RESEARCH ARTICLE

Optimization of Liver Decellularization

Maintains Extracellular Matrix Micro-
Architecture and Composition Predisposing
to Effective Cell Seeding
Panagiotis Maghsoudlou1, Fanourios Georgiades1, Holly Smith2, Anna Milan1,
Panicos Shangaris1, Luca Urbani1, Stavros P. Loukogeorgakis1, Benedetta Lombardi3,
Giuseppe Mazza4, Charlotte Hagen5, Neil J. Sebire6, Mark Turmaine7, Simon Eaton1,
a11111 Alessandro Olivo5, Jasminka Godovac-Zimmermann3, Massimo Pinzani4, Paul Gissen2,
Paolo De Coppi1*
1 Department of Stem Cells and Regenerative Medicine, UCL Institute of Child Health and Great Ormond
Street Hospital, 30 Guilford Street, London, WC1N 1EH, United Kingdom, 2 MRC Laboratory for Molecular
Cell Biology, University College London, London, WC1E 6BT, United Kingdom, 3 London Center for
Nephrology, University College London, London, WC1N 1EH, United Kingdom, 4 Institute for Liver and
Digestive Health, University College London, London, WC1N 1EH, United Kingdom, 5 Department of
Medical Physics & Bioengineering, University College London, London, WC1N 1EH, United Kingdom,
OPEN ACCESS
6 Department of Histopathology, UCL Institute of Child Health and Great Ormond Street Hospital, London,
Citation: Maghsoudlou P, Georgiades F, Smith H, WC1N 1EH, United Kingdom, 7 Division of Bioscience, University College London, London, WC1N 1EH,
Milan A, Shangaris P, Urbani L, et al. (2016) United Kingdom
Optimization of Liver Decellularization Maintains
Extracellular Matrix Micro-Architecture and * [email protected]
Composition Predisposing to Effective Cell Seeding.
PLoS ONE 11(5): e0155324. doi:10.1371/journal.
pone.0155324
Abstract
Editor: Feng Zhao, Michigan Technological
University, UNITED STATES Hepatic tissue engineering using decellularized scaffolds is a potential therapeutic alternative
Received: January 8, 2016 to conventional transplantation. However, scaffolds are usually obtained using decellulariza-
tion protocols that destroy the extracellular matrix (ECM) and hamper clinical translation. We
Accepted: April 27, 2016
aim to develop a decellularization technique that reliably maintains hepatic microarchitecture
Published: May 9, 2016
and ECM components. Isolated rat livers were decellularized by detergent-enzymatic tech-
Copyright: 2016 Maghsoudlou et al. This is an nique with (EDTA-DET) or without EDTA (DET). Histology, DNA quantification and proteo-
open access article distributed under the terms of the
mics confirmed decellularization with further DNA reduction with the addition of EDTA.
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any Quantification, histology, immunostaining, and proteomics demonstrated preservation of
medium, provided the original author and source are extracellular matrix components in both scaffolds with a higher amount of collagen and gly-
credited. cosaminoglycans in the EDTA-DET scaffold. Scanning electron microscopy and X-ray phase
Data Availability Statement: All relevant data are contrast imaging showed microarchitecture preservation, with EDTA-DET scaffolds more
within the paper and its Supporting Information files. tightly packed. DET scaffold seeding with a hepatocellular cell line demonstrated complete
Funding: The authors acknowledge support by repopulation in 14 days, with cells proliferating at that time. Decellularization using DET pre-
grants from the Medical Research Council, the Royal serves microarchitecture and extracellular matrix components whilst allowing for cell growth
College of Surgeons of England, the Sparks
for up to 14 days. Addition of EDTA creates a denser, more compact matrix. Transplantation
Childrens Medical Charity, the British Foreign Office
for the UK/USA Stem Cell Collaboration Award and of the scaffolds and scaling up of the methodology are the next steps for successful hepatic
the Mittal Research Fund. AO and CKH are funded tissue engineering.
by the EPSRC (grants EP/G004250/1, EP/I021884/1
and EP/L001381/1). HS and PG are supported by the
Wellcome Trust and ERC (WT095662MA, ERC-

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 Liver Decellularization Protocol Optimized

2013-StG - 337057). SE and PDC are supported by Introduction

Great Ormond Street Hospital Childrens Charity. The
authors would also like to thank the Royal Society/ Decellularized tissues have provided an option for engineering tissue both for transplantation
Wolfson Foundation for the tissue engineering and for disease modeling. However, ideal scaffolds should have architectural and mechanical
laboratory refurbishment grant obtained for the characteristics allowing migration and proliferation of introduced cells, a defined biodegrada-
Pediatric Surgery Department at UCL Institute of tion profile, and a minimal immune response [1,2]. For complex organs, such as the liver, scaf-
Child Health. The funders had no role in study
fold choice is limited to decellularized materials, wherein cell removal from the whole-organ
design, data collection and analysis, decision to
publish, or preparation of the manuscript. yields a three-dimensional extracellular matrix (ECM) [1,3].
Rat liver decellularization was initially performed using increasing concentrations of sodium
Competing Interests: The authors have declared
dodecyl sulphate (SDS), followed by Triton-X 100 (TX100) [4]. This was succeeded by methods
that no competing interests exist.
using increasing concentrations of TX100, followed by SDS [5], a combination of trypsin,
EDTA and TX100 [6], and a combination of TX100 and ammonium hydroxide [7]. The inclu-
sion of ion-chelating agents, such as EDTA and EGTA, was derived from their routine use for
hepatocyte isolation. The general methodology based on detergents such as TX100 and SDS
has been duplicated in many laboratories with slight variations [812]. Decellularization based
on SDS and TX100 has also been scaled-up to larger animals [1316]. However, current decel-
lularization protocols cause substantial harm to the ECM and may render the vasculature too
porous for successful transplantation. This is effectively shown in vascular casting images in rat
livers decellularized by 1% SDS and 1% TX100 that demonstrate destruction of the blood vessel
network [9].
We have previously decellularized intestine, lung and esophagus [1720] using deionized
water (dH2O), a low concentration of a mild detergent (sodium deoxycholate; SDC) and an
enzyme to remove DNA remnants. This detergent enzymatic treatment (DET) [21] preserves
scaffold microarchitecture and the microvascular network and has allowed successful clinical
transplantation of human tracheas [22].
The aim of this study was to develop a decellularization protocol for rat liver that will pre-
serve microarchitecture and ECM components. We aim to examine the interplay between the
scaffold structural proteins and the DET and EDTA chemicals so as to create a scaffold that
will allow long-term transplantation.

