Technical Reports

Organ reengineering through development of

a transplantable recellularized liver graft using
decellularized liver matrix
Basak E Uygun1, Alejandro Soto-Gutierrez1,6, Hiroshi Yagi1,6, Maria-Louisa Izamis1, Maria A Guzzardi1,2,
Carley Shulman1, Jack Milwid1, Naoya Kobayashi3, Arno Tilles1, Francois Berthiaume1,4, Martin Hertl5,
Yaakov Nahmias1,6, Martin L Yarmush1,4 & Korkut Uygun1
© 2010 Nature America, Inc. All rights reserved.

Orthotopic liver transplantation is the only available treatment
for severe liver failure, but it is currently limited by organ
shortage. One technical challenge that has thus far limited the
development of a tissue-engineered liver graft is oxygen and
nutrient transport. Here we demonstrate a novel approach to
generate transplantable liver grafts using decellularized liver
matrix. The decellularization process preserves the structural
and functional characteristics of the native microvascular
network, allowing efficient recellularization of the liver matrix
with adult hepatocytes and subsequent perfusion for in vitro
culture. The recellularized graft supports liver-specific function
including albumin secretion, urea synthesis and cytochrome
P450 expression at comparable levels to normal liver in vitro.
The recellularized liver grafts can be transplanted into rats,
supporting hepatocyte survival and function with minimal
ischemic damage. These results provide a proof of principle
for the generation of a transplantable liver graft as a potential
treatment for liver disease.
About thirty million people in the United States have liver dis­
order, with roughly 27,000 deaths registered annually due to liver
disease1. The only definitive treatment for severe hepatic failure
is transplantation. However, critical shortage of organs causes a
growing deficit of approximately 4,000 livers per year 2. Hepatocyte
transplantation offers an alternative way to treat patients with liver
diseases3,4, and years of laboratory and clinical studies have demon­
strated its efficacy 5–7. However, a limited cell supply and minimal
engraftment efficiency have limited this approach3,8,9. Whereas the
cell supply limitation can potentially be resolved by a number of
alternative cell sources 10–13, low engraftment efficiency 14 remains
a major concern.
Tissue engineering approaches have thus far been able to partially
improve cell engraftment in animal models by enhancing cell-to-cell
contact and providing nonimmunogenic matrices before transplan­
tation15. However, these approaches have been limited by oxygen
and nutrient diffusion to thin, two-dimensional structures that are
transplanted into highly vascularized regions16–18. Practical consid­
eration of primary hepatocyte oxygen consumption requires that
any hepatic mass, sufficiently large to provide metabolic function,
contains an extensive microvascular network and be connected
to the blood supply, or the internal mass of the cells will suffer
ischemic damage19,20.
Decellularization is an attractive technique for scaffold preparation
in tissue engineering, as the resulting material can potentially retain
the architecture of the original tissue, including the functional aspects
of the native microvasculature21. The potential applications of decellu­
larized matrix in tissue engineering have been demonstrated for a
number of tissues, including bladder22, artery23, esophagus24, skin25
and trachea26. More recently, another group reported the decellulari­
zation of an entire heart through perfusion, preserving the original
architecture and original microvascular network and allowing for
extensive recellularization27.
In this work, we modified and applied their perfusion decellulariza­
tion technique27 to prepare whole liver grafts, and we further intro­
duced perfusion-seeding and culture techniques for the preparation
of recellularized liver matrix for transplantation. A key advantage of
this methodology is the preservation of liver-specific extracellular
matrix and three-dimensional architecture, providing crucial cues
for hepatocyte engraftment, survival and long-term function. Most
importantly, the decellularized liver matrix (DLM) has the underlying
matrix of the vascular network, which can be readily connected to
the circulation, facilitating rapid oxygen and nutrient delivery after

1Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Shriners Hospitals for Children, Boston, Massachusetts, USA.
2Sector of Medicine, Scuola Superiore Sant’Anna, Pisa, Italy. 3Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Shikata-cho,
Okayama, Japan. 4Department of Biomedical Engineering, Rutgers University, Piscataway, New Jersey, USA. 5Transplantation Unit, Massachusetts General Hospital,
Boston, Massachusetts, USA. 6Current addresses: Center for Innovative Regenerative Therapies, Department of Surgery, Transplantation Section, Children’s Hospital
of Pittsburgh, McGowan Institute for Regenerative Medicine and University of Pittsburgh, Pittsburgh, Pennsylvania, USA (A.S.-G.), Department of Surgery, Keio
University School of Medicine, Shinjuku-ku, Tokyo, Japan (H.Y.) and the Selim and Rachel Benin School of Computer Science and Engineering, The Hebrew University
of Jerusalem, Jerusalem, Israel (Y.N.). Correspondence should be addressed to K.U. (kuygun@partners.org).

