Tissue and Organ Decellularization in Regenerative Medicine

Abstract

The advancement and improvement in decellularization methods can be attributed to the increasing demand for tissues and organs for transplantation. Decellularized tissues and organs, which are free of cells and genetic materials while retaining the complex ultrastructure of the extracellular matrix (ECM), can serve as scaffolds to subsequently embed cells for transplantation. They have the potential to mimic the native physiology of the targeted anatomic site. ECM from different tissues and organs harvested from various sources have been applied. Many techniques are currently involved in the decellularization process, which come along with their own advantages and disadvantages. This review focuses on recent developments in decellularization methods, the importance and nature of detergents used for decellularization, as well as on the role of the ECM either as merely a physical support or as a scaffold in retaining and providing cues for cell survival, differentiation and homeostasis. In addition, application, status and perspectives on commercialization of bioproducts derived from decellularized tissues and organs are addressed.
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Tissue and Organ Decellularization in Regenerative Medicine
Rajesh Guruswamy Damodaran
Laboratoire de bio-ingénierie et de biophysique de l’Université de Sherbrooke, Department of Chemical and Biotechnological Engineering,
Université de Sherbrooke, 2500 Boul. de l’Université, Sherbrooke, QC, J1K 2R1, Canada
Pharmacology Institute of Sherbrooke, Faculté de médecine et des sciences de la santé, 3001 12
ième
Avenue Nord, Sherbrooke, Québec,
J1H 5N4, Canada
Research Centre on Aging, Institut universitaire de gériatrie de Sherbrooke, 1036 rue Belvédère Sud, Sherbrooke, Québec, J1H 4C4,
Canada
Patrick Vermette
Laboratoire de bio-ingénierie et de biophysique de l’Université de Sherbrooke, Department of Chemical and Biotechnological Engineering,
Université de Sherbrooke, 2500 Boul. de l’Université, Sherbrooke, QC, J1K 2R1, Canada
Pharmacology Institute of Sherbrooke, Faculté de médecine et des sciences de la santé, 3001 12
ième
Avenue Nord, Sherbrooke, Québec,
J1H 5N4, Canada
Research Centre on Aging, Institut universitaire de gériatrie de Sherbrooke, 1036 rue Belvédère Sud, Sherbrooke, Québec, J1H 4C4,
Canada
DOI 10.1002/btpr.2699
Published online 0, 2018 in Wiley Online Library (wileyonlinelibrary.com)
The advancement and improvement in decellularization methods can be attributed to the
increasing demand for tissues and organs for transplantation. Decellularized tissues and organs,
which are free of cells and genetic materials while retaining the complex ultrastructure of the
extracellular matrix (ECM), can serve as scaffolds to subsequently embed cells for transplanta-
tion. They have the potential to mimic the native physiology of the targeted anatomic site. ECM
from different tissues and organs harvested from various sources have been applied. Many tech-
niques are currently involved in the decellularization process, which come along with their own
advantages and disadvantages. This review focuses on recent developments in decellularization
methods, the importance and nature of detergents used for decellularization, as well as on the role
of the ECM either as merely a physical support or as a scaffold in retaining and providing cues
for cell survival, differentiation and homeostasis. In addition, application, status, and perspectives
on commercialization of bioproducts derived from decellularized tissues and organs are
addressed. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018
Keywords: decellularization, recellularization, artificial organs, extracellular matrix (ECM),
commercialization
Introduction
Increased demand for tissues and organs for transplantation
has expanded the fields of research on decellularization. Prep-
aration of decellularized tissues and organs involves removal
of cells and genetic materials from the tissue or organ and
retention of the complex ultrastructure of the extracellular
matrix (ECM) to serve as a natural scaffold. The ECM is pro-
duced by the native cells and serves as a mechanical support
and cues for cell migration, attachment, differentiation and
function.
1,2
Many tissues and organs have been successfully
decellularized and used for research and commercial purposes.
Different techniques including physical, chemical and biologi-
cal methods are currently applied in decellularization and
these have their own pros and cons.
The two important criteria to be fulfilled during a decellu-
larization procedure are: (i) retention of the native structures
and (ii) removal of a maximum of cell components. Several
tissues and organs such as the small intestine, bladder,
placenta, and liver, only to name a few, have been decellular-
ized and used as ECM scaffolds.
3–6
Recent advances in regen-
erative medicine and tissue engineering have paved the path
for investigating the use of whole decellularized organs for
transplantation. Many in vitro and in vivo studies support that
ECM-based scaffolds made of decellularized tissues and
organs have beneficial effects on cells, including stem cells, in
terms of their attachment, proliferation, viability and function-
ality. In favor to that, these scaffolds have a low immunoge-
nicity, which facilitates their in vivo applications.
6,7
Over the past 20 years, decellularized tissues and organs
have been commercially available for wound healing. Compa-
nies are working to commercialize ECM-based products for
organ transplantation, which some are in clinical trials.