Materials and Methods

Harvest of organs
This study was carried out in accordance with the recommendations in the Animal (Scientific
Procedures) Act 1986. The Home Office approved the study protocol (licence number 70/
2716). 250300 g Sprague-Dawley rats (n = 100) were sacrificed by CO2 inhalation. The abdo-
men was sterilized with 70% ethanol and a U-shaped incision was performed to expose the
abdominopelvic cavity. The abdominal inferior vena cava (IVC) and portal vein (PV) were
identified and the PV was cannulated with a 24G cannula (BD, UK), which was secured in
place with a 30 silk suture (Ethicon, UK). The abdominal IVC was ligated using silk sutures
proximal to the right renal vein and the IVC was sectioned. The diaphragm was used as a hold-
ing point to release the whole liver from the supporting tissue. The whole procedure was car-
ried out with special caution not to damage the Glissons capsule, which surrounds the organ.

Decellularization
For DET treatment, the PV was connected to a peristaltic pump (Masterflex, UK) and perfused
with dH2O (18.2 mO/cm) for 36 hours at 4C. For the EDTA-DET treatment, rat livers were
perfused with 2mM EDTA (Sigma, UK) for 15 minutes followed by dH2O for 36 hours at 4C.
Both DET and EDTA-DET rat livers were then transferred at room temperature and perfused

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 Liver Decellularization Protocol Optimized

with 4% SDC (Sigma, UK) for 6 hours followed by perfusion of 500 kU/ml DNase-I (Sigma,
UK) in 1M sodium chloride (NaCl; Sigma, UK) for 3 hours. Flow rate was 4.5 ml/min (dH2O)
and 6.5 ml/min (SDC and DNase).

Histology
Samples were fixed in 4% paraformaldehyde (PFA; Sigma, UK), dehydrated in graded alcohol,
paraffin embedded and sectioned at 5m. Tissue slides were stained with haematoxylin and
eosin (H&E; Leica, Germany), Massons Trichrome (MT; Leica, Raymond A Lamb, BDH
Chemicals Ltd), Picrosirius Red (PR; Polysciences Inc., Germany), Elastin Van Gieson (EVG;
VWR, Leica, Raymond A Lamb), and Alcian Blue (AB; BDH Chemicals Ltd, Cellpath Ltd)
stains. Appropriate positive controls were used to ensure histological stains were performed
correctly (small intestine for MT, PR and AB, skin for EVG). For all histological samples three
biological and technical replicates were assessed using standard H&E prior to any special stains
to ensure homogeneity. All images shown are representative samples.

Immunohistochemistry
Following rehydration of the paraffin-embedded slides, and quenching of endogenous peroxi-
dase activity using 1% H2O2 (Sigma, UK), antigen retrieval was performed using pepsin
(Sigma, UK) at a concentration of 1 mg/ml in 1N HCl (Sigma, UK), at 37C for 60 minutes.
Slides were then permeabilized with 0.2% Triton X-100 (Sigma, UK) for 60 minutes and
blocked in 1% BSA (Sigma, UK) for 30 minutes. Primary antibodies were used against fibro-
nectin (Abcam, UK), laminin (Abcam, UK), collagen I (Abcam, UK), collagen III (Abcam,
UK), and collagen IV (Abcam, UK) at dilutions of 1:100, in BSA 1% overnight. Staining was
visualized using the ImmPRESS (Vector Laboratories, CA, USA) detection system. Once dia-
minobenzidine (DAB substrate kit, BD Pharmingen) staining was developed, sections were
dehydrated and mounted in Eukitt mounting medium (Fluka, Sigma, UK). Appropriate fresh
tissue and no primary antibody controls were used to ensure appropriate tissue positivity.
Three biological and technical replicates were assessed. All images shown are representative
samples.

DNA quantification
DNA was isolated using a tissue DNA isolation kit (PureLink Genomic DNA MiniKit, Invitro-
gen, UK) following the manufacturers instructions. Briefly, the samples were digested over-
night using Proteinase K and a digestion buffer. DNA samples were purified using alcohol
washes and measured spectrophotometrically (Nanodrop, Thermo Scientific, US). Optical den-
sities at 260nm and 280 nm were used to estimate the purity and yield of nucleic acids. For all
quantifications at least three biological and technical replicates were obtained.

Collagen quantification
The collagen content of native and decellularized tissue was quantified using the total collagen
assay kit (QuickZyme Biosciences, The Netherlands). Briefly for collagen, the samples were
hydrolyzed in 6N HCl at 95C for 20 hours, the hydrolysates were mixed with a chromogen
solution staining hydroxyproline residues and color was developed at 60C for 1 hour. The
absorbance for each sample was determined at 555 nm using a Infinite 200 Pro microplate
reader (Tecan, US) and the collagen quantity was calculated from a standard curve from
known concentrations of pure collagen hydrolysates. For all quantifications at least three bio-
logical and technical replicates were obtained.

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 Liver Decellularization Protocol Optimized

Elastin quantification
The elastin content of native and decellularized tissue was quantified using the FASTIN elastin
assay (Biocolor, UK) according to the manufacturers instructions. Briefly, the samples were
homogenized, and elastin was solubilized in 0.25 M oxalic acid. Two consecutive incubations
were performed at 95C to ensure complete extraction of elastin. Extracts were incubated with
5,10,15,20-tetraphenyl-21H,23H-porphine tetrasulfonate (TPPS) dye, and absorbance was
determined at 555 nm spectrophotometrically (Tecan Infinity, US). Elastin concentrations
from a standard curve were used to calculate the elastin content of the tissue. For all quantifica-
tions at least three biological and technical replicates were obtained.