Received 1 July 2009; accepted 3 February 2010; published online 13 June 2010; doi:10.1038/nm.2170

814 VOLUME 16 | NUMBER 7 | JULY 2010  nature medicine

 Technical Reports

a b c d e

f H&E Collagen I Collagen IV Fibronectin Laminin

Normal liver Normal liver Normal liver Normal liver Normal liver

H&E Collagen I Collagen IV Fibronectin Laminin

© 2010 Nature America, Inc. All rights reserved.

Decellularized liver Decellularized liver Decellularized liver Decellularized liver Decellularized liver

Figure 1  Decellularization of ischemic rat livers. (a–e) Representative images of ischemic rat livers during decellularization process at 0 h (a), 18 h
(b), 48 h (c), 52 h (d) and 72 h (e). The livers were perfused through the portal vein using SDS as described in the Online Methods. (f) Comparison of
normal liver (top) and DLM (bottom). Left to right: H&E, collagen I (red), collagen IV (red), fibronectin (red) and laminin (red) staining. Sections were
counterstained with DAPI (blue). Scale bars: 10 mm (a–e) and 100 μm (f).

transplantation. The large pool of donor organs unsuitable for trans­
plantation, such as donors after cardiac death, provides a source for
decellularized liver matrices28, rendering this approach feasible.
Our work demonstrates the decellularization of ischemic rat ­livers
by portal vein perfusion and its characterization. We show the preser­
vation of both structural and basement membrane–based compo­
nents of the native liver extracellular matrix, as well as the retention
of a functional microvascular network. DLM supported efficient
engraftment and in vitro metabolic function of adult primary hepato­
cytes. Retention of vascular structures allowed for transplantation
of ­recellularized liver graft in rats, and post-transplantation analysis
demonstrated preservation of hepatocyte structure and function with
minimal indications of ischemic damage. Preliminary recellulariza­
tion with endothelial cells also indicated attachment and viability
within the recellularized matrix in vitro. Overall, the methods and
techniques developed in this study present what is to our knowledge
the first step toward manufacture of auxiliary liver grafts as an alter­
native source of organs for liver transplantation.
RESULTS
Characterization of decellularized liver matrix
We achieved whole-organ decellularization by portal perfusion with
SDS, an anionic detergent that lyses cells and solubilizes cytoplasmic
components27. After 72 h of decellularization, a translucent acel­
lular scaffold, which retained the gross shape of liver, was generated
(Fig. 1a–e). Histological evaluation revealed no nuclei or cyto­
plasmic staining in DLM compared to normal rat liver (Fig. 1f).
Immunostaining for four extracellular matrix (ECM) proteins,
collagen type I, collagen type IV, fibronectin and laminin-β1, indi­
cated that both structural and basement membrane components of
the ECM were retained similarly to native liver (Fig. 1f). A lack of
DAPI staining in DLM confirmed the absence of cells (Fig. 1f). We
observed both collagen type IV and fibronectin in ­ decellularized
sinusoidal spaces, whereas we saw laminin-β1 in the basement
membrane of the larger vessels (Fig. 1f). We found that 100% of the
fibrillar collagen and approximately 50% of the glycosaminoglycans
of native liver were retained after decellularization (Supplementary
Table 1). Residual DNA content in the DLM was less than
3% (Supplementary Table 1).
To show that the decellularization protocol was successful in retain­
ing a functional vascular bed, we characterized its perfusion with
Allura Red dye. Structural components of the vascular tree were
clearly apparent in the translucent matrix (Fig. 2a,b). The dye injected
through the portal vein flowed as would be expected inside the vas­
cular network, gradually moving from larger vessels to smaller capil­
laries, suggesting the microvasculature remained intact. We further
characterized the intricate vascular tree by generating a corrosion cast
of DLM (Fig. 2c,d). Portal (red) and venous (blue) vasculature casting
of both normal (Fig. 2c) and decellularized (Fig. 2d) livers showed
that larger portal and venous circulation system of vessels and the vast
majority of the smaller microcirculatory branches were preserved,
indicating that physiologic flow could be achieved by traversing the
portal venous system and emptying into the systemic venous circu­
lation via the hepatic vein and inferior vena cava (IVC). Moreover,
scanning electron microscopy (SEM) images confirmed the presence
of large vessels within DLM (Fig. 2e), and we also observed remnants
of small vessels closely resembling portal triad (Fig. 2f). The honey­
comb of about 30-μm diameter gaps in the ECM is thought to be the
footprint of hepatocytes removed during decellularization (Fig. 2g).
Recellularization of decellularized liver matrix
The presence of a functional vascular bed in the DLM offers the abil­
ity to control hepatocyte engraftment and characterize liver-specific
metabolic function in vitro before transplantation of the ­recellularized