The objective of this review is to give an update on the
most important tissues and organs that are used for decellulari-
zation, the importance of the ECM as a complete set of bio-
molecules, the techniques and chemicals applied in
decellularization operations, rationale behind using ECM-
based bioproducts, as well as applications, perspectives and
path to commercialization.
Correspondence concerning this article should be addressed to
P. Vermette at: patrick.vermette@usherbrooke.ca
© 2018 American Institute of Chemical Engineers 1

Major tissues and organs used for decellularization
Liver
The liver is one of the important organs, which has been
decellularized for regenerative medicine. The first successful
liver decellularization was done by surface treatment with
detergents and the resulting bioproduct was recellularized with
rat hepatocytes to assess functionality.
8
Surface treatment
refers to decellularizing a tissue or an organ by immersion in
decellularizing solutions as opposed to perfusion decellulariza-
tion, which involves perfusing solutions within the tissue or
organ. Studies on liver decellularization report the perfusion
of detergents through the network of veins and arteries, then
its recellularization and in vivo transplantation.
9–11
For exam-
ple, seeding rat hepatocytes into decellularized liver and sub-
sequent transplantation in rats showed that less damages
occurred to the cells following an 8 h transplantation, as
revealed by TUNEL staining.
9
In a separate study, when
human liver stem cells were seeded in decellularized rat liver,
differentiation into functional hepatocytes was observed as
well as the presence of epithelial and endothelial-like cells
reported.
12
Research on achieving human-sized whole liver
has also been attempted with pigs by decellularizing the organ
with SDS (sodium dodecyl sulfate).
13,14
Heart
Heart decellularization techniques started initially with heart
valves by surface decellularization using detergents and subse-
quent recellularization with different cell types.
15–17
Intro-
duced in 2008, decellularization of whole hearts from rats was
done by perfusion methods and the resulting scaffolds were
seeded with cardiac and endothelial cells; contraction in cell
patches was observed after 4 days.
18
Recent advances in heart
decellularization aim to improve perfusion processes during
decellularization and cell seeding with progenitors as well as
to achieve decellularization of organs from larger animals.
19–23
Allo-transplanted porcine heart seeded with mesenchymal
stem cells showed thrombosis in arteries as well as the pres-
ence of inflammatory cells and the observation was similar to
decellularized heart transplanted without mesenchymal stem
cells.
24
In addition, a 14-day culture of human cardiomyocytes
in human decellularized heart showed visible contraction when
electrically stimulated.
25
Lungs
Decellularized lungs were considered as a source of bioartifi-
cial organs for transplantation in end-stage lung diseases. The
first successful perfused decellularization was reported in 2010
following methods initially applied on heart and liver.
26
Recent
works on decellularizing lung involve the improvement of
decellularization process and cell seeding methods, as well as
studies on the role of stem cells.
26–28
Attempts to decellularize
human lungs are step closer to fill the gap in the demand for
organ transplantation.
29,30
Stabilization of endothelial cells by
seeding adipose-derived stem cells into decellularized lungs
could be a potential solution to regenerate the vasculature of
decellularized lungs.
31
Seeding mesenchymal stromal cells in
decellularized lung in suspension bioreactor resulted in differen-
tiation of the cells into collagen-1 alpha-1 producing cells.
32
In
another study, human-induced pluripotent stem cells (iPSCs)-
derived endothelium and ventralized iPSC were seeded in
decellularized lungs resulting in retention of epithelial progeni-
tors phenotype by expressing Nkx21 and endothelial phenotype
by expressing CD31.
33
Transplantation of decellularized rat
lung seeded with epithelial and endothelial cells maintained the
animals alive without ventilation for 6 h.
26
Pancreas
The first perfused decellularization of pancreas was carried
out in 2009 with a porcine organ,
34
and then applied in
mouse.
35
The decellularized tissues were used to seed cells and
islets and their in vitro functions were studied as well as the
biocompatibility of these bioartificial pancreata was investigated
in vivo.
34–37
Later, rat pancreata were decellularized and a new
method was introduced to infuse islets into the ducts, veins and
arteries.
38
Decellularized pancreata were seeded with human
stem cells, an AR427 acinal cell line, MIN-6 cells and islets,
and cell attachment as well as basic cell functions was
studied.
35–38
Recently published work from our group demon-
strated that, islets infused into the ductal system of decellular-
ized pancreata were functional by releasing insulin in response
to glucose stimulation after 48 days in vitro.
39
Small intestinal submucosa
One of the first usages of small intestinal submucosa (SIS)
was during 1989, as a vascular graft for dogs.
40
SIS biomate-
rials are widely used in regenerative medicine, for example to
reconstruct urethra, cornea, esophageal, and heart.