Glycosaminoglycan (GAG) quantification

The GAG content of native and decellularized tissue was quantified using the Blyscan GAG
Assay Kit (Biocolor, UK). Briefly, the tissues were digested with papain at 65C for 18 hours
and aliquots of each sample were mixed with 1,9-dimethyl-methylene blue dye and reagents
from the GAG assay kit. The absorbance at 656 nm was measured spectrophotometrically
(Tecan Infinity, US) and compared to standards made from bovine tracheal chondroitin-4-sul-
fate to determine the GAG content. For all quantifications at least three biological and technical
replicates were obtained.

Scanning Electron Microscopy (SEM) and quantification

Samples were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (PBS; Sigma, UK) and
left for 24 hours at 4C. Following washing with 0.1 M PBS, they were cut into segments of
approximately 1 cm length and cryoprotected in 25% sucrose (Sigma, UK), 10% glycerol in
0.05 M PBS (pH 7.4) for 2 hours, then fast frozen in Nitrogen slush and fractured at approxi-
mately -160C. The samples were then placed back into the cryoprotectant at room tempera-
ture and allowed to thaw. After washing in 0.1 M PBS (pH 7.4), the material was fixed in 1%
OsO4 / 0.1 M PBS (pH 7.3) at 3C for 1 hours and washed again in 0.1 M PBS (pH 7.4). After
rinsing with dH2O, specimens were dehydrated in a graded ethanol-water series to 100% etha-
nol, critical point dried using CO2 and finally mounted on aluminium stubs using sticky car-
bon taps. The material was mounted to present the fractured surfaces across the parenchyma
to the beam and coated with a thin layer of Au/Pd (approximately 2nm thick) using a Gatan
ion beam coater. Images were recorded with a 7401 FEG scanning electron microscope (Jeol,
US). For quantification purposes a blinded observer assessed images taken at 200x using the
measure tool in ImageJ (NIH, US). For all samples examined with SEM three biological and
technical replicates were assessed. Prior to SEM analysis tissue homogeneity was assessed using
standard histology.

Synchrotron-based x-ray phase contrast imaging (XPCI)

Measurements were performed at the biomedical beamline (I17) of the European Synchrotron
Radiation Facility in Grenoble, France. The samples were placed approximately 150 m from
the source (a 21-pole, 15 cm period wiggler), to ensure spatially coherent illumination. The
beam was monochromatized by a fixed-exit Laue/Laue silicon (111) double crystal to 26 keV
(E/E~0.02%) and filtered using 0.8 mm of copper and 3 mm of aluminium. The sample had
been placed on a PI miCos rotation stage (PI mi-Cos GmbH, Eschbach, Germany) to enable
tomographic acquisitions. Images were recorded by a FReLoN CCD camera coupled to a
47 m thick Gd3Ga5O12scintillator. The effective pixel size was 3.5x3.5 micron2. 2000 projec-
tions over 360 were acquired, with an angular step of 0.18. Images were phase-retrieved using

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 Liver Decellularization Protocol Optimized

the single-distance method developed by Paganinet al, and 3D reconstructions performed

using standard filtered back-projections [23]. For all samples examined with XPCI two biologi-
cal and technical replicates were assessed. Prior to XPCI analysis tissue homogeneity was
assessed using standard histology.

Vascular network imaging

Vascular network imaging was carried out on scaffolds as described previously [18]. For visual-
ization of the vascular tree, 1% Trypan Blue (Sigma, UK) was perfused into the scaffold at a
rate of 2 ml/min. An iPhone 4S (Apple, US) was used to film the infusion and iMovie to sepa-
rate still shots. The movies were uploaded on Fiji (NIH, US), the borders of the scaffolds delin-
eated, and following a threshold placement for the white background, intensity and area
covered by the infused dye measured across the stillshots. For all vascular network imaging at
least three biological and technical replicates were obtained. Analyses shown in the figures are
representative samples.

Protein extraction, separation and in-gel protein digestion

A proof-of-concept proteomics analysis was performed comparing fresh liver, DET and
EDTA-DET scaffolds (n = 1). Prior to proteomics analysis a punch biopsy and standard histol-
ogy confirmed appropriate decellularization. Proteins extracted from fresh liver samples,
EDTA-DET and DET treated liver samples were separated by 12% SDS-PAGE, under reducing
conditions. Proteins were visualized by silver staining (ProteoSilver Plus, Sigma, UK), and
bands (32 horizontal slices for each sample) excised from the gel lane and destained using a
solution containing 100 mM sodium thiosulphate and 30 mM potassium ferricyanide in ratio
1:1. Samples were reduced by 10 mM DTT and alkylated with 100 mM iodoacetamide using
the ProGest Investigator Instrument (DigiLab, Genomics Solutions, Cambs, UK) according to
the established protocol [24]. Finally, each dry gel piece was rehydrated in 30 L of 50 mM
ammonium bicarbonate solution containing 250 ng of Trypsin Gold, Mass Spectrometry grade
(Promega, Madison, USA), and incubated at 37C overnight. The trypsinolysis was stopped
with 0.1% formic acid (FA), and tryptic peptides were eluted, vacuum dried, and dissolved in
0.1% FA for LCMS/MS.

Mass spectrometry
LCMS/MS analysis was performed with an LTQ-Velos mass spectrometer (Thermo Fisher
Scientific, UK). Peptide samples were loaded using a Nanoacquity UPLC (Waters, UK) with
Symmetry C18 180 m 20 mm (Waters part number 186006527) trapping column for desalt-
ing and then introduced into the MS via a fused silica capillary column (100 m i.d.; 360 m o.
d.; 15 cm length; 5 m C18 particles, NikkyoTechnos CO, Tokyo, Japan) and a nanoelectros-
pray ion source at a flow rate at 0.42 L/min. The mobile phase comprised H2O with 0.1% FA
(buffer A) and 100% acetonitrile with 0.1% FA (buffer B). The gradient ranged from 1% to 30%
buffer B in 95 min followed by 30% to 60% B in 15 min and a step gradient to 80% B for 5 min
with a flow of 0.42 L/min. The full scan precursor MS spectra (4001600 m/z) were acquired
in the Velos-Orbitrap analyzer with a resolution of r = 60 000. This was followed by data
dependent MS/MS fragmentation in centroid mode of the most intense ion from the survey
scan using collision induced dissociation (CID) in the linear ion trap: normalized collision
energy 35%; activation Q 0.25; electrospray voltage 1.4 kV; capillary temperature 200C; and
isolation width 2.00. The targeted ions were dynamically excluded for 30 s, and this MS/MS
scan event was repeated for the top 20 peaks in the MS survey scan. Singly charged ions were

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 Liver Decellularization Protocol Optimized

excluded from the MS/MS analysis, and XCalibur software version 2.0.7 (Thermo Fisher Scien-
tific, UK) was used for data acquisition.

