Research paper

Organogenesis 6:2, 134-136; April/May/June 2010; 2010 Landes Bioscience

Method for the decellularization of intact rat liver

Thomas Shupe,1,* Matthew Williams,2 Alicia Brown,1 Bradley Willenberg3 and Bryon E. Petersen1,4
Department of Pathology, Immunology and Laboratory Medicine; College of Medicine; 2J. Crayton Pruitt Family Department of Biomedical Engineering;
3
Department of Materials Science and Engineering; 4Program for Stem Cell Biology; Shands Cancer Center; University of Florida; Gainesville, FL USA

Key words: bioartificial liver, liver transplant, liver matrix, liver progenitor cell, artificial liver, organ engineering, decellularization
Abbreviations: BAL, bioartificial livers; IDL, intact decellularized livers; IVC, inferior vena cava; SVC, superior vena cava; FBS,
fetal bovine serum

We have developed a method for the decellularization of whole rat livers by perfusion with increasing concentrations of
detergents. This procedure resulted in an intact, decellularized organ with an intact liver capsule. These decellularized
organs were analyzed by immunohistochemistry, and retained an appropriate distribution of extracellular matrix
components. The laminin basement membranes of the liver vasculature also remain intact. These acellular vessel
remnants were strong enough to be cannulated, providing a convenient means for the delivery of cells to areas deep
within the decellularized organ. Cannulation of the extrahepatic vessel remnants allow for media to be circulated through
the decellularized organ. These decellularized livers provide a natural matrix for research in the fields of bio-artificial
livers and liver engineering.

Introduction
Liver pathologies, ranging from viral hepatitis to inborn metabolic disorders to injury resulting from alcohol abuse, often
result in the need for liver transplantation. An insufficient supply of organs suitable for transplantation has limited our ability
to cure many instances of these diseases. Approximately 6,500
liver transplants were performed in the United States during the
year 2005.1 Over 17,000 Americans are currently waiting for a
liver transplant.2 Unfortunately, many of these patients will succumb to their disease before a suitable organ becomes available.
Patients who do receive liver transplant must contend with lifelong immunosuppression. Even with appropriate immunosuppression, the transplanted organ has a finite lifespan. These facts
have driven research in fields that provide alternatives to orthotopic liver transplant.
Bioartificial livers (BAL) are not a permanent alternative to
liver transplant. Instead, they would be used to sustain a critically ill patient until a suitable donor organ became available.
BAL generally take the form of closed, ex vivo systems containing
functional liver cells grown on a synthetic matrix.3-5 In theory,
a patients blood could be passed through these systems, allowing for the detoxification of xenobiotics and ammonia as well as
supplying plasma proteins and glucose. While this concept may
seem straight forward, complications in maintaining functional
hepatocytes in three-dimensional culture have limited the development of these systems.3-6 Synthetic matrices have proven to be
sub-optimal in regards to both the extent of colonization as well
as the long term maintenance of cell functionality and viability.

Ex Vivo engineering of a transplantable liver would be a permanent alternative to donor liver transplant. The discovery that
liver stem cells may be derived from bone marrow open the possibility of colonizing a matrix scaffold with a patients own cells.7
We have developed a method for removing all of the cells from
an intact liver. This process leaves behind the extracellular matrix
and liver capsule, as well as the laminin basement membranes of
the extrahepatic and extrahepatic vasculature. We feel that these
intact decellularized livers (IDL) represent an exceptional tool for
studies related to BAL and engineered livers.
Results
Following perfusion of the liver with PBS to clear the blood,
Triton X-100 was used to solublize lipids (Fig. 1A and B).
Analysis of sections from livers treated only with Triton X-100
indicated clearing of all cells. However, DAPI staining revealed
intact nuclear cages containing DNA remained within the
matrix (Data not shown). Subsequent perfusion of the acellular
organ with solution containing SDS resulted in the clearance of
all DNA (Fig. 1C). H&E staining of formalin fixed sections of
IDL demonstrate a fine web of matrix remains within the IDL
capsule (Fig. 2A). Immunohistochemical staining of the matrix
indicated the presence of collagen IV within the matrix (Fig.
2B). Additionally, laminin was shown to be present within the
remaining basement membrane of the vessels and surrounding
the acellular remnants of the hepatic cords (Fig. 2C).
Following decellularization, the acellular vessel remnants were
strong enough to maintain cannulation. The rat liver progenitor cell