nature medicine  VOLUME 16 | NUMBER 7 | JULY 2010 815

 Technical Reports

markers in engrafted hepatocytes was similar

a b c d to that in normal livers (Fig. 3h).
To assess the metabolic activity of engrafted
hepatocytes, we quantified hepatocyte albu­
min production and urea synthesis. The
cumulative urea amounts in the recellular­
ized liver system were statistically higher than
in hepatocyte sandwich culture during the
e f g 5-d culture period (P = 0.0017) (Fig. 4a,b).
The difference in cumulative albumin levels
was statistically not significant (P = 0.0176)
(Fig. 4a,b). The albumin production rate by
the recellularized liver graft was 27.6 ± 7.0 μg
million cells per day as compared to 140 μg
million cells per day by the normal adult
rat liver29.
Figure 2  DLM retains intact lobular structure and vascular bed. (a) Representative photograph Analysis of the expression of drug metabo­
of decellularized left lateral and median lobes of rat liver, with the vascular tree visible. (b) The lism enzymes (Supplementary Table 2) via
vascular tree, after perfusion with Allura Red AC dye. (c,d) Corrosion cast model of left lobe of a quantitative RT-PCR at 2 d revealed that
© 2010 Nature America, Inc. All rights reserved.

normal liver (c) and the DLM (d), with portal (red) and venous (blue) vasculature. (e–g) SEM images
expression levels of drug metabolism enzymes
of a vessel (e), a section featuring bile duct–like small vessels (arrows) (f), extracellular matrix
within the parenchyma (g), with hepatocyte-size free spaces. Scale bars: 10 mm (a,b), 5 mm (c,d) in the recellularized liver were similar to those
and 20 μm (e–g). measured in sandwich hepatocyte cultures
(P = 0.05) (Fig. 4c,d). As expected at this
stage in culture, gene transcription levels were
graft. We introduced primary rat hepatocytes via portal vein per­ overall much lower than those of freshly isolated hepatocytes (Fig. 4c).
fusion recirculation. Roughly 12.5 million cells were introduced at However, Cyp2c11 (encoding cytochrome P450, subfamily 2, polypep­
each step, for a total of four steps, with 10-min intervals between each tide 11) Gstm2 (glutathione S-transferase mu 2), Ugt1a1 (encoding
step (Fig. 3a). Cell viability and distribution in the parenchyma after UDP glucuronosyltransferase-1 family, polypeptide A1) and Cyp1a1
this four-step seeding protocol was superior to single-step infusion (encoding cytochrome P450, family-1, subfamily a, polypeptide 1)
of 50 million cells for 40 min (Supplementary Fig. 1a). Engraftment were expressed in the recellularized liver at similar levels to those in
efficiency with the four-step protocol was 95.6% ± 3.4%. After seed­ normal liver (Fig. 4e–h). Adh1 (encoding alcohol dehydrogenase-1)
ing, we transferred the recellularized liver grafts (Fig. 3b,c) into a and Cyp3a18 (encoding cytochrome P450, family 3, subfamily a,
specially designed perfusion chamber (Supplementary Fig. 1b) for polypeptide 18) expression levels were higher in recellularized liver
in vitro culture. The perfusion chamber features two hermetically than in sandwich plate culture control, although they were much lower
sealed silicon sheets, forming a pouch filled with culture medium; than in normal liver (Fig. 4i,j). Primary hepatocytes are known to
this design avoids rigid surfaces, preventing development of pressure require between 7 and 10 d in culture before their metabolic activity
spots, while enabling sterile culture of the recellularized grafts up to and gene transcription levels stabilize30.
2 weeks in vitro (data not shown). Histological staining of recellular­
ized sections at 4 h and at 1 d, 2 d and 5 d of in vitro perfusion culture Further scalability of recellularization methodology
revealed that at 4 h, the majority of the cells remain in and around Ultimately, reconstruction of liver grafts in vitro requires the addi­
the vessels, whereas at 1 d and 2 d, they leave the vessels and get tion of nonparenchymal cells. As a preliminary test, we attempted to
­distributed throughout the matrix (Supplementary Fig. 1c). We did seed a nonparenchymal component to the recellularized liver graft
not observe any change in the distribution of the cells within the by incorporating microvascular endothelial cells to the hepatocyte-
matrix at 5 d compared to previous days (Supplementary Fig. 1c). repopulated graft and tested it by perfusion culture for up to 5 d to
We continuously perfused the recellularized liver graft for 5 d. allow for endothelial cell engraftment. Histological analysis showed
Hepatocyte viability was maintained during culture, and quantifica­ that endothelial cells were capable of lining the vasculature encir­
tion of TUNEL-positive cells revealed that less than 20% of the cells cled by hepatocytes at 3 d of culture (Supplementary Fig. 2a–d).
were apoptotic on the first 2 d of culture (Fig. 3d,e). Lactate dehydro­ Hepatocytes remained viable, as indicated by TUNEL staining and
genase (LDH) release during perfusion was comparable and statis­ immunohistochemistry (Supplementary Fig. 2e–i).
tically not different between groups, indicating minimal cell death To test the scalability of the seeding approach, as well as the limits
(P = 0.0455) (Fig. 3f). SEM and histological analysis (Fig. 3g) showed of the perfusion-culture system, we repopulated the median lobe
that hepatocytes engrafted around the larger vessels, populating the of DLM with 200 million hepatocytes and observed the perfusion
surrounding parenchyma, suggesting the cells migrated beyond the culture for 10 d. This cellular amount corresponds to approximately
matrix barrier to reach decellularized sinusoidal spaces. We assessed 20% of a rat’s liver mass, more than double the minimum hepatic mass
the functional characteristics of engrafted hepatocytes in the decellu­ necessary for therapeutic intervention6. In these studies, we injected
larized matrix via immunostaining at 2 d of culture of UDP glucuron­ 50 million hepatocytes at each of the four steps mentioned above. We
osyltransferase 1 family, polypeptide A1 (Ugt1a), a sensitive enzyme assessed the metabolic activity of the recellularized liver graft through
with a short half-life (about 50 min) whose presence indicated hepato­ albumin production, urea secretion and total bile acid synthesis, and
cyte viability and function, glucose-6-phosphatase, catalytic subunit the cumulative levels of production of these meta­bolites were found
(G6pc) and albumin (Fig. 3h). The level of immunostaining for these to be at similar levels to those of static sandwich-culture controls

