific binding with 1% BSA for
30 min and then incubated in 0.1% Triton X-100 for
Artif Organs, Vol. , No. , 2015

5 min at 37C. Then, the sections were incubated with

rabbit anti-rat ALB (Santa, Cruz Biotechnology,
1:50) overnight at 4C and washed with PBS. Next, the
sections were incubated with Alexa Fluor 488conjugated donkey anti rabbit (Proteintech, Wuhan,
China, 1:200) at room temperature for 1 h. In addition, the slides were stained with Hoechst (SigmaAldrich Corp., St. Louis, MO, USA) to label the
nucleus for 10 min. Slides were visualized and
recorded under an Olympus fluorescence microscope.
EPC isolation and culture from the rat
bone marrow
Bone marrow mononuclear cells (BMMNCs) were
isolated from rat bones as described previously by our
group (57). The femur and tibia of hind legs
were removed from 4-week-old SD rats after
heparinization and euthanasia. The bones were repeatedly washed with endothelial basal medium (EBM2,
Lonza, Allendale, NJ, USA) until the bone marrow
cavity became white. The washing fluid was filtered
through a 40 m mesh (Franklin Lakes, NJ) to obtain a
single-cell suspension. Single-cell suspensions were
lysed with ACK lysing buffer (Gibco) and then washed
with EBM2 twice. The BMMNCs were then suspended
in endothelial growth medium (EGM2-MV, Lonza)
containing VEGF, hFGF, IGF, hEGF, ascorbic acid,
hydrocortisone, and 5% FBS and plated in 10 g/mL
human fibronectin (Sigma) coated T25 plates (Corning,
Shanghai, China). The cells were cultured in an incubator under 5% CO2 at 37C. After 24 h, the nonadherent
cells were transferred to a new plate coated with
fibronectin to remove rapidly adherent cells. After
another 3 days, the nonadherent cells were aspirated
and the EGM2-MV medium was changed every day
until day 7 and on alternate days thereafter. When the
cells reached 8090% confluency, they were digested
with 0.25% trypsin/EDTA (Gibco) and passaged at the
ratio of 1:2. EPCs were seeded on fibronectin-coated
slides after digestion and cultured for 24 h in EGM2MV. The slides were blocked with 1% BSA for 30 min
to prevent nonspecific antibody-antigen binding. Then,
the slides were incubated with the following primary
antibodies: rabbit anti-rat CD133 (Abcam, 1:100) and
goat anti-rat CD31 (Santa, Cruz Biotechnology, 1:50)
overnight at 4C and washed with PBS. Next, the slides
were incubated with the following secondary antibodies
(Proteintech, 1:200): Alexa Fluor 488-conjugated
donkey anti-rabbit for CD133 and TRITC-conjugated
donkey anti-goat for CD31 at room temperature for 1 h.
In addition, the slides were stained with Hoechst
(Sigma) to label the nucleus for 10 min. Slides were
visualized and recorded under an Olympus fluorescence
microscope.

RECELLULARIZATION OF HEPATOCYTES AND EPCs

Endothelialization of the decellularized
scaffold with EPCs
Decellularized liver scaffolds were perfused with
EGM2 through the PV before endothelialization. A
total of 5 106 EPCs were suspended in 1 mL of
EGM2 and seeded through the PV very slowly. After
stationary culture for 4 h, the scaffold was transferred
to the chamber of the circulation perfusion device.
The circulation culture was performed at the flow
rate of 1 mL/min for another 3 days. Finally, the
scaffold was harvested for H&E staining and
immunofluorescence for CD31.
Statistical analysis
Statistical analysis was performed using SPSS 22
(SPSS Inc., Chicago, IL, USA). Statistical differences
were evaluated by the KruskalWallis test. A P value
<0.05 was considered significant. The mean values of
DNA, GAG, albumin synthesis, and total bile acid
secretion were expressed as the mean standard
deviation (SD).
RESULTS
Perfusion decellularization of rat livers
Whole liver decellularization was achieved by
portal perfusion with Triton X-100 and other
reagents. The PV was successfully cannulated with a
22-G cannula and fixed with 3-0 silk sutures (Fig. 1a).
The liver color turned soft red after the blood was
washed out (Fig. 1b,c). After rinsing with distilled
water, the liver turned yellowish brown and showed
mild swelling (Fig. 1d). During the perfusion of 1%
(w/v) Triton X-100/0.1% ammonium hydroxide, liver
cells were washed out in great numbers, and the liver
color quickly became semi-transparent. At the end of
the process, the liver was transparent and the
acellular scaffold retained the gross shape of the liver
(Fig. 1e). The vascular system was visualized clearly
through the capsule of the acellular scaffold (Fig. 1f).