41–44
Recently, SIS seeded with urothelial cells showed formation
of new vessels, proliferation of smooth muscle cells, epitheli-
zation and maintenance of the opening of urethra when com-
pared with unseeded scaffold.
45
Reconstruction of cornea was
accomplished successfully in 106 cases by preserving vision
at third month of postsurgery using porcine SIS.
46
Seeding
autologous oral mucosa epithelial cells along with SIS resulted
in muscular regeneration and re-epithelization after 8 weeks of
surgery and the results were superior to SIS with no cells.
47
Applying Food and Drug Administration (FDA)-approved SIS
ECM seeded with mesenchymal stem cells resulted in a lower
response of the adaptive immune system in a porcine model,
when compared with SIS–ECM with no cells.
48
Bladder
Urinary bladder has been one of the first organs selected for
decellularization. Recent studies on ECM derived from decel-
lularized bladder have indicated a beneficial effect on neuron
survival, stem cells, spinal cord, and open wounds.
49–52
Uri-
nary bladder-derived ECM has shown greater advantage than
cardiac-derived ECM in remodelling and for attracting site-
specific cells (cardiomyocytes) after implantation.
53
The
implantation of large segments (>24cm
2
) of urinary bladder
into porcine bladder showed infiltration of smooth muscle
cells and promoted angiogenesis after 30 days.
54
Vesicles
derived from urinary bladder ECM had a positive effect on
neuron neurite growth and on spinal cord injury repair.
50,51
Urinary bladder-derived ECM also had a better effect on the
treatment of open wounds when compared with conventional
therapies.
49
Bioproducts made from urinary bladder-derived
ECM are available in different formulations such as sheets
(Cytal
®
Burn Matrix, also marketed as MatriStem
®
Burn
Biotechnol. Prog., 20182

Matrix, ACELL (Columbia, USA); the product is applied
directly on wounds), powder (MicroMatrix
®
, ACELL, Colum-
bia, USA) and hydrogels (not available commercially).
Kidneys
The initial method of decellularization of whole kidneys
was achieved by perfusion techniques, and the resulting prod-
ucts were used to seed and differentiate stem cells.
34,55
Advances in kidney decellularization include in vitro and
in vivo studies on rat and mouse kidneys to investigate the
functionality of recellularized kidneys with endothelial, epithe-
lial and renal progenitor cells.
56,57
Bones and cartilages
The successful removal of cells from bones and cartilages
was achieved in 2011.
58
Recent advancements in cartilage and
bone repair include utilizing adipose-derived stem cells, multi-
potent stromal cells seeded on decellularized cartilage ECM to
study the mechanical properties and bone regenerative
capacity.
59,60
Plants
Recent advancements in tissue and organ decellularization
have extended their boundaries to apply perfused decellulari-
zation protocols on plant leaves and eventually recellularize
them with cardiomyocytes resulting in contractile function
successfully for 21 days.
61
Recent decellularization methods
Over the years, several studies have been conducted to
understand the utility of decellularized tissues and organs in
regenerative medicine. Table 1 provides a summary of recent
studies highlighting the various techniques and their corre-
sponding results on decellularized tissues and organs.
Chemicals used for decellularization
Any decellularizing procedure involves disruption of the
ECM. The selection of decellularizing agents causing the least
possible damage to the ECM is vital. The choice of decellular-
izing agents depends on the nature of the tissue or organ. A
decellularization operation involves notable changes in the tis-
sue and organ color, starting from its original color to become
translucent. The process consists in removing cellular compo-
nents and nuclear materials. Destruction of the cell membrane,
dissolution of cellular components and removal of nucleic acid
materials are the key steps.
Alkalis and acids dissolve cellular components and wash
nucleic acids.
80
Detergents solubilize cell membranes and
remove cellular components and nucleic acids.
81
Detergents are soluble amphiphiles, which can solubilize
biological membranes. They have both hydrophilic and hydro-
phobic groups with a more hydrophilic nature than biological
membranes. They can be classified according to their charges:
nonionic, anionic, cationic, and zwitterionic detergents.
82,83
Triton X-100, a nonionic detergent, is commonly used for cell
lysis and permeabilization.
84,85
The hydrogen bonds in lipid
bilayers are disrupted by the polar head group of Triton X-
100, breaking the cell plasma membrane.
82
In some studies,
Triton X-100 has been reported to be the best decellularizing
agent compared with other detergents and that for many tis-
sues and organs including ligaments, small intestine, annulus
fibrosus, liver and aortic valves.
42,86–89
On the other hand,
some studies demonstrated that Triton X-100 had poor cell
removal capacity for many tissues and organs such as veins,
cornea, urethra, heart and kidneys.
18,90–93
A detergent commonly used in decellularization is the SDS.