Protein identification
96 Raw MS files (32 for each experimental condition tested) were loaded and assembled by
MaxQuant (version 1.4.1.2) and visualized by Perseus (version 1.4.1.3) software platform. The
peak list generated by Quant.exe (the first part of MaxQuant) was searched using the Androm-
eda search engine against rat FASTA files (RAT.fasta.gz) downloaded from the UNIPROT
Web site: ftp://ftp.uniprot.org/pub/databases/uniprot/current_release/knowledgebase/
proteomes, last modified 19/2/2014. Selected parameters for Max-Quant analysis included the
trypsin enzyme specificity, 2 missed tryptic cleavages, oxidation of methionine and acetylation
of protein N-terminal as variable modifications and cysteine carbamidomethylation as fixed
modification. In addition, the following parameters were applied to perform the database
search: a minimum peptide length of 6 amino acids, a minimum of 1 peptide identified, a mini-
mum of 1 razor + unique peptide and minimum 1 of unique peptide, top 6 MS/MS peaks per
100 Da, a peptide mass tolerance of 10 ppm for precursor ions, and a tolerance of 0.5 Da for
MS/MS peaks. All proteins were filtered according to a false discovery rate (FDR) of 0.01%
applied at both peptide and protein levels and a maximum peptide posterior error probability
(PEP) of 1. MaxQuant output files were subsequently uploaded into Perseus in order to visual-
ize and combine in one matrix the results obtained for the 3 different experimental conditions
as well as to add GO term association for each protein group and to obtain data for the Venn
Diagram.

Cell culture and scaffold seeding

HepG2 cells were cultured using DMEM/F12 (Life Technologies, UK) supplemented with 10%
FCS (Sigma, UK) and trypsinized and counted prior to seeding. DET liver scaffolds (n = 3)
were divided in lobes (n = 15) and placed in individual petri dishes with high glucose DMEM
and 1% penicillin/streptomycin (Sigma, UK). Cells were resuspended in 1 106/ml concentra-
tion and 2 106 injected per lobe multifocally (~30 injections/lobe) using a 33G needle (TSK
laboratory, Japan). Seeded scaffolds were maintained in hepatocyte medium in a humidified
environment at 37C with 5% CO2 for up to 14 days. Medium was changed every 2 days. At
days 1, 4 and 14 following seeding, the scaffolds were placed in 4% PFA and assessed by histol-
ogy and immunofluorescence.

Immunofluorescence
Seeded scaffolds were fixed in 4% PFA overnight, paraffin embedded and sectioned at 5m.
Sections were rehydrated, permeabilized (0.2% TX100, 30 min) and heat-mediated antigen
retrieval was performed using a citrate buffer. Primary antibodies were used against Ki67
(Abcam, UK) and cleaved-caspase 3 (Cell Signalling, UK) (dilution 1:100) overnight at 4C.
Sections were incubated with Alexa-488 conjugated secondary antibody (Life Technologies)
(dilution 1:100) at room temperature for 1 hour. Nuclei were counterstained with DAPI (dilu-
tion 1:1000) and Sudan Black was used to quench auto fluorescence. For quantification, a mini-
mum of 500 cells over 810 fields of view at x63 magnification were counted for each time
point in a blinded experiment. For all quantifications three biological and technical replicates
were assessed.

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 Liver Decellularization Protocol Optimized

Results
Following the first step of the DET (dH2O), the livers acquired a blanched appearance. Treat-
ment with SDC and DNase led to translucency with vessels visible internally. There were no
macroscopic differences observed between the rat livers pre-treated with EDTA and those
treated with DET only (Fig 1A and 1B). Absence of cells was observed in sections stained with
H&E (Fig 1C), while DNA quantification revealed a significant reduction in DNA amount
between the fresh liver and liver subjected to either decellularization treatment (p<0.001; Fig
1D). Addition of EDTA further reduced the DNA amount compared to the DET treatment
(p<0.001). Quantitative shotgun proteomics confirmed decellularization showing an absence
of selected nuclear (Ntf2, H2A) and cytoplasmic (Rpl39, Tim9) markers from both EDTA--
DET and DET only treated liver samples (Fig 1E and 1F). Moreover, EDTA-DET treatment
was more efficient in removing cellular proteins as the nuclear protein Sub1 and the cyto-
plasmic protein Tim13 could be identified in the DET sample (Fig 1E and 1F).
ECM components were assessed by means of staining and quantification. MT staining dem-
onstrated that the cellular component was detectable only in the fresh tissue (Fig 2A). PR stain-
ing showed that the connective tissue was mostly composed of collagen fibers and was more
compact closer to blood vessels in both fresh and decellularized tissue (Fig 2B). EVG staining
confirmed maintenance of elastin fibers in the inner surface of blood vessels in both fresh and
decellularized livers (Fig 2C). AB staining confirmed the presence of GAG in the fibers of both
DET and EDTA-DET scaffolds (Fig 2D). Collagen quantification of the DET scaffolds demon-
strated no significant changes when compared to fresh tissue (Fig 2E). EDTA-DET scaffolds
had increased collagen compared to both fresh tissue (p<0.05) and DET scaffolds (p<0.01; Fig
2E). Elastin quantification demonstrated a decrease in both DET (p<0.001) and EDTA-DET
(p<0.01) scaffolds to approximately 2040% of fresh tissue (Fig 2F). In similar fashion to colla-
gen, the elastin content of the EDTA-DET scaffolds was higher than DET scaffolds (p<0.05;
Fig 2F). GAG content of both DET and DET-EDTA scaffolds was significantly reduced com-
pared with fresh tissue (p<0.001; Fig 2G); however, there was no difference in content between
DET and DET-EDTA scaffolds. Immunostaining demonstrated collagen I in fresh liver as fine
strands in the parenchymal space, as well as around the blood vessels (Fig 3A). This was pre-
served following decellularization with a strong signal from vascular structures both in DET
and EDTA-DET scaffolds. Immunostaining against collagen III and IV demonstrated a similar
distribution in both fresh and decellularized tissues (Fig 3B and 3C). Weak parenchymal stain-
ing was observed, with a strong signal surrounding vascular and biliary structures. Strong
staining of fibronectin was observed around the main blood vessels in fresh tissue with a more
distributed signal pattern in decellularized scaffolds (Fig 3D). Immunostaining against laminin
was positive only around major blood vessels in both fresh and decellularized tissues (Fig 3E).
Proteomic analysis revealed 679 proteins in common amongst fresh tissue, DET and EDTA--
DET scaffolds, while 231 proteins were uniquely identified in the decellularized scaffolds (Fig
2H). The main ECM constituents were identified in the decellularized samples and could not
be detected in fresh tissue. For example, proteins belonging to the collagen family (IV, VI2
and XVIII), glycoproteins such as fibulin (Fb5), nidogen (Nd1) and the laminin family of gly-
coproteins were detected in DET and EDTA-DET scaffolds, but were undetectable in the fresh
sample (Fig 4A4C). The same trend was observed in the ECM proteins (Fig 4D). A full list of
identified proteins with the indication of the intensity measured in each sample is summarized
in the supplementary information (S1 Table).
SEM further confirmed the complete absence of cells, while the scaffolds consisted of a
three-dimensional network of connective tissue fibers arranged in a honeycomb-like structure.
The portal triad structure was identified clearly at low magnification in fresh, DET and