816 VOLUME 16 | NUMBER 7 | JULY 2010  nature medicine

 Technical Reports

a –30 min 0 min 10 min 20 min 30 min 40 min d Recellularized liver Negative control Positive control

Perfusion with Inject Inject Inject Inject Incubation

culture medium 12.5 million 12.5 million 12.5 million 12.5 million starts
starts cells cells cells cells

b c e

TUNEL-positive cells (%)

(ng per 106 cells per 24 h)

100
f 20

Lactate dehydrogenase
Recellularized liver
80 Static sandwich culture
15
60
10
40
5
20
0 0
0 1 2 3 4 5 6

N ive

h
iv

24

48
t
at
si
Time (d)

eg
Po
g h H&E Albumin G6pc Ugt1a
© 2010 Nature America, Inc. All rights reserved.

Normal liver Normal liver Normal liver Normal liver

H&E Albumin G6pc Ugt1a

Recellularized liver Recellularized liver Recellularized liver Recellularized liver

Figure 3  Repopulation of rat DLM with adult rat hepatocytes. (a) Recellularization scheme of the DLM. (b,c) Decellularized whole liver matrix (b) and
same liver after recellularization with about 50 million hepatocytes (c). (d) TUNEL staining of recellularized liver grafts. Left to right: recellularized
graft at 48 h in culture (arrows indicate positive cells), fresh isolated liver (negative control), DNase-treated normal liver (positive control). Insets show
Hoechst 33258 counterstain of the same sections. (e) TUNEL-positive cell percentage in recellularized liver grafts as a function of perfusion-culture
time. (f) LDH release from recellularized livers during perfusion culture (P = 0.0455 by Friedman’s test). (g) SEM micrographs of recellularized liver
graft after 2 d in culture. (h) Immunohistochemical staining of the recellularized liver graft (bottom) in comparison to normal liver (top); left to right:
H&E, albumin (red), glucose 6-phosphatase (red) and Ugt1a (green). Sections were counterstained with Hoechst 33258 (blue). Scale bars: 20 mm
(b,c), 200 μm (d) and 100 μm (h). All error bars represent s.e.m. (n = 6).