*Correspondence to: Thomas Shupe; Email: shupe@pathology.ufl.edu

Submitted: 07/12/09; Accepted: 02/18/10
Previously published online: www.landesbioscience.com/journals/organogenesis/article/11546
134

Organogenesis

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Research Paper

Research paper

Figure 1. Decellularization process. (A) Blood is removed from the organ by perfusion with PBS. (B) Triton X-100 is used to solublize cellular membranes. (C) SDSs used to clear the remaining nuclear cages and DNA from the matrix.

line WB344 in RPMI medium was infused into the IDL through
the cannulated IVC. H&E sections from within the center of the
IDL contained cells indicating that the intrahepatic vasculature
was able to traffic cells from the IVC to these areas (Fig. 2D).
Discussion
We have developed a simple method for removing the cellular
component from intact rat livers. Triton X-100 was sufficient to
clear the cells, but the nuclei remained. It is likely that Triton
X-100 treatment spares the nuclear cage that is tethered to the
matrix by the cytoskeleton. Perfusion with 0.1% SDS for 1 hour
completely cleared all DNA from the IDL. Supplementation of
all perfusion solutions with antibiotics/antimycotics prevented
microbial growth, and the IDL could be stored at 4C for several
weeks. The extrahepatic vessel remnants maintained sufficient
strength for cannulation. This should provide a continent means
to deliver media to the internal regions of the IDL; supplying
oxygen and nutrients for long term culture experiments.
The matrix within the IDL may be beneficial for inclusion in
BAL studies. Because the distribution of proteins is maintained
during decellularization, the resulting matrix should be quite
close to that found in normal liver. The IDL matrix may also be
useful in studies involving the epigenetic control of cell phenotype. For instance, IDL may be produced from livers containing
experimentally induced tumors. Sections of IDL from these livers

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may be used to test the phenotype of tumor cells on tumor matrix

as compared to normal adjacent matrix. It is also possible that
IDL may be recellularized by mature or progenitor cells. This
would likely involve the stepwise repopulation of the vascular
basement membranes with endothelial cells followed by colonization of the hepatic cord remnants with hepatocytes. It is possible
that equilibration of IDL with serum would allow rebinding of
growth factors to the matrix. This may help to direct the proliferation and differentiation of progenitor or stem cells and result
in a tissue architecture similar to normal liver.
Materials and Methods
Animals. Fisher 344 rats ranging in age from 6 weeks to 8 months
have been successfully used for the production of IDL. The animals are maintained in conventional housing on standard diet
prior to treatment. All animal procedures are conducted under
guidelines approved by the University of Florida IACUC.
Liver decellularization. Rats received a lethal (100 mg/kg)
dose of sodium pentobarbital. The inferior vena cava (IVC) was
cannulated, the portal vein severed and the superior vena cava
(SVC) was clamped as previously described.8 The liver was first
perfused with 100 mL PBS (pH 7.4) to clear blood from the
organ. The liver was then perfused with biological detergents to
solublize cell membranes. Isotonic solutions (PBS) of 1, 2 and
3% (w/v) Triton X-100 (300 mL each) were perfused through

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Figure 2. H&E staining of IDL. (A) Extracellular matrix within the IDL. (B) Collagen IV staining of the IDL matrix. (C) Immunohistochemistry for laminin
indicates intact basement membrane of the vessels as well as appropriate deposition of the matrix component between hepatic cords. (D) Cryosection of WB344 rat liver progenitor cells delivered to the center of the IDL through the cannulated IVC remnant.

the organ by peristaltic pump at a flow rate of 5 mL/minute.

This was immediately followed by perfusion with 300 mL each
of PBS containing 0.1% SDS (w/v). Detergent containing solutions were cleared from the liver by perfusion with 300 mL PBS.
Finally, 10 mL fetal bovine serum was pumped into the organ.
For experiments involving administration of cells to the IDL, 106
cells were delivered to the organ through the IVC. The IVC and
SVC were then tied off and the liver was excised intact. All perfusion solutions (including FBS) contained 1% antibiotic/mycotic
(Invitrogen, Carlsbad, CA).
WB344 cell line. The rat liver progenitor cell line WB344
was kindly provided by Dr. William Coleman (Dept. Pathology,
University of North Carolina, Chapel Hill). These cells were cultured in RPMI 1640 (Cellgro, Herndon, VA) medium containing
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Supported by PF-05-165-01 from the American Cancer Society.

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