Hearts beating through decellularized scaffolds: whole-organ engineering for cardiac regeneration and transplantation

Abstract

Abstract Whole-organ decellularization and tissue engineering approaches have made significant inroads during recent years. If proven to be successful and clinically viable, it is highly likely that this field would be poised to revolutionize organ transplantation surgery. In particular, whole-heart decellularization has captured the attention and imagination of the scientific community. This technique allows for the generation of a complex three-dimensional (3D) extracellular matrix scaffold, with the preservation of the intrinsic 3D basket-weave macroarchitecture of the heart itself. The decellularized scaffold can then be recellularized by seeding it with cells and incubating it in perfusion bioreactors in order to create functional organ constructs for transplantation. Indeed, research into this strategy of whole-heart tissue engineering has consequently emerged from the pages of science fiction into a proof-of-concept laboratory undertaking. This review presents current trends and advances, and critically appraises the concepts involved in various approaches to whole-heart decellularization and tissue engineering.

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Crit Rev Biotechnol, Early Online: 1–11
!2015 Informa Healthcare USA, Inc. DOI: 10.3109/07388551.2015.1007495
REVIEW ARTICLE
Hearts beating through decellularized scaffolds: whole-organ
engineering for cardiac regeneration and transplantation
Sonia Zia
1
*, Masoud Mozafari
2
*, G. Natasha
3,4
, Aaron Tan
3,4
, Zhanfeng Cui
5
, and Alexander M. Seifalian
3,6
1
Department of Cardiothoracic Transplantation & Vascular Surgery, Hannover Medical School, Hannover, Germany,
2
Bioengineering Research
Group, Nanotechnology & Advanced Materials Department, Materials & Energy Research Center (MERC), Tehran, Iran,
3
Research Department of
Nanotechnology, UCL Division of Surgery & Interventional Science, Centre for Nanotechnology & Regenerative Medicine, University College London
(UCL), London, UK,
4
UCL Medical School, University College London (UCL), London, UK,
5
Department of Engineering Science, Oxford Centre for
Tissue Engineering & Bioprocessing, Institute of Biomedical Engineering, University of Oxford, Oxford, UK, and
6
Royal Free London NHS Foundation
Trust Hospital, London, UK
Abstract
Whole-organ decellularization and tissue engineering approaches have made significant
inroads during recent years. If proven to be successful and clinically viable, it is highly likely that
this field would be poised to revolutionize organ transplantation surgery. In particular, whole-
heart decellularization has captured the attention and imagination of the scientific community.
This technique allows for the generation of a complex three-dimensional (3D) extracellular
matrix scaffold, with the preservation of the intrinsic 3D basket-weave macroarchitecture of
the heart itself. The decellularized scaffold can then be recellularized by seeding it with cells
and incubating it in perfusion bioreactors in order to create functional organ constructs for
transplantation. Indeed, research into this strategy of whole-heart tissue engineering has
consequently emerged from the pages of science fiction into a proof-of-concept laboratory
undertaking. This review presents current trends and advances, and critically appraises the
concepts involved in various approaches to whole-heart decellularization and tissue
engineering.
Keywords
Cardiac decellularization, cardiac
regeneration, cardiac tissue engineering,
whole-organ decellularization, whole-heart
decellularization, whole-organ tissue
engineering
History
Received 16 September 2014
Revised 25 November 2014
Accepted 18 December 2014
Published online 5 March 2015
The heart of the problem
Heart failure is one of the leading causes of hospitalization
and death, affecting up to 15 million Europeans (Heidenreich
et al., 2011). An advanced phase of heart failure, also known
as end-stage heart failure, is increasing and this trend presents
itself as a challenge to both clinicians and healthcare systems.
Heart failure is defined as having cardiac output that is
inadequate for biological requirements, and prognosis is
particularly poor, with around 25–50% of patients dying
within 5 years of diagnosis. The management of heart failure
is multifaceted, often requiring a multimodal approach
including lifestyle changes, pharmacological agents, implan-
table devices and even heart transplantation surgery.
Fortunately, it is expected that remarkable improvements in
the treatment of heart failure will be realized in the near future
(Friedrich & Bo
¨hm, 2007). Although the gold standard
treatment for end-stage heart failure is still heart transplant-
ation surgery, the main problems associated with it are the