(P = 0.5249, P = 0.5271 and P = 0.0114, respectively), indicating
the scalability of the techniques we introduce here, as well as the
usability of the perfusion-culture system as an in vitro model
(Supplementary Fig. 3).
Transplantation of recellularized liver grafts
An available functional vascular structure offers the opportunity to
transplant the recellularized liver graft by connecting the graft to the
blood supply, allowing for the transplantation of a critical hepatocyte
mass while avoiding ischemic damage due to poor graft perfusion.
Briefly, recipient rats underwent unilateral nephrectomy to prepare a
viable site for auxiliary liver graft transplantation. We used the renal
vein and artery as ports to create blood flow within the graft. Upon
unclamping of the artery, the graft was perfused, quickly filling with
blood, and appropriate efflux was established within 5 min (Fig. 5a).
The recellularized graft was kept in vivo for 8 h before harvesting
for further analysis. TUNEL staining revealed that there was mini­
mal damage to the hepatocytes due to the arterial blood flow and the
resulting shear stress during 8 h of transplantation (19.7% ± 13.7% of
TUNEL-positive cells after transplantation versus 21.4% ± 5.8% of
TUNEL-positive cells before transplantation, P = 0.55, Supplementary
Fig. 4). Histological staining indicated that the hepatocytes retained
both normal morphology and their parenchymal positions (Fig. 5b).
Immunohistochemical staining for albumin, G6pc and Ugt1a
c­ onfirmed that hepatic function was also retained in the transplanted
grafts, with minimal indications of ischemic damage (Fig. 5b).
To investigate early graft function beyond 8 h, we adopted an
ex vivo whole-blood perfusion technique as a surrogate model of trans­
plantation, which has been shown to be representative of early graft
performance and to predict primary graft failure in an orthotopic rat
liver transplant model31,32. The ex vivo perfusion system was similar
to the in vitro perfusion system, except that the perfusate consisted of
whole rat blood diluted with perfusion medium (hematocrit 20%). The
recellularized liver graft was perfused for 24 h, during which the blood
was replenished three times, once every 8 h to keep the hematocrit
level at 20% (Supplementary Fig. 5b). TUNEL staining and histologi­
cal analysis showed that hepatocytes remained viable (22.6% ± 13.8%
TUNEL-positive cells, Supplementary Fig. 5c) and preserved their
morphology and parenchymal position at the end of ex vivo blood per­
fusion (Supplementary Fig. 5d). In addition, hepatocytes remained
metabolically active during the 24 h ex vivo blood perfusion, as evi­
denced by urea and albumin secretion (Supplementary Fig. 5e,f).
DISCUSSION
These results present the first step toward development of recellular­
ized liver matrix as an auxiliary graft for transplantation. Previous
attempts, including those of our group, have failed in creating ­complex
tissue-engineered constructs for liver transplantation16,18. Here we

nature medicine  VOLUME 16 | NUMBER 7 | JULY 2010 817

 Technical Reports

Figure 4  Hepatic function of the recellularized

liver graft in vitro. (a,b) Albumin synthesis (a)
a c e f
1.2 1.2

Normalized mRNA

Normalized mRNA
and urea secretion (b) in comparison to static 250 Recellularized liver 1.0 1.0

Albumin (µg per 106 cells)

Static sandwich culture

ce ult . liv r
0.8 0.8

lls ur er
sandwich culture (P values are 0.0176 for

C ll e
R al ls
200

e v
m l

level

level
ed
ec li
or e
0.6 0.6

N hc
albumin and 0.0017 for urea, by Friedman’s

es
150 0.4 0.4

Fr
test). (c) Gene expression analysis of hepatocytes 0.2 0.2
in the recellularized liver graft (2 d) compared 100 0 0

el ltu ich

el ltu ch

er
or liv zed
er
or ve d
to normal liver, fresh hepatocytes and sandwich

lu re

lu re
N li rize
50

al r
ec u i

liv
m r
liv
ec u w

R c ndw

m e
ri
R c d

al

la
la
n
culture hepatocytes (2 d) for phase 1 and phase 2

sa

sa
0

ic

ic

N
drug metabolism enzymes. (d) Scatter plot com­

at

at
St

St
0 1 2 3 4 5 6
paring gene expression of phase 1 and phase 2
g h

Phase 1
Time (d)
drug metabolism enzymes at 2-d recellularized
b 1.6 50

Normalized mRNA
Normalized mRNA
liver graft and cultured hepatocytes (P = 0.0499 500 1.4
Recellularized liver
by Friedman’s test). (e–j) Normalized gene 1.2

Urea (µg per 106 cells)

Static sandwich culture 1.0
400

level
level
expression of Cyp2c11 (e), Gstm2 (f), Ugt1a1 (g), 0.8 40
0.6 1
Cyp1a1 (h), Adh1 (i) and Cyp3a18 (j). All error 300 0.4
bars represent s.e.m. (n = 3). 0.2
200 0 0

el ltu ch

el ltu ch
er

or ve d
or iv d

er
N li ize
N l rize
lu re

lu re
liv

m r
ec u i

m er

ec u i

liv
100

R c ndw

R c ndw

r
al

al
la
la
successfully demonstrate the preservation

sa

sa
Phase 2
0

ic

ic
at

at
of the liver’s three-dimensional architecture,

St

St
0 1 2 3 4 5 6
functional vasculature and native matrix d 100 Time (d)
i j
© 2010 Nature America, Inc. All rights reserved.