Histological and immunohistochemical

examinations of the scaffold
H&E staining of the decellularized liver scaffolds
showed the absence of cells compared with the native
liver (Fig. 2a,d). Massons trichrome staining confirmed these result. Most of the collagen fiber components (stained blue) were retained and maintained
a tubular structure (Fig. 2b,e). Sirius red staining
showed that the collagen structure of the vascular
walls was retained after the decellularization process
(Fig. 2c,f). Immunohistochemical analysis of the
native liver and scaffold showed that collagen I
(Fig. 2h,m), collagen IV (Fig. 2i,n), fibronectin
(Fig. 2j,o), and laminin (Fig. 2k,p) were preserved
and distributed around the vascular structures and
parenchymal areas.
SEM examination
SEM showed the three-dimensional ECM microstructure of the decellularized liver scaffolds and confirmed the absence of cell components. The space
surrounded by the collagen fibers in the ECM was
thought to be the original position of hepatocytes
(Fig. 3a,b).
DNA and GAG content quantification
Quantitative DNA assay showed that the DNA
content of the decellularized liver scaffolds was
48.6 17.1 ng/mg dry weight, and the total DNA
content of the normal liver was 5263.1 336.9 ng/mg dry
weight (Fig. 4a). The GAG content in the decellularized
liver scaffolds was 28.2 0.79 ng/mg wet weight. Normal
liver GAG content was 34.2 0.61 ng/mg wet weight.
These results show that about 80% of GAG was preserved in the decellularized live scaffold during the
decellularization process (Fig. 4b).
Biocompatibility assay
After the operation, the mice behaved normally
regarding eating and drinking. On days 5, 7, 10, 14,
and 21 postsurgery, the scaffolds were removed. The

FIG. 1. Sequential whole-organ decellularization progress. (a) The PV was cannulated with a 22-G cannula and (b) after 50 mL PBS was
perfused through the PV to clear blood. (c) The liver was harvested. (d) After rinsing with distilled water, the liver turned yellowish brown
and swelled a little. (e) After perfusion of EDTA and 1% (w/v) Triton X-100/0.1% ammonium hydroxide, the liver became transparent, and
the acellular scaffold retained the gross shape of the liver. (f) The vascular system was intact.
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P. ZHOU ET AL.

FIG. 2. H&E staining of the normal liver (a) and decellularized liver scaffold (d). Massons trichrome staining of the normal liver (b) and
decellularized liver scaffold (e). Sirius red staining of the normal liver (c) and decellularized liver scaffold (f). Immunohistochemical analysis
of the normal liver and decellularized liver scaffold: negative control (g, l), collagen I (h, m), collagen IV (i, n), fibronection (j, o), and laminin
(k, p).

FIG. 3. ECM microstructure of the decellularized liver scaffolds (a, b). Recellularized scaffold showed that BRL cells engrafted into the
parenchymal area of the scaffold (c, d).
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RECELLULARIZATION OF HEPATOCYTES AND EPCs

FIG. 4. (a) DNA content of the native liver versus the decellularized liver scaffolds (P < 0.001). (b) GAG content of the native liver versus
the decellularized liver scaffold (P < 0.001).

FIG. 5. Histologic change at 5th, 7th, 10th, 14th, 21st day postsurgery. Inflammatory cells began to infiltrate into the scaffold in small
amount at day 5 and reached the peak at day 7, 10. Inflammatory cells began to fade away at day 14, and only a few inflammatory cells
can be seen in the view at day 21. The scaffold retained almost the original structure as before.

scaffolds retained their appearance and texture. The

results of H&E staining are shown in Fig. 5. Inflammatory cells began to infiltrate into the scaffold in
small amounts on day 5. On days 7 and 10, the
number of inflammatory cells began to increase,
reaching a peak value before starting to decrease,
showing obviously reduced numbers on day 14. On
day 21, few inflammatory cells were present in the
scaffold. In addition, the scaffold retained its original
structure. No macrophagocytes or other pathological
signs of graft rejection were observed, suggesting that
the decellularized liver scaffolds were biocompatible.