shortage of donor organs, and even if a matching donor is
found, there are risks associated with this major surgery and
postsurgical complications could arise. As with any other
organ transplantation surgery, long-term immunosuppressants
must be administered to patients. Immunosuppressants can
cause a variety of side effects including immunodeficiency,
malignancy, infection, hypertension, diabetes and renal
insufficiency (Halloran & Gourishankar, 2001). Infection is
a common complication after heart transplantation surgery,
and is a common cause of death in the first year postsurgery.
The risk of malignancy is also well-documented, and is
caused by impaired immunoregulation acting in tandem with
carcinogens such as ultraviolet light exposure, and oncogenic
viruses such as papilloma virus and Epstein-Barr virus (Penn,
2000). Indeed, malignancies account for around 24% of death
after 5 years posttransplantation (Taylor et al., 2006).
Given the problems seen in allogenic human heart
transplantation surgery, an attractive solution might be to
‘‘grow a heart in the lab’’. Although still in its infancy,
there have been small but nonetheless significant steps made
in the field of cardiac tissue engineering, geared toward the
concept of ‘‘growing a heart in a lab’’. Decellularization is a
*These authors contributed equally to this work.
Address for correspondence: Prof. Alexander M. Seifalian, Centre for
Nanotechnology & Regenerative Medicine, University College London
(UCL), Royal Free London NHS Foundation Trust Hospital, London
NW3 2QG, UK. Tel: +44 207 830 2901. E-mail: a.seifalian@gmail.com
Dr. Masoud Mozafari, Bioengineering Research Group, Nanotechnology
& Advanced Materials Department, Materials & Energy Research
Center, P.O. Box 14155-4777, Tehran, Iran. E-mail: mozafari.masoud@
gmail.com
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promising and new technique that has been successfully used
in a variety of tissue engineering applications, including
cardiac tissue engineering. It is a process by which immuno-
genic cells are discharged from an organ via physical,
chemical or enzymatic means, while leaving the architectural
framework intact. This framework can subsequently be used
to as a scaffold to seed host cells on, with the extracellular
matrix (ECM) providing instructional cues to guide cell
infiltration, proliferation and migration. Decellularized scaf-
folds can be either allogenic or xenogenic. However, the
availability of decellularized human allografts is restricted
due to the lack of donors. Although attempts have been made
to manufacture and repair various components of the heart
such as using nanocomposite polymers for bioartificial heart
valves (Kidane et al., 2009), injectable hydrogels that deliver
therapeutic payloads to the heart (Chiu & Radisic, 2011),
scaffold-free constructs to deliver cardiac cells (Miyahara
et al., 2006), conventional cell-scaffold constructs to mimic
the anisotropy of the native heart (Engelmayr et al., 2008),
these techniques all require the existence of a functioning
myocardium and does not take into account the need of
‘‘whole heart engineering’’. Thus, decellularized xenogeneic
tissues have emerged as an attractive scaffold material for
whole-heart cardiac tissue engineering (Zhou et al., 2013).
The main advantage of using decellularized scaffolds is that
the engineered tissue is capable of remodeling. Furthermore,
as the architectural framework is preserved, the shape of the
tissue is a close replica of the native tissue (Lynch & Ahearne,
2013). Owing to these unique advantages, decellularization
has been used as a technique to engineer numerous tissues and
organs, including the liver (Uygun et al., 2010), lung
(Petersen et al., 2010), kidney (Song et al., 2013), cornea
(Hashimoto et al., 2010), bladder (Yang et al., 2010),
vasculature (Quint et al., 2011), articular cartilage
(Elder et al., 2009), intestine (Totonelli et al., 2012) and
heart (Ott et al., 2008). There has been a surge of research in
into this field, exemplified by the publication growth rate on
the Scopus database between 1994 and 2014 (Figure 1).
This trend highlights the interest within the scientific
community. Notably, a major portion of the available
literature is devoted to the decellularization of cardiovascular
tissues including the aortic and pulmonary valves, vasculature
and even whole heart decellularization. More importantly,
elucidation of the mechanisms behind the biological and
structural cues that the intact ECMs provide could form the
basis on which this exciting field can further progress – from
a scientific proof-of-concept to a clinical reality.
Principles of cardiac decellularization
Overview of heart decellularization methodology
As artificial matrices have not been fully able to reproduce
the complex structure of a