composition upon decellularization. Further,

Recellularized liver

1.2 1.20

Normalized mRNA

Normalized mRNA
10
1.0
we present a technique for efficient in vitro 1 0.8 1.00
recellularization of the graft, which main­ 0.6

level

level
0.1 0.80
0.02
tains cell viability and function. Finally, we 0.01
0.2

demonstrate the feasibility of transplanting 0.001 0 0

these recellularized liver grafts in vivo with 0.001 0.01 0.1 1 10 100

el ltu ich

el ltu ich
er
or iv d

or iv d

er
N l rize

N l rize
lu re

lu re
m er
liv

m er
liv
ec cu dw

ec u w
R c nd
al
Static sandwich culture

la
minimal ischemic damage.

la

al
n
sa

sa
ic

ic
Oxygen and nutrient transport limitations

at

at
R
St

St
have thus far restricted the development of
tissue-engineered liver constructs to the diffusion limit, about 200 μm. distance from the nearest blood vessel17. It is a crucial configuration
The high rates of oxygen consumption 33–36 by primary hepato­ that should ideally be replicated in vitro before transplantation.
cytes result in ischemic damage in cells found further away from This report builds on the previously reported technique developed
the blood within minutes of transplantation. Owing to high oxygen for heart perfusion decellularization27, demonstrating its applicabil­
demands, the liver is the only organ that receives both arterial and ity for rat liver decellularization and further extending its innova­
venous blood supply, whereas hepatocytes are located less than 2 μm tive contributions by introducing a perfusion-seeding method and
an extracorporeal perfusion-culture system.
Additionally, we further optimize the decel­
a lularization for liver.
Our work shows that portal vein perfusion
decellularization creates a translucent liver
matrix while preserving the three-dimensional
architecture, the native structural and basement

Figure 5  Transplantation of the recellularized

liver graft. Decellularized and recellularized rat
liver was transplanted as auxiliary heterotopic
graft with portal vein arterialization, and
graft viability was determined 8 h after
transplantation. (a) Representative images of
graft transplantation; top, left to right: transplant
b Normal liver Normal liver Normal liver Normal liver site, recellularized graft at the transplant site,
transplanted graft before blood reperfusion,
the graft right after declamping the renal
artery; bottom, left to right: transplanted graft
1 min, 2 min and 4 min after declamping of
H&E Albumin G6pc Ugt1a the renal artery, and auxiliary graft in contrast
Recellularized liver Recellularized liver Recellularized liver Recellularized liver with the native liver. (b) Immunohistochemical
staining of recellularized auxiliary graft 8 h after
transplantation (bottom) compared to native liver
(top); left to right: H&E, albumin (red), glucose
6-phosphatase (red), Ugt1a (green). Sections
were counterstained with Hoechst 33258 (blue).
H&E Albumin G6pc Ugt1a
Scale bars: 10 mm (a) and 100 μm (b).