SDS is an anionic detergent, used in commercial applications
such as in cleaning and in the making of cosmetics. SDS is
also well known in biochemistry for its use in the SDS-PAGE
technique. Its application in decellularization can be counterin-
tuitive when aiming to preserve the tissue or organ ECM
structure intact, as SDS is known to denature proteins.
94
SDS
solubilizes membrane proteins and thus helps effectively in
the decellularization process. SDS incorporates into the outer
membrane layer by increasing its curvature, creating a stress
which results in the membrane disruption.
95
SDS proved to be
superior when compared with other detergents in removing
cells and genetic materials in many tissues and organs includ-
ing aorta, veins, heart, kidneys and urethra.
18,90,92,93,96
Triton
X-100 and SDS have also shown negative influence on tissues
and organs used for decellularization but, on the other hand,
when these two detergents were used in combination, the mix-
ture allowed efficient decellularization and preservation of
ECM structures and this has been tested on tendons, kidneys,
SIS, and liver.
11,43,97,98
Another ionic detergent/bile salt frequently used in decellu-
larization is sodium deoxycholate (SDC). After adhering to
the membrane, SDC incorporates its cholate moiety into the
lipid membrane, resulting in pore formation and membrane
disruption.
99
SDC, alone or in combination with other deter-
gents, has shown to be effective in removing cells while
retaining ECM structures in decellularized tissues.
16,100
CHAPS, 3-([3-cholamidopropyl]dimethylammonio)-
1-propanesulfonate hydrate, is another detergent used in decel-
lularization. CHAPS is a zwitterionic detergent with a hydro-
philic side and a hydrophobic back. CHAPS does not result in
pore formation, instead the molecules seem to directly break
the lipid membrane.
101
CHAPS has proven to be milder and
to have greater ability to retain mechanical strength in
lungs.
27,30,102
Other chemicals applied in decellularization protocols are
acids and bases. Both acids and bases had a negative impact
on ECM mechanical strength.
103
Acetic acid-treated bovine
pericardia had reduced tensile strength, but on the other hand,
it supported the growth of human mesenchymal stem cells and
hence proved to be biocompatible.
104
Peracetic acid along
with ethanol was used to sterilize decellularized scaffold mate-
rials, a well-known and FDA-approved method for medical
devices.
6
However, the use of peracetic acid for decellulariza-
tion resulted in incomplete cell removal from tumor ECM,
porcine liver and kidneys.
105,106
Decellularization of skin
using NaOH had a cytotoxic effect on fibroblasts, however,
prolonged washing of the scaffold helped in decreasing cyto-
toxicity.
107
Alcohols or acetone solutions can be used in pre-
treatment of tissues for lipid removal. It is easier to lyse and
remove cells after eliminating lipid contents.
108
Acetone/alco-
hol combinations had a negative effect on tissue dehydration
and greater influence on modifying mechanical properties and
morphology of temporomandibular joint from pig jaws, as
compared with SDS-treated tissues.
109
Biotechnol. Prog., 2018 3

Table 1. Recent Studies Involving Decellularization Methods and Their Outcomes
Organ or
Tissue Species Applications Methods Chemicals Results References
Liver Mouse Culture of mouse hepatocytes Nonthermal irreversible
electroporation
- Cell integration into the host liver
parenchyma
62
Rat Hepatocyte seeding and
transplantation into rat
Perfusion through portal vein SDS (0.01, 0.1 and 1%) Preservation of functional and
structural characteristics of
microvascular network and display
of liver-specific functions
9
Decellularization using
increasing detergent
concentration
Perfusion through portal vein 1, 2 and 3% Triton X-100 +
0.1% SDS
Retention of laminin in the basement
membrane and collagen IV
63
Minimizing ECM damage Arterial perfusion under
oscillation pressure
conditions
1% Triton X-100 + 1% SDS Fast, homogenous, higher
concentration of hepatocyte growth
factor compared with the native
tissue, high GAG concentration and
gentle method of decellularization
64
Pig Comparison of three detergent
mixtures for decellularization
and recellularization with rat
hepatocytes
Perfusion decellularization and
freeze–thaw
1% SDS;
1% Triton X-100 + 1% SDC;
1% SDC +1% SDS
Cell removal, preservation of ECM
structure and biocompatibility,
achieved by 1% Triton X-100 + 1%
SDC
11
Kidneys Pig Study on decellularization
methods
Perfusion using a
high-throughput
decellularization apparatus
water and 0.5% SDS Preservation of important ECM
components, intact vasculature tree,
in vitro biocompatibility for cells
and preservation of gross anatomy
after 2 weeks of implantation
65
Heart Rat Recellularization with aortic
endothelial cells and rat
neonatal cardiomyocytes
Coronary perfusion 1% SDS Performance of basic functions 66
Zebra fish
+ Mouse
In vitro proliferation and
migration of human cardiac
precursor cells
Freeze–thaw cycles - Exhibition of pro-proliferative and
chemotactic effect with human
cardiac precursor cells by zebra fish
ECM including structural
preservation and cardiac contractile
function
67
Pig Comparison of two detergents Surface treatment 1% Triton X-100 + nucleases +
trypsin and SDC with and
without nucleases
Complete cell removal and
preservation of ECM structures by
Triton X-100 + nucleases + trypsin
68
Myocardium Pig Comparison of decellularizing
agents and recellularization
with rat myocardial
fibroblasts
Surface treatment 1% SDS;
1% Triton X-100; 0.5% trypsin
Effective decellularization by SDS and
retention of ECM microstructures.