of the seeding cells. The infused cells tended to block

the vessel lumen or were washed out of the scaffolds
(Fig. 7d). The multipositional parenchymal injection
method resulted in a better engraftment rate
(93.2 4.0%), and cells were dispersed in the parenchymal space after 24 h (Fig. 7e). Therefore, the
second method was selected in our study. After a
stationary culture of 40 min, the scaffold was transferred to the chamber for dynamic perfusion culture

BRL cell growth

BRL cells were cultured in two types of medium
for 48 h, and cell proliferation was assessed in each
medium (Fig. 6). The proliferation curves showed
that the extract liquid of the decellularized liver scaffolds did not inhibit the growth of BRL cells, and
instead showed mild growth promoting activity.
In vitro recellularization of decellularized
live scaffolds
Two recellularization methods were used, namely
multipositional parenchymal injection and the infusion method. The infusion method resulted in a low
engraftment rate (83.4 5.2%) and poor distribution

FIG. 6. The proliferation curve of BRL cells cultured in two kinds

of medium.
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FIG. 7. (a) The multiposition parenchymal injection method. (b, c) The circulation perfusion device which consisted of a peristaltic pump,
oxygenator, and chamber. (d) H&E staining after 24 h by infusion method. (e) H&E staining after 24 h by multiposition parenchymal
injection method. (f, g) H&E staining of the normal liver and the recellularized liver scaffold. (h, i) Immunofluorescence of ALB of the normal
liver and the recellularized liver scaffold.

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RECELLULARIZATION OF HEPATOCYTES AND EPCs

FIG. 8. Function of the BRL cells in the recellularized liver matrix. (a) Albumin secretion over time during the 7 days. (b) Total bile Acid
(TBA) secretion over time during the 7 days.

with daily culture media changes. To assess the metabolic function of the engrafted BRL cells, we detected
albumin production and total bile acid secretion daily.
The results showed that albumin production in the
scaffold was lower than that in the 2-D dish culture
for the first 3 days, whereas it was higher from day
4 to day 7. The 7-day cumulative albumin production
in the scaffold was significantly higher than that
in the 2-D dish culture (48.14 3.97 g/106 cells vs.
42.76425 0.448 g/106 cells, P < 0.05) (Fig. 8a). Total
bile acid secretion in the scaffold was higher than that
in the 2-D dish culture for the first 4 days, whereas it
was lower from day 5 to day 7. The difference in the
cumulative total bile acid secretion was not statistically significant (Fig. 8b). The production of albumin
and total bile acid increased gradually. These results
indicated that the decellularized live scaffold is suitable for BRL cell attachment and growth. After 7 days
of perfusion culture, the scaffold was harvested. H&E
staining, immunofluorescence staining for albumin,
and SEM were performed. H&E staining and albumin
immunofluorescence revealed that the BRL cells
were distributed in the matrix in a pattern resembling
the structure of the normal liver (Fig. 7fi). SEM
showed that BRL cells engrafted into the parenchymal area of the scaffold (Fig. 3c,d).
Morphology and characteristics of EPCs
Nonadherent rat BMMNCs cultured for 24 h were
harvested and cultured in EGM2. On day 4,
nonadherent cells were aspirated and adherent cells
formed scattered colonies of spindle-shaped cells
(Fig. 9a). After 68 days, the colonies expanded
quickly and formed clusters (Fig. 9b,c). With increasing passages, the shape of cells gradually changed
from spindle-shaped to round. After passage 2, the
endothelial cell-like cobblestone morphology
began to be observed (Fig. 9d). EPCs were positive