818 VOLUME 16 | NUMBER 7 | JULY 2010  nature medicine


membrane matrix, and the functional vascular network of the original
organ. Portal vein dye perfusion, corrosion casting, SEM and immuno­
staining show the presence and function of the microvascular network
of the liver post decellularization. Quantification of extracellular matrix
proteins, SEM analysis and immunostaining suggests that the matrix
of both the large vessels (collagen type I, laminin-β1) and the sinusoids
(collagen type IV, fibronectin) remained relatively intact. We note that
the main structural protein of the liver, collagen type I, was retained in
its entirety during the decellularization process. However, we also note
the marked loss of half of the glycosaminoglycan content of the liver
during decellularization. This loss is not surprising, as many glycosamino­
glycans are associated with cellular membranes that are solubilized in the
decellularization process.
We have found that the DLM can be seeded with primary rat
hepatocytes via a multistep perfusion technique, with efficiency
greater than 90%. As expected, this grafting efficiency is much
higher than in vivo levels of engraftment after hepatocyte trans­
plantation, possibly as a result of the lack of endothelial barrier and
the abundant empty space. We seeded 50 million primary rat hepato­
© 2010 Nature America, Inc. All rights reserved.
cytes, roughly 5% of the cells in native rat liver, as this amount was
previously shown to be sufficient to restore liver function in animal
models6. We have also shown that this number can be increased
up to 200 million to account for 20% of the native rat liver hepatic
mass, corresponding to repopulation of approximately 50% of the
original hepatic mass within the lobe used for transplantation.
After engraftment, hepatocyte viability and metabolic function was
maintained in perfusion culture, as assessed by TUNEL staining,
LDH release, synthetic activity and gene expression. Engrafted cells
showed albumin production and gene expression equivalent to cells
from the same isolation cultured in a collagen-sandwich configu­
ration, a culture configuration previously shown to maintain high
liver-specific function for up to a month in vitro37. Overall, albu­
min production and gene expression were roughly 20% and 30% of
in vivo levels, respectively.
We have shown that rat livers can be depleted of their cellular com­
ponents and used to engineer sophisticated hepatic structures that
may recover many functions of normal liver. Despite its advantages,
further improvements of the decellularization and recellularization of
liver matrix are necessary. Engineering of an entire liver will require
addition of nonparenchymal cells such as liver sinusoidal endothelial
cells, stellate cells, biliary epithelial cells and Kupffer cells. Particularly,
we observed the presence of hepatocytes outside the microvasculature
system, mainly distributed in the parenchymal spaces, possibly owing
to loss of sinusoidal integrity in the absence of the liver sinusoidal
endothelium and other nonparenchymal cells. We speculate that the
nonparenchymal cells of the liver will be essential for reacquisition of
vascular integrity. Our ongoing studies are directed toward optimizing
seeding strategies, re-endothelialization and biliary tract reconstruc­
tion. However, our preliminary results on re-endothelialization of the
graft indicate the feasibility of this approach. Addition of nonparen­
chymal cells and modification of the perfusate may further enhance
the reconstitution of a functional liver graft and even lead to hepatic
repopulation by imitating the regenerative behavior of the liver. Thus,
we envision the possible use of DLM ultimately as a model for the
study of both liver development and liver regeneration.
In summary, this report provides a foundation for efficient develop­
ment of auxiliary liver grafts for transplantation and practical, unique
techniques to prepare recellularized liver graft. Further studies are
required to determine whether the techniques described here can be
scaled up for use in humans.
nature medicine  VOLUME 16 | NUMBER 7 | JULY 2010 819
Technical Reports
Methods
Methods and any associated references are available in the online ­version
of the paper at http://www.nature.com/naturemedicine/.
Note: Supplementary information is available on the Nature Medicine website.
Acknowledgments
This work was supported by grants from the US National Institutes of Health,
R01DK59766 and R01DK084053 to M.L.Y., K99/R00 DK080942 to K.U. and
K99DK083556 to A.S.-G., and US National Science Foundation CBET-0853569 to
K.U. We thank the support of the Shriners Hospitals for Children to B.E.U. (grant
no. 8503), the American Liver Foundation to A.S.-G. and Massachusetts General
Hospital Junior Faculty Grant to K.U. and the Shriners Hospitals for Children. We
would like to thank A. Vitalo, C. Calhoun and B. Crowther for technical support.
AUTHOR CONTRIBUTIONS
K.U. had full access to all of the data in the study and takes responsibility for the
integrity of the data and the accuracy of the data analyses. B.E.U., A.S.-G., H.Y.
and K.U., study concept and design; B.E.U., A.S.-G., H.Y., M.A.G., acquisition of
data; B.E.U., A.S.-G., Y.N. and K.U., analysis and interpretation of data; M.-L.I.,
C.S. and B.E.U., rat liver harvest, decellularization and hepatocyte isolation; J.M.
and B.E.U., design and construction of the recellularized liver chamber; B.E.U.,
A.S.-G., H.Y. and M.A.G., recellularization; A.S.-G. and H.Y., histology and
transplantation studies; B.E.U., A.S.-G., H.Y. and K.U., drafting of the manuscript;
B.E.U., A.S.-G., H.Y., M.L.Y., Y.N., A.T., F.B., M.H., N.K. and K.U., critical revision
of the manuscript for intellectual content; B.E.U. and K.U., statistical analysis;