Display of different beating
magnitudes of seeded cells: largest
in trypsin-, moderate in SDS- and
none in Triton X-100-treated
scaffolds
69
Teeth Human Comparison of
decellularization methods
Surface treatment 10% formaldehyde, PBS +
EDTA +2.5% sodium
hypochlorite; PBS + EDTA
+ 40 v hydrogen peroxide;
PBS + 2.5% sodium
hypochlorite + Ryozime
®
;
PBS + EDTA + 40 v
hydrogen peroxide +
Ryozime
®
Maximum removal of biological
particles and less damage to the
structures obtained with
PBS + EDTA +40 v hydrogen
peroxide + Ryozime
®
70
Biotechnol. Prog., 20184

Bones and
cartilages
Rat Calvaria regeneration in vivo
and recellularization with
mesenchymal stem cells
Surface treatment 0.5% SDS + 0.1% ammonium
hydroxide
Formation of new bone and merging
with the host bone after 3 months.
Proliferation and osteogenic
differentiation of mesenchymal stem
cells
71
Pig Tissue characterization and
recellularization with murine
fibroblasts and porcine
chondrocytes
Surface treatment 1% SDS Retention of compatible tension
properties of intact tissue but
reduction in compression. No
cytotoxicity on recellularized cells
72
Bovine Tissue characterization and
recellularization with human
adipose mesenchymal stem
cells
Surface treatment 10 mM
ethylenediaminetetraacetic
acid disodium salt dihydrate
(Na
2
EDTA, pH 7.2–7.4)
Chondrogenic differentiation of human
adipose mesenchymal stem cells
73
Skeletal
Muscle
Pig Comparison of
decellularization methods
and formulation of hydrogel
scaffolds
Surface treatment 1% SDS; 1% SDS + 0.2%
SDC; 1% SDS + 1% Triton
X-100; 1. 0.2%
Trypsin/0.1% EDTA,
2. 0.5% Triton X-100, 3. 1%
Triton X-100/0.2% SDC
Formation of jelly-like hydrogel by
using 0.2% trypsin/0.1% EDTA,
0.5% Triton X-100, and 1% Triton
X-100/0.2% SDC treatment
74
Nerve Rat Reconstruction of long gap
nerve injury
Surface treatment (Dulbecco’s modified Eagle
medium +10% fetal bovine
serum and 4% penicillin/
streptomycin/amphotericin
B) + (DMEM with 10% fetal
bovine serum and 2%
penicillin/streptomycin/
amphotericin) + PBS
Exhibition of functional nerve
regeneration in vivo
75
Skin Mouse Comparison of detergent-free
decellularization with ionic
and anionic detergents for
decellularization
Surface treatment (50 nM latrunculin B +
Dulbecco’s modified Eagle
medium) + 0.6 mol/L
potassium chloride +1.0 M
potassium iodide
Retention of matrix composition and
biomechanical properties with
detergent-free decellularization
method, but all methods resulted in
comparable bio-functionality
76
Trachea Rabbit In vivo transplantation Freeze-drying, detergent and
sonication
1% SDS Exhibition of necrosis and animal
death in 7–24 days and integration
of decellularized scaffold into the
host structure was observed
77
Spleen Rat Recellularization of bone
marrow mesenchymal stem
cells (BMSCs).
Freeze–thaw cycles and
perfusion decellularization
0.1% trypsin, 0.05% EDTA,
3% Triton X-100
Differentiation of BMSCs into
functional hepatocyte-like cells
78
Culture of rat hepatocytes Perfusion decellularization 0.1% SDS Survival and secretion of urea and
albumin for 10 days in culture
79
Pancreas Mouse Recellularization of MIN-6
cells and AR42J
Perfusion decellularization 0.5% SDS Upregulation of insulin gene in vitro,
less immune reaction and
angiogenesis after 14 days in vivo
35
Recellularization of MIN-6
cells and in vivo
implantation
Freeze–thaw at −80Cfor
1 day and followed by
perfusion decellularization
1% Triton X-100 + 0.1%
ammonium hydroxide
Biocompatibility and angiogenesis
induction
37
Rat Three perfusion routes for
decellularization and
recellularization of islets
Perfusion decellularization 1% Triton X-100 + 0.5% SDS Reach of islets into parenchymal space
of the pancreas after infusion
through ductal system
38
BMSCs, bone marrow mesenchymal stem cells; ECM, extracellular matrix; GAG, glycosaminoglycans; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate.