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 Liver Decellularization Protocol Optimized

Fig 1. Decellularization of the rat liver is achieved following one cycle of DET and EDTA-DET
treatments. (A) Timeframe and infusion of solutions for the DET and EDTA-DET protocols. (B) Macroscopic
appearance of the liver scaffolds showed no difference between the DET and EDTA-DET scaffolds.
Following dH2O addition, the livers became blanched, with SDC and DNase addition resulting in the livers
becoming transparent. (C) H&E staining demonstrated absence of cells in sections from DET and EDTA-DET
scaffolds. (D) DNA quantification reduced DNA in DET and EDTA-DET scaffolds compared with fresh tissue
(p<0.001). (E-F) Histograms showing the signal intensities, associated to relevant nuclear proteins (E) and
cytoplasmic proteins (F); : p<0.001, compared to fresh tissue, #: p<0.001, compared to DET scaffold, scale
bar in macroscopic images: 2cm, scale bar on histology: 100m.
doi:10.1371/journal.pone.0155324.g001

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 Liver Decellularization Protocol Optimized

Fig 2. Decellularization preserves ECM components, with a higher amount of collagen and elastin
present in the EDTA-DET scaffold. (A) MT staining demonstrated acellularity in both scaffolds, showing
composition only by connective tissue. (B) PR staining demonstrated composition mostly by collagen fibers.
(C) EVG staining revealed maintenance of elastin fibers in the inner surface of blood vessels. (D) AB staining
confirmed the presence of GAG in both scaffolds. (E) Collagen was significantly increased in EDTA-DET
scaffolds (p<0.01) compared with fresh tissue. (F) Elastin significantly decreased in DET (p<0.001) and
EDTA-DET (p<0.01) scaffolds compared with fresh tissue. Elastin was higher in EDTA-DET scaffolds
compared with DET scaffolds (p<0.05). (G) GAG quantification showed that while both DET and EDTA-DET
scaffolds had significantly reduced GAG amount, EDTA-DET scaffolds had significantly less when compared
to DET scaffolds (p<0.001). (H) Venn Diagram showing the number and the distribution over sample types of
proteins identified in Fresh, DET and EDTA-DET treated liver tissues; *: p<0.05, : p<0.01, : p<0.001,
compared to fresh tissue, : p<0.05, ||: p<0.01, compared to DET scaffold, scale bar: 100m.
doi:10.1371/journal.pone.0155324.g002

EDTA-DET livers (Fig 5A5C, asterisk). Interestingly, scaffolds prepared with EDTA-DET
were more tightly packed compared to the DET scaffolds, as the porous structure appeared to
be compressed (Fig 5C). Quantification confirmed the change in microarchitecture, demon-
strating a reduction of the hepatocyte pocket from 20.90.5 m to 11.30.3 m (p<0.001; Fig
5D). Synchrotron-based XPCI also showed denser tissue in the EDTA-DET scaffolds com-
pared to DET (Fig 5E and 5F). While the major vessels can clearly be seen in both scaffolds, the

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 Liver Decellularization Protocol Optimized

Fig 3. Immunostaining demonstrates the preservation of ECM proteins in the decellularized scaffolds.
(A) Collagen I staining in fresh tissue was positive as fine strands in the parenchymal space as well as around
the blood vessels (asterisk). This was preserved following decellularization with a strong signal from vascular
structures both in DET and EDTA-DET scaffolds. (B, C) Collagen III and collagen IV staining demonstrated a
similar distribution and preservation in fresh tissue and decellularized scaffolds. Weak parenchymal staining
was observed, and a strong signal surrounding vascular and biliary structures. EDTA-DET scaffolds
demonstrated slightly increased staining in the parenchymal space when compared to DET scaffolds. (D)
Fibronectin showed strong staining around the main blood vessels in fresh tissue with a more distributed
signal pattern in decellularized scaffolds. (E) Laminin showed strong staining around the main blood vessels
in fresh tissue with a more distributed signal pattern in decellularized scaffolds; scale bar: 100 m.
doi:10.1371/journal.pone.0155324.g003

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 Liver Decellularization Protocol Optimized