M.L.Y., K.U. and A.S.-G., obtained funding; M.-L.I., C.S., J.M., Y.N., A.T. and
F.B., administrative, technical or material support. All authors contributed to the
preparation of the report.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Published online at http://www.nature.com/naturemedicine/.
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/.
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 ONLINE METHODS
Rats. Female Lewis rats (150–200 g) (Charles River Laboratories) were used
for hepatocyte isolation and liver harvest. Male Lewis rats (250–300 g) (Charles
River Laboratories) were used as recipients. The rats were cared for in accord­
ance with the guidelines set by the Committee on Laboratory Resources, US
National Institutes of Health, and Subcommittee on Research Animal Care and
Laboratory Animal Resources of Massachusetts General Hospital.
Liver decellularization. Frozen livers were thawed at 4 °C and washed with
PBS overnight through perfusion via portal vein at 1 ml min−1. Liver was per­
fused with SDS (Sigma) in distilled H2O for 72 h starting with 0.01% SDS for
24 h and then with 0.1% SDS for 24 h and with 1% SDS for 24 h. Subsequently,
liver was washed with distilled H2O for 15 min and with 1% Triton X-100
(Sigma) for 30 min to remove residual SDS. Decellularized livers were washed
with PBS for 1 h. Unless otherwise mentioned, caudal, left lateral and right
lateral lobes were ligated and resected, and the median lobe was kept for recel­
lularization. DLM was sterilized in 0.1% peracetic acid (Sigma) in PBS for 3 h.
DLM was washed extensively with sterile PBS and preserved in PBS containing
antibiotics at 4 °C for up to 7 d.
Hepatocyte seeding. DLM was connected to the perfusion system through
© 2010 Nature America, Inc. All rights reserved.
portal vein cannulation. The inferior vena cava and superior vena cava were
left open for outlet. DLM was kept immersed in medium in a perfusion
chamber while it was perfused through the portal vein. We recirculated 50 ml
medium in the perfusion system. Portal pressure was maintained between
10 and 15 cm H2O. The perfusate consisted of Williams’ E (Sigma), 5% FBS
(Hyclone), 0.5 U ml−1 insulin (Eli Lilly), 20 ng ml−1 epidermal growth factor
(Invitrogen), 14 ng ml−1 glucagon (Bedford Laboratories), 7.5 μg ml−1 hydro­
cortisone (Pharmacia), 100 U ml−1 penicillin and 100 μg ml−1 streptomycin
(Invitrogen). For recellularization with 200 million hepatocytes (10-d culture
period), the perfusate was high-glucose DMEM and 10% FBS with other sup­
plements remaining the same to support survival and function of hepatocytes
for more than 7 d in vitro37. After a 30-min perfusion with medium, a total of
50 or 200 million primary adult rat hepatocytes with over 90% viability were
infused into the circuit in four steps at 10-min intervals and were recirculated
in the system at 25 °C. At the end of 40 min, the perfusate was collected and the
number and viability of cells not retained in the liver was determined with a
hemacytometer and trypan blue exclusion. The total number of cells retained
in DLM was the difference between the initial number of cells seeded and the
number of cells present in the perfusate after seeding.
doi:10.1038/nm.2170
Recellularized liver graft perfusion system. For in vitro perfusion-culture
experiments, the recellularized liver was transferred to a clean chamber, part
of a perfusion system previously developed for donors-after-cardiac-death
organ resuscitation31 that consisted of a peristaltic pump, bubble trap and
oxygenator. The system was placed in an incubator for temperature control,
and the oxygenator was connected to atmospheric gas mixture. The graft was
continuously perfused through the portal vein at 15 ml min−1 with continuous
oxygenation that delivered an inflow partial oxygen tension of ~300 mm Hg.
The medium was changed daily.
Heterotopic recellularized liver graft transplantation. The left renal artery
and vein of the recipient rat were isolated, and the left ureter was ligated
and transected. After clamping the left renal artery and vein at their origin,
nephrectomy was performed. The recellularized graft was placed in the left
renal space of the recipient. One milliliter of saline containing 100 U of heparin
and 0.25 mg per kg body weight of the Fab fragment of a chimeric monoclonal
antibody, a glycoprotein IIb/IIIa inhibitor (ReoPro, Eli Lilly), was injected
into the penile vein to prevent coagulation. An 8-mm stent (0.3-mm inner
diameter) was introduced into the donor’s portal vein, and another 8-mm
stent (0.6-mm inner diameter) was introduced and secured in the inferior vena
cava. The stent in the portal vein was inserted into the left renal artery, allow­
ing for arterial-venous anastomosis with standardized flow-regulated inflow
rates38. The portal vein and renal artery were connected with two sutures. The
donor’s inferior vena cava and the stent were introduced and sutured in the
left renal vein. The arterial clamp and then the venous clamp was removed
to start reperfusion.
Statistical analyses. Statistical differences of collagen, glycosaminoglycans
and DNA in DLM and comparison of TUNEL-positive cells were evaluated by
two-tailed unpaired Student’s t test at α = 0.01. Statistical analyses of albumin,
urea, total bile acid and LDH in the recellularized grafts were performed with
Friedman’s test in MATLAB (v7.7.0), a nonparametric alternative to two-way
analysis of variance, which does not assume normal distribution, with multiple
replicates at α = 0.01.
Additional methods. Detailed methodology is described in the Supplementary
Methods.
38. Schleimer, K. et al. Improved technique of heterotopic auxiliary rat liver
transplantation with portal vein arterialization. Langenbecks Arch. Surg. 391,
102–107 (2006).
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