Biotechnol. Prog., 2018 5

Along with chemicals, the use of enzymes in decellularizing
techniques can be advantageous. However, it is not possible to
decellularize tissues or organs only with enzymes. Enzymes
can be used in the removal of cell debris. The most commonly
used enzymes are trypsin, nucleases, collagenase, thermolysin,
α-galactosidase and dispase.
103,110
Rationale for using ECM-based products
ECM proteins have shown positive influence on cell
growth, function, and differentiation and examples of biomi-
metic surfaces have been reviewed by us.
111
An important class of ECM-derived proteins is that of colla-
gens, as reviewed elsewhere.
112
Collagen is in fact an abun-
dant source of ECM proteins. Collagen microspheres can
differentiate oligodendrocyte progenitors into oligodendro-
cytes and collagen-immobilized nanowires had positive influ-
ence on human microvascular endothelial cells.
113,114
Fibronectin and laminin showed positive effects on cell sur-
vival and functions both in vitro and in vivo.
115,116
In the past
years, our group has verified the positive effects of various
ECM peptides and proteins. For example, immobilization of
fibronectin, fibrinogen and fibrin on polymer surfaces
enhanced the proliferation of smooth muscle cells.
117
Also,
embedding pancreatic islets and endothelial cells in fibrin
showed increased insulin secretion and preservation of islet
integrity.
118
Culturing young porcine islets in fibrin exhibited
the protective nature of fibrin towards hydrogen peroxide.
119
In addition to whole ECM proteins, functional peptides
derived from the ECM can influence cell behaviors.
120–123
Peptide motifs from collagen I (GTPGPQGIAGQRGVV), col-
lagen IV (MNYYSNS), fibronectin (PHSRN), laminin
(YIGSR) can enhance cell attachment.
124,125
Arginine-glycine-
aspartic acid (RGD), a peptide sequence found in fibronectin,
vitronectin, laminin, collagen types I and IV, has a high affin-
ity for integrin-mediated cell attachment and spreading, func-
tionality and tissue development.
126–130
The RGD sequence
co-immobilized with the SVVYGLR sequence was used to
enhance the adhesion of endothelial cells.
123
Three ECM-
derived peptides, laminin (IKLLI and PDSGR) and cadherin
(HAVDI) supported the adhesion and survival of insulin-
containing cultures for 5 days.
122
Rat insulinoma INS-1 cells
cultured on immobilized ECM proteins and peptides (RGD
and CDPGYIGSR) showed higher glucose-stimulated insulin
secretion compared with the surfaces bearing no peptides.
120
Hyaluronic acid, a polysaccharide, helped in neuron sur-
vival and controlled self-renewal and differentiation of human
embryonic stem cells.
131,132
Many studies demonstrated that
proteoglycans from the ECM are involved in molecular events
including cell adhesion, proliferation and migration.
133
These are just examples of the effects ECM components
can have on cells allowing appreciating the potential impact of
bioproducts made from decellularized tissues and organs. As
reviewed by us and others,
111,134,135
other ECM proteins have
been applied to produce biomimetic materials and surfaces
and these include: collagen types I, III, and IV, gelatin, fibro-
nectin, vitronectin, polysaccharide nanofibers, proteoglycans,
nidogen, and laminins, as well as ECM-mimicking peptides
including YIGSR,RGD, DGEA, KRSR, IKVAV, P15, and
GFOGER, to list a few.
Furthermore, following their implantation, decellularized
scaffolds are repopulated by host cells and even degraded,
resulting in a functional tissue containing site-specific cells.
136
Decellularized tissues or organs, with or with no cells, can
chemo-attract progenitor cells towards the implantation
site.
137,138
The degrading ECM molecules are also involved in
various biological activities and growth factor signaling.
139,140
Therefore, these matrices made from decellularized tissues and
organs act as bioactive scaffolds by modifying the host envi-
ronment to create a more suitable solution for healing, repair,
and even regeneration.
ECM scaffolds made from decellularized tissues and organs
also provide mechanical support in many conditions. They
possess different physical characteristics, which are often diffi-
cult to reproduce with synthetic materials.
141
The ECM pro-
vides a mechanical support for the recellularizing cells to
attach to the matrix. It is very important for scaffolds made
from decellularized tissues and organs to have certain mechan-
ical properties matching the site of implantation of the tissue
or organ to be substituted. Creating less damage to the tissue
during decellularization could yield better mechanical proper-
ties and more intact vasculature structure, facilitating the
induction of angiogenesis post-implantation.
37
Also, the pres-
ence of ECM proteins and functional peptides in 3D scaffolds
allows recreating environments more physiological for cells,
as opposed to traditional 2D culture systems on flat surfaces.