Fig 4. Proteomics demonstrate the preservation of ECM components in the decellularized scaffolds.
Histograms showing the signal intensities, based on the summed extracted ions counts, measured for each
peptide identified and associated to proteins belonging, accordingly to the GOCC classification, to the class
of Collagens (A), Glycoproteins (B), Laminins (C) and Extracellular Matrix Proteins (D).
doi:10.1371/journal.pone.0155324.g004

more compact nature of the tissue makes the shade of grey in the image of the EDTA-DET
scaffold overall more intense, which, in a phase-retrieved image, correlates directly with
increased density (S1 and S2 Videos).
Trypan blue perfusion through the PV demonstrated maintenance of the vascular network
with no leakage of the dye to the surrounding tissue, as the dye flowed to the IVC (Fig 5G, S3
and S4 Videos). Macroscopically, the DET scaffolds seemed to possess a more compact vascu-
lar network with the dye diffusing through the vessels more readily (Fig 5G). Quantitative anal-
ysis of the dye distribution using Fiji (NIH, USA, 1.47h) confirmed this. Intensity of the dye
infusion through the DET scaffolds followed an S-shaped curve, reaching a maximum point at
approximately 35 seconds (Fig 5H). Dye intensity within the EDTA-DET scaffold increased in
a less steep S-shaped manner with the exponential part lasting 45 seconds instead of 15 (Fig
5H). Maximum intensity was reached at 75 seconds. These results were paralleled by the quan-
tification of surface area of dye distribution with the point of maximal surface area coverage
having a difference of 40 seconds between the two scaffolds (Fig 5I). This is in keeping with the
data from SEM and XPCI showing a more compact appearance of the tissue following EDTA-
DET treatment.

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 Liver Decellularization Protocol Optimized

Fig 5. Addition of EDTA to the protocol makes the matrix more compact in the parenchyma and
vasculature. (A-C) SEM confirmed acellularity in the scaffolds, demonstrating composition by a three-
dimensional network of connective tissue fibers arranged in a honeycomb-like manner. The structure of the
portal triads was identified clearly at low magnification (asterisk). (C) Scaffolds prepared with EDTA-DET
were more tightly packed compared to the DET scaffolds, as the porous structure appeared to be
compressed. (D) Quantification of the hepatocyte pockets in the EDTA-DET scaffold showed a reduction to
approximately 50% of the size in the DET scaffolds. (E, F) Synchrotron-based x-ray phase contrast imaging
corroborated these results, demonstrating a denser scaffold in scaffolds pre-treated with EDTA. (G) Infusion
of trypan blue dye via the portal vein showed maintenance of the vascular network architecture. There was no
dye leakage through the walls to the surrounding tissue; the dye followed the regular pathway of flow to the
IVC. Macroscopically, the DET scaffolds were seen to possess a denser vascular network with the dye
diffusing through the vessels more readily. (H) Dye intensity within the DET scaffolds followed an S-shaped
curve, reaching a maximum point at approximately 35 seconds. Dye intensity within the EDTA-DET scaffold
increased in a less steep S-shaped manner with the exponential part lasting 45 seconds instead of 15.
Maximum intensity was reached at 75 seconds. (I) These results were paralleled by the quantification of

PLOS ONE | DOI:10.1371/journal.pone.0155324 May 9, 2016 12 / 19

 Liver Decellularization Protocol Optimized

surface area of dye distribution with the point of maximal surface area coverage having a difference of 40
seconds between the two scaffolds; #: p<0.001, compared to DET scaffold, scale bar: 1cm.
doi:10.1371/journal.pone.0155324.g005

Having identified the DET scaffolds as a more suitable option for tissue engineering due to
better preservation of micro-architecture and ECM components, the scaffold biocompatibility
was assessed with seeding of a hepatocellular cell line. HepG2 cells were microinjected in a
multifocal manner in the DET scaffolds and seeded constructs were harvested at day 1, 4 and
14 (Fig 6A). H&E staining demonstrated a wide distribution of cellular engraftment across the
scaffold at day 1 and complete repopulation at 2 weeks (Fig 6B). At higher magnification, his-
tology showed the cells repopulating the empty hepatic spaces and forming colonies (Fig 6C).
Immunofluorescence staining against ki67 demonstrated high numbers of proliferating cells at
the first two time-points and only a few ki67-positive cells at 14 days (Fig 6D). Quantification
by a blinded observer showed 6.41%, 16.91.3% and 7.71.8% of ki67 positive cells at days 1,
4 and 14 respectively (Fig 6F). Staining for CC-3 demonstrated a few apoptotic cells across the
three time-points (Fig 6E). Quantification of the CC-3-positive cells showed a non-significant
increase from 2.20.8% to 41% to 4.10.8% at days 1, 4 and 14 (Fig 6G).

Discussion
Hepatic tissue engineering has so far had limited success in animal transplantation studies, in
part due to the vascular leakiness of the decellularized scaffolds. We describe a rat hepatic scaf-
fold obtained using a gentle decellularization process that preserves microarchitecture, ECM
components, and maintains a suitable environment for growth of reseeded cells.
We have firstly described an effective DET decellularization in pig trachea [25] and, in addi-
tion to our successful clinical application of DET-decellularized trachea in a child [22], we have
more recently explored this simple treatment in more complex organs such as the lung and the
intestine [1719]. The DET uses mild decellularization agents at low concentrations, however,
due to the compact and highly cellular nature of hepatic tissue, more challenges could poten-
tially be present and therefore additional treatments may be necessary. We thus compared
decellularization using our normal DET agents versus a protocol in which EDTA was also
used.
The decellularization protocol was slightly modified when compared to our work in the tra-
chea, esophagus, intestine, and lung. We used longer perfusion steps of dH2O and SDC, the lat-
ter being determined by the time required to reach transparency. In order to keep the liver fully
inflated during perfusion, the perfusion speed was increased from 4.5 ml/min (in the dH2O
step) to 6.5 ml/min (in the SDC and DNase-I steps), with the complete protocol lasting 45
hours. Protocols published so far have used a variety of perfusion speeds with protocols
lasting from 2 to 52 hours [416,26,27]. The main factors of a decellularization protocol that
determine its duration are the chemicals used, their concentration and the flow rate. Decellu-
larization with preservation of microarchitecture should involve mild chemicals, at low con-
centrations and correspondingly low flow rates. Shorter protocols (23 hours) either had high
perfusion rates(1030 ml/min), high concentrations of SDS and TX100, or both [5,8,11,12]. At
45 hours we achieved decellularization using a flow rate of 56.5 ml/min. This admittedly may
be difficult for all species, however, we have recently demonstrated for the first time that com-
plete decellularization of whole human liver with a preserved architecture still requires both
SDS and TX100 [26]. The need for gentler decellularization methodologies that avoid SDS and
TX100 led to our comparison of the DET and EDTA-DET protocols. In rat liver, addition of
EDTA further reduced the DNA content compared with DET alone. The concentration of
EDTA we applied (2mM) has been previously used by other laboratories [12,13,16]. Cheng