for the endothelial cell markers CD133 and CD31

(Fig. 9e,f).
Endothelialization of the decellularized scaffold
The vascular tree structure of the scaffold was
observed by phase contrast microscopy. For
endothelialization of the scaffold, EPCs from passage
2 were seeded via the PV and cultured for 3 days.
H&E (Fig. 10a,b) and immunofluorescence staining
for CD31 (Fig. 10bd) showed that EPCs covered
the internal surface of the tubular structures in the
scaffold.
DISCUSSION
Scaffold materials composed of ECM derived from
decellularized organs are increasingly being used for
regenerative medicine and tissue engineering. The
ultimate goal of organ decellularization is maximizing
the removal of cellular material while minimizing
alterations in the composition, biologic activity, or
mechanical integrity of the ECM. The efficacy of
decellularization methods depend on certain factors
as follows: (i) the density of the organ, (ii) the cellular
density of the organ, (iii) the lipid content of the
organ, and (iv) the thickness of the organ. Unlike
diffusion techniques used for the decellularization of
simple and thin tissues, solid organs require antegrade
or retrograde perfusion techniques to efficiently
remove cellular components (5861). Commonly
used methods of decellularization include the combination perfusion of acids and bases, hypotonic and
hypertonic solutions, detergents, alcohols, enzymes,
and nonenzymatic agents. In the present study, considering that the liver is an organ with a complicated
structure that is formed of an abundance of cells and
contains many cellular enzymes, we selected continuous perfusion at low speed with detergent containing
EDTA, a hypotonic solution, Triton X-100, and
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P. ZHOU ET AL.

FIG. 9. (ac) Morphologic characteristics of the EPCs after separation from the bone marrow. (d) Morphology of cobblestone-shaped
EPCs. (e, f) Cobblestone-shaped EPCs were positive for CD133 and CD31.

ammonium hydroxide for decellularization. EDTA

aids in cell dissociation from ECM proteins by
chelating metal ions (62). Hypotonic solutions are
used to lyse the cells within the organs. Triton X-100 is
the most widely used nonionic detergent to disrupt
lipidlipid and lipidprotein interactions without
affecting proteinprotein interactions (63). Sodium
dodecyl sulfate (SDS), an ionic detergent, is more
effective than Triton X-100. However, studies have
shown that SDS tends to damage the component of
the ECM (20,31,64). We therefore chose the mild
detergent Triton X-100 instead of SDS. The hypotonic ammonium hydroxide solution was used to lyse
cell membranes.
After the decellularization process, it is necessary
to evaluate the efficiency of the decellularization
method. The removal of cellular material can be
Artif Organs, Vol. , No. , 2015

confirmed by the lack of nuclear material in scaffold

sections stained with H&E and DNA content
assays. Visualization of the gross appearance allows
assessment of the integrity of the vascular structure.
The retention of ECM components is visualized by
histological staining, such as Massons trichrome
and Sirius red stains for collagens. In addition, SEM
is often performed to examine the ultrastructure
within the decellularized scaffolds and to verify the
absence of cellular material. The protection of collagen types I and IV, fibronectin, and laminin is
confirmed by immunohistochemical examinations.
Collagen type I is the most abundant form of collagen in the body, whereas collagen type IV is found
in the basement membrane and provides bioactive
signals to endothelial cells in the vasculature (59).
Fibronectin is an ECM glycoprotein that plays a

RECELLULARIZATION OF HEPATOCYTES AND EPCs

11

FIG. 10. (a, b) H&E staining showed that EPCs covered the internal surface of the tubular structures in the scaffold. (ce)
Immunofluorescence of CD31 staining of the EPCs in the vessels of the scaffold.

role in cell adhesion, growth, migration, and differentiation (65). Laminin is another protein that exists
on the basement membrane and the vasculature of
most organs. GAG is important for the protection
of free growth factors from enzymatic degradation,
as free growth factors are anchored to GAG
(66).
Graft rejection, which occurs as a response to the
antigenic components of xenogeneic tissues, is the
main barrier to the use of xenogeneic scaffolds in
translational applications (67). Xenogeneic antigens
are usually recognized as foreign by the host and
cause a destructive inflammatory response (68). To
test the biocompatibility of the decellularized scaffold, transplantation was performed in heterologous
animals. Decellularized liver scaffolds derived from
SD rats were embedded in the subcutaneous
adipose layer of the back of C57BL/6 mice. Inflammatory cellular infiltration and the original structure
were monitored at different time points. Twenty-one
days after transplantation, the inflammatory cells
were not obvious, and the structure was preserved.
However, the mechanisms of constructive remodeling and degradation of the ECM are only partially
understood. The ECM can promote a switch from
Th1 effector cells to Th2 effector cells and evoke an
M2 phenotypic macrophage immune response (69).
Both Th2 and M2 are responsible for antiinflammatory, wound-healing, and constructive
remodeling responses. Ineffective decellularized
scaffolds containing both DNA and cellular
epitopes, such as the Gal epitope, will promote a