Immunogenicity of ECM-based scaffolds
Most decellularization protocols involve removing cell
materials, which could cause the major immunogenic reac-
tions: hyperacute, acute immune, and chronic immune rejec-
tion as well as inflammatory reactions. Hyperacute rejection is
a severe reaction of vascularized xenografts, triggered a few
minutes to a few hours after implantation.
142,143
This hypera-
cute rejection is mainly caused by the α-Gal epitope
(galactose-α[1,3]-galactose) expressed on many cell surfaces,
and is less common in human and old-world monkeys. Pigs
have been modified genetically to remove the Gal epitope to
avoid xenograft rejections, but the application of this method
is resulting into an acute immune rejection by non-Gal anti-
bodies circulating at low levels, a few days to a few weeks
after implantation.
144
Chronic immune rejection is associated
with allografts transplantation by introducing donor-specific
antibodies.
145
Also, the presence of DNA in decellularized tis-
sues and organs can potentially cause inflammatory
reactions.
146
Collagens with lower immunogenicity is achieved by
removing N- and C-terminal telopeptides by pepsin type-I
treatment, which are referred to as atelocollagens.
147
Atelocol-
lagens have been clinically applied in wound healing and as
bone substitutes.
147
Host response towards bioproducts made from ECM mate-
rials are different and are based on the product properties, spe-
cies and methods of preparation; five different commercially
available products were tested on rats and resulted in distinct
morphological appearance of the implantation site.
148
Simi-
larly, the immune reaction from the decellularized heart tissue
(SynerGraft
®
) had a mixed reaction after implantation. In one
study,
149
SynerGraft
®
exhibited much less human leukocyte
antigen Classes I and II antibodies, as claimed by the com-
pany; however, in another study,
110
macrophage infiltration
after implantation was observed into the tissue decellularized
using the SynerGraft
®
process.
142
In addition, porcine SIS had
both positive and negative inflammatory responses when
implanted into rodents.
150–152
Biotechnol. Prog., 20186

Another important factor to be investigated is the response
of the host innate and adaptive immune responses towards
implanted xenograft materials. The polarization of T helper
cells 1 (Th1) and macrophages (M1) results in a proinflamma-
tory response and T helper cells 2 (Th2) and macrophages
(M2) are involved in an anti-inflammatory response, which
leads to constructive tissue remodelling.
153
Many studies have different conclusions on host-
decellularized tissues/organs interactions and responses.
Therefore, a detailed scientific understanding of the outcomes
following decellularized products implantation including the
responses of host tissues towards the implanted decellularized
materials is vital.
154
Applications, status, and perspective of decellularized
tissues and organs
Engineered substitutes could be a possible cure for patients
in the last stage of tissue or organ failure. The increasing
demand for tissues and organs for transplantation and the
encountered immune rejection of xenograft transplantations
have pushed researchers to find alternative tissue and organ
sources.
Decellularized tissues and organs have a great potential to
serve as carriers for transplanting autologous tissue made from
the host cells or site-specific functional cells. Regardless of
the availability of genetically engineered animals to harvest
tissues or organs for transplantation, the rejection of the trans-
planted graft by the host appears inevitable.
155
This situation
can potentially be solved with scaffolds and other bioproducts
made from decellularized tissues and organs since antigenic
elements of cell components are supposed to be completely
removed. Recent advances involving recellularization of
human-iPSCs in decellularized scaffolds could be a way to
avoid the intake of immunosuppressive drugs required after
organ transplantation.
33,156
Achieving a functional tissue or organ is the goal of any
decellularized scaffolds carrying either functional cells or stem
cells. The absence of re-endothelialization of the organ with a
functional endothelium is one of the main reasons for organ
loss in transplantation and a major hindrance in developing
functional decellularized scaffolds. However, many studies
have shown promising results on re-endothelialization and
could be considered as a potential solution to solve re-
endothelialization problems.
157,158
Materials and scaffolds made from decellularized tissues
and organs have been commercially available for many appli-
cations. ECM-derived products from animal sources have been
commercially available for more than 20 years and are applied
in the treatment of many tissue regeneration processes and sta-
tistically, many years ago, over 200,000 patients have been
implanted with xenogeneic decellularized scaffolds.