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 Liver Decellularization Protocol Optimized

Fig 6. Seeding of the DET scaffolds by microinjection demonstrates complete repopulation at 14

days. (A) DET scaffolds were microinjected with HepG2 cells in a multifocal manner and the seeded
constructs harvested at day 1, 4 and 14. (B) H&E staining demonstrated a wide distribution across the
scaffold at day 1 and a complete repopulation at 14 days (B). At higher magnification, cells were seen
repopulating the hepatic spaces, forming colonies and secreting their own ECM at 14 days (C, asterisk). Ki67
staining demonstrated proliferation at days 1 and 4, with only a few ki67-positive cells at 14 days (D).

PLOS ONE | DOI:10.1371/journal.pone.0155324 May 9, 2016 14 / 19

 Liver Decellularization Protocol Optimized

Quantification of ki67-positive cells as a percentage to DAPI-positive cells (F). Staining for CC-3 showed a
few positive cells across the three time-points (E), with quantification demonstrating a non-significant
increase (G); scale bar: 100m.
doi:10.1371/journal.pone.0155324.g006

et al used a higher concentration at 0.2%, while Soto-Guttierez et al preferred the use of EGTA,
a similar compound that preferentially binds calcium ions [6,27]. Even though decellulariza-
tion with EDTA-DET still retained approximately 10% of DNA, a slightly higher value when
compared to published results demonstrating reduction to 0.55%, histological evidence dem-
onstrates cell removal in both DET and EDTA-DET scaffolds [416,26,27].
Following demonstration of acellularity, we sought to assess the maintenance of the ECM
components in both DET and EDTA-DET scaffolds. Histological examination using MT
showed preservation of the portal and lobular connective tissue structure. PR, EVG, and AB
staining demonstrated preservation of the collagen, elastin and GAG components respectively.
Histological stains did not indicate any gross differences between the two scaffolds; however,
we investigated the ECM components further using quantitative assays. Collagen was main-
tained to the same level as fresh tissue following DET treatment, as has been shown for SDS
and TX100 protocols [4,11]. Interestingly, addition of EDTA increased the collagen content by
300%. The increase in collagen content compared with fresh tissue is probably due to normali-
zation by wet weight (that includes the weight of cellular components in fresh tissue) coupled
with the more compact nature of EDTA-DET scaffolds as shown by SEM, XPCI and vascular
network analysis. We have previously demonstrated an increase in collagen following the
decellularization process [17,26]. No protocols containing EDTA have quantified ECM com-
ponents to compare with [6,12,13,27]. Similar results were seen with elastin quantification,
with DET and EDTA-DET treatments reducing the elastin content to 30% and 40% respec-
tively compared to fresh tissue, which is also comparable to the 1050% reported in other pub-
lished work [11,26]. GAG quantification is more complex since approximately 30% of GAG
are associated with cell membranes [28,29], suggesting that any GAG loss is associated primar-
ily with the process of decellularization and only secondarily with removal of GAG attached to
ECM fibers. GAG are important for decellularized matrices as they are associated with factors
that stimulate cell growth and differentiation that are essential for the successful transplanta-
tion of the seeded scaffolds. GAG quantifications demonstrated a reduction to around 20% of
the fresh tissue values with EDTA having a slightly lower value. GAG in the EDTA-DET scaf-
fold may be lower than that in DET due to the improved decellularization that removes more
cellular membrane remnants and thus any associated GAG.
Since scaffold microarchitecture is dependent on the presence of collagen and elastin we
investigated these proteins using immunohistochemistry and proteomics. Localization of spe-
cific ECM proteins (collagens I, III, IV, fibronectin and laminin) showed preservation follow-
ing decellularization with no detectable differences between DET and EDTA-DET scaffolds.
The collagens showed strong staining around the main blood vessels and weak parenchymal
staining. Laminin staining was only positive around the major blood vessels, both in fresh and
decellularized tissue, as reported previously [7,8,12,15]. Fibronectin staining was also consis-
tent with the literature, appearing around blood vessels and within parenchyma [7,8,12]. Prote-
omics confirmed the presence of collagens, laminin, and fibronectin detected by IHC but not
for collagen III. This may be due to low abundance, poor ionization or antibody specificity.
Previous work that demonstrated the presence of growth factors following decellularization
used semi-quantitative fluorometric assays [30]. No growth factors were detected using our
shotgun mass spectrometric analysis due to the use of an SDS gel that limits the detection of
low molecular weight proteins. Growth factors are also difficult to detect using a shotgun

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 Liver Decellularization Protocol Optimized

proteomic approach due to their low concentration. The preservation of laminins, fibronectin,
nidogen-1 and type IV collagen suggests that the basement membrane of the decellularized
scaffolds is maintained. However, the decrease in laminin and nidogen-1 levels in the EDTA-
DET scaffolds suggests that addition of EDTA may partly degrade the basement membrane. A
number of ECM proteins were not detected in fresh tissue. This is probably to a combination
of reasons [31,32]: (i) ion suppression of peptides originating from ECM by peptides originat-
ing from the more abundant cellular components; (ii) incomplete in-gel digestion of ECM
components in the presence of cellular proteins; (iii) limitations on the sensitivity and sequenc-
ing throughput of MS instruments; (iv) inadequate identification of peptides in MS/MS data;
(v) insufficient pre-resolution of the peptides presented to MS/MS sequencing. It should be
borne in mind that proteomic methods allow preferential identification of more abundant, eas-
ily digested proteins and ECM proteins provide a particular challenge for proteomics [33]. Our
proteomics results are in keeping with recent work by Li et al who demonstrated uniquely iden-
tified ECM proteins in decellularized rat livers [34].
Electron microscopy and XPCI proved to be useful at revealing scaffold structure, as previ-
ously described in intestine and lung [17,18]. EDTA addition produced a denser scaffold with
reduced space between the collagen fibers, which would account for the increased collagen and
elastin levels in the tissue, since the matrix is more compact. Hepatic scaffolds with a denser
structure have previously been produced, and even though these may retain ECM components,
they may not be optimal for repopulation due to the collapse of cellular spaces [8,27,30]. The
scaffold resulting from DET treatment retained the architecture of the portal tract as well as
hepatocyte spaces surrounded by collagen fibers. This compares well to work performed by
other groups who found that collagen fibers are weak and the general microarchitecture is
poorly preserved after decellularization [9,13]. XPCI overcomes a basic limitation of conven-
tional x-ray imaging, in which soft tissue contrast is lacking [35]. In this study, we used the
simplest XPCI approach, free-space propagation [36] in its gold-standard synchrotron imple-
mentation, which has already been validated in vivo [37]. Later, translation to more compact,
laboratory-based methods will be pursued [38].
The preservation of the vascular network is of paramount importance for liver tissue engi-
neering, since a porous or destroyed ECM would result in the tissue being unacceptable for
transplantation. This has previously been assessed by dye infusion though the PV/IVC
[7,13,16,30], or by creation of vascular casts [4,6,9]. High concentrations of TX100 and SDS led
to the destruction of the vascular network [9]. We have shown the preservation of the capillary
network, which, interestingly, differed between DET and EDTA-DET scaffolds. Dye infusion
was quantified and it was shown that dye intensity along with surface area coverage was
increased in DET scaffolds. EDTA-DET scaffolds followed a two-stage increase in dye cover-
age. This may be due to the collapsed vascular spaces owing to the denser ECM. Following the
build-up of sufficient pressure the distal vessels open up, allowing the passage of the dye.
We chose the DET scaffold as the optimal scaffold for cell seeding as although EDTA addi-
tion slightly increased the decellularization efficiency, it worsened architecture preservation as
assessed by SEM, XPCI, and vascular network analyses. Previous cell seeding methodologies
for the liver have focused on intravascular seeding of hepatic and/or endothelial cells
[4,7,11,14,27]. Following seeding, cells were found to have organized around central vascular
structures. However, movement across the blood vessel walls would require extravasation
across the basement membrane. The mechanisms suggested for such cell migration include
proteolytic digestion, mechanical force and variability in basement membrane composition
[39]. In scaffolds where there are no cells to repair the ECM following proteolytic and/or
mechanical degradation, the blood vessels may become perforated and leaky, preventing its
use for transplantation. We sought to avoid this by performing seeding using multifocal

PLOS ONE | DOI:10.1371/journal.pone.0155324 May 9, 2016 16 / 19

 Liver Decellularization Protocol Optimized

microinjection, allowing wide and homogeneous cell distribution, engraftment and survival in
a three-dimensional matrix culture condition. Cells were alive and proliferating 2 weeks follow-
ing in vitro seeding, completely repopulating the scaffold, similarly to published results with
intravascular cell infusion [4,7,11,14,27]. The scaffolds architecture and composition enabled
cell seeding and adhesion that, coupled with oxygen and nutrient exchange, allowed cellular
expansion and scaffold repopulation. We aim to expand on this by performing concomitant
intravascular seeding of endothelial cells.

Conclusions
The present work demonstrates the production of a natural acellular matrix that can be
obtained from rat liver by DET decellularization while preserving microarchitecture and ECM
components. Addition of EDTA creates a denser, compact matrix, and decreases residual DNA
content. The DET scaffold is biocompatible, allowing cell adhesion and growth for up to 14
days. The fine-tuning of the protocol including scaling up of the methodology, optimization of
cellular reseeding and transplantation studies are critical for the long-term goal of providing a
potential therapeutic alternative to conventional transplantation.

Supporting Information
S1 Table. Proteomics full data table. Master table of MS data summarizing all the proteins
identified in each sample (Fresh, DET, and EDTA-DET).
(XLSX)
S1 Video. Synchrotron-based XPCI of DET scaffold.
(DIVX)
S2 Video. Synchrotron-based XPCI of EDTA-DET scaffold.
(DIVX)
S3 Video. Vascular network imaging of DET scaffold.
(AVI)
S4 Video. Vascular network imaging of EDTA-DET scaffold.
(AVI)

Acknowledgments
The authors acknowledge support by grants from the Medical Research Council, the Royal Col-
lege of Surgeons of England, the Sparks Childrens Medical Charity, the British Foreign Office
for the UK/USA Stem Cell Collaboration Award and the Mittal Research Fund. AO, and CKH
are funded by the EPSRC (grants EP/G004250/1, EP/I021884/1 and EP/L001381/1). HS and
PG are supported by the Wellcome Trust and ERC (WT095662MA, ERC-2013-StG337057).
The authors are grateful to the personnel of beamline ID17 at the European Synchrotron Radi-
ation Facility (especially Dr. A. Bravin and Dr. E. Brun) and to Dr. P.C. Diemoz for their help
in setting up and running the synchrotron experiment. The authors would like to thank Dr.
Bertrand Vernay for his help with the quantitation in the vascular network imaging. SE and
PDC are supported by Great Ormond Street Hospital Childrens Charity. We would also like
to thank the Royal Society/Wolfson Foundation for the tissue engineering laboratory refurbish-
ment grant obtained for the Pediatric Surgery Department at UCL Institute of Child Health.

PLOS ONE | DOI:10.1371/journal.pone.0155324 May 9, 2016 17 / 19

 Liver Decellularization Protocol Optimized

Author Contributions
Conceived and designed the experiments: PM SE AO JGZ MP PG PDC. Performed the experi-
ments: PM FG HS AM PS LU SPL BL GM CH NJS MT. Analyzed the data: PM HS LU BL CH
PG PDC. Contributed reagents/materials/analysis tools: NJS MT SE AO JGZ MP PG PDC.
Wrote the paper: PM FG HS AM PS LU SPL BL GM CH NJS MT SE AO JGZ MP PG PDC.

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