more M1-type response and induce rejection

responses after implantation (69). These results indicate that the decellularized scaffold did not induce
apparent xenograft rejection and shows good biocompatibility.
In the present study, we used the BRL cell line for
recellularization, which has been rarely reported.
Continuous perfusion of medium through the scaffold imitated the blood perfused through the liver in
vivo. The results showed that BRL cells attached and
functioned well in the scaffold. After 7 days of
culture, the BRL cells were laid out in a streak
pattern that was comparable to the hepatocyte
arrangement in the native liver.
Revascularization remains the major challenge for
creating an artificial organ. The intact vascular structure of the artificial organ can be directly connected
to the circulation of the recipient. Decellularized
whole-organ scaffolds retain the vasculature;
however, the use of this vascular system to direct
blood in vivo without thrombus formation requires
proper endothelialization. EPCs can be induced to
differentiate into mature endothelial cells in vitro
and in vivo and have been used to coat the vascular
grafts to produce an intact surface (69). We used
EPCs to revascularize the decellularized liver scaffold. However, there is controversy regarding the
identification and method of culture of this cell
type. Amiel et al. (70) first reported that CD34+
mononuclear cells from the peripheral blood could
be differentiated into EPCs with endothelial structure and function. Asahara et al. (71) showed that
Artif Organs, Vol. , No. , 2015

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P. ZHOU ET AL.

CD34 /CD133+ subpopulations can also be differentiated into EPCs. As reliable cell surface markers
for the detection of EPCs have not been identified,
the separation of EPCs by magnetic-activated cell
sorting is not accurate. Currently, the isolation of
EPCs is achieved by a differential attachment
method. Recent studies (7274) showed that slow
adherent BMMNCs exhibit the genetic phenotype
of both immature and endothelial lineage cells and a
high potential for forming tube-like structures,
whereas fast adherent BMMNCs are less likely to
show the genetic phenotype of endothelial cells and
low tube-like structure forming activity, which are
considered to be monocyte/macrophage or
inflammation-related cell-rich BMMNC populations. As a result, we discarded the 24-h adherent
cell population and harvested the nonadherent cell
population for further culture. Cells were positive
for CD133 and CD31. The EPCs were repopulated
into the vascular lumen through the PV by slow
infusion, and medium was perfused through the vascular system by pressure gradient. The large vessels
had a higher pressure than the microvessels, resulting in more medium perfused than that in
microvessels. As a result, large vessels were better
endothelized than microvessels. To obtain better
results, we may try to infuse fibronectin before
repopulating EPCs, which is beneficial for the settlement and proliferation of EPCs.
CONCLUSIONS
In summary, the present study showed that whole
rat decellularized liver scaffolds can be successfully
obtained by continuous perfusion of EDTA, a hypotonic solution, and Triton X-100/ammonium hydroxide. Analyses verified the preservation of the
ultrastructure and biomechanical properties of the
extracellular matrix. Heterologous transplantation
showed good biocompatibility of the scaffold, which
laid the foundation for xenografts. BRL cells
attached and functioned well in the threedimensional scaffold. Endothelial progenitor cells
were successfully used for re-endothelialization of
the vessels of the scaffolds. Although the present
study was limited to rat livers, we have also successfully applied the decellularization method to porcine
livers, which could be scaled up to human liver size
(data not shown). Additional experiments on
co-reseeding the nonparenchymal liver cells with
hepatocytes are necessary, and the efficiency of
endothelialization needs to be optimized before the
bioengineered liver can be transplanted and function
in large animals. Ultimately, future clinical studies
Artif Organs, Vol. , No. , 2015

could develop such an artificial liver by combining

xenogeneic liver scaffolds with autologous stem cellderived hepatocytes. Taken together, our results
indicated that the decellularized scaffold is a promising choice for liver tissue engineering and regenerative medicine.
Acknowledgments: This work was supported by
the National Natural Science Foundation of China
(no.81471801), Jiangsu provinces key project of
health department (K201101), and the Science and
Technology Innovation Project of Jiangsu Province
and Nantong University for postgraduates. We
acknowledge the technical assistance from Surgical
Comprehensive Laboratory (Affiliated Hospital of
Nantong University), Pathology Department (Affiliated Hospital of Nantong University), and Key
Laboratory
of
Neuroregeneration
(Nantong
University).
Conflict of Interest: None.
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