159
Many
animal and human tissue-derived decellularized commercial
products are applied in wound healing and these include:
Oasis
®
(porcine small submucosa, Cook Biotech, Inc.,
Indiana, USA), GraftJacket
®
(human dermis, Acelity L.P. Inc.,
Texas, USA), DermACELL
®
(human dermis, Novadaq Tech-
nologies Inc., Mississauga, Canada), Alloderm
®
(human der-
mis, Allergan plc [NYSE: AGN, Dublin, Ireland]),
NeoForm™(human dermis, California, USA), Strattice™
(porcine dermis, Allergan plc [NYSE: AGN, Dublin, Ireland]),
Restore™(porcine small intestine [DePuy Orthopedics, Inc.,
Indiana, USA]), Prima™Plus (porcine heart valve, Edwards
Life Sciences LLC, California,USA), AlloSkin™AC (human
dermis, AlloSource, Centennial, CO, USA), MatriStem
®
(mucosa of urinary bladder, ACell, Columbia, USA),
Biodesign
®
(small intestine, COOK MEDICAL INC., Bloom-
ington, IN, USA), Lyoplant
®
(pericardium, Aesculap, Inc.,
Center Valley, PA, USA).
160,161
Most of the biological scaffolds are marketed as surgical
mesh devices.
161
Scaffolds made from decellularized tissues or
organs have a potential to be marketed under the category of
510(K) of the FDA.
162
Under a 510(K) clearance, a company
must register to notify (PreMarket Notification) the FDA
90 days in advance to market a medical device. FDA would
assess the notified device to make sure it falls under already
existing three classifications of devices in the market, for which
a premarketing approval is not required. The claim must be
made by the company to support their device is substantially
equivalent to the ones already available in the market. 510
(K) devices can fall under three classes: Classes I–III, depend-
ing on the assurance of safety and effectiveness (Table 2).
Oasis
®
, MiroMesh
®
, and PhotoFix
®
are few examples of
510(K)-cleared commercially available bioproducts derived
from decellularized tissues and organs.
160,164
Decellularized tissues which are intended to be implanted
or transplanted have a potential to be marketed under the cate-
gory of Human Cells, Tissues, and Cellular and Tissue-Based
Products (HCT/Ps) by FDA. HCT/Ps are the products regu-
lated by two different divisions namely the Center for Devices
and Radiological Health and the Center for Biologics Evalua-
tion and Research, or sometimes as a combination of both.
165
Table 2. Regulatory Requirements for 510(k) Devices
163
Regulatory Requirements Class I Class II Class III
General controls All general controls are
complied with and provide
reasonable assurance
General controls are
insufficient to provide
reasonable assurance
General controls are insufficient to
provide reasonable assurance
Secondary controls No further secondary controls
are required
Information required to
perform secondary controls
are sufficient and hence
secondary controls are
performed for gaining
reliable assurance of the
device
Information required to perform
secondary controls are
insufficient and hence
secondary controls cannot be
performed for gaining reliable
assurance of the device
Premarket notification/
approval
Eligible directly for premarket
notification
Eligible directly for premarket
notification
Requires premarket approval prior
to notification
Device/product risk Low Medium High
Note: General controls refer to a comprehensive set of regulatory authorities to be complied for by any medical device to be marketed. Special controls
are controls to be complied where general controls are insufficient.
Biotechnol. Prog., 2018 7

DermACELL
®
(a decellularized human dermis) and Allopatch
HD
®
(an acellular human dermis) are examples of HCT/Ps
products.
160,166
Miromatrix Medical Inc. (Eden Prairie, Min-
neapolis), a USA-based biotechnology company, is involved
in developing fully functional human organs by perfusion
technology, primarily applied to the liver and upon completion
extended to the kidneys. Miromesh™and Miroderm™
(derived from porcine liver) are products launched by the
company. AxoGen Inc. (Alachua, Florida) from USA,
develops a technology to regenerate peripheral nerve injuries.
AxoGuard
®
is derived from pigs and is used as a nerve con-
nector. Another USA-based company, Humacyte Inc.
(Morrisville, North Carolina), develops acellular grafts to
repair vascular damages; some of their products are under
phase III clinical trials.
Conclusions
The selection of a technique or chemicals to decellularize a
specific tissue or organ is an important decision to achieve
complete decellularization and to retain the ECM as intact as
possible. Perfusion decellularization has allowed fast and com-
plete decellularization of many whole tissues and organs using
either SDS or Triton X-100. Many whole organs have been
successfully decellularized and subsequently seeded with dif-
ferent cell types. Recent advances in recellularizing decellular-
ized tissues and organs involve the use of iPSCs to attain the
native function of the tissue. Decellularized tissues or organs
offer both mechanical support and cues from the ECM for cell
growth and function. Many bioproducts derived from decellu-
larized tissues and organs are commercially available, along
with that, regenerative medicine companies are working to
market functional whole organs to meet the demand. Extract-
ing purified proteins from ECM or synthesizing new motifs
are costly affairs compared with decellularizing a tissue, there-
fore, decellularized tissues and organs could be a potential
source of substitutes for transplantation, provided they are
devoid of cellular components, they maintain the necessary
functionality and they have acceptable immunogenicity.
Acknowledgments
This research project was supported by the Université de
Sherbrooke and NSERC through a Discovery Grant awarded
to Patrick Vermette (Grant no. 250296-2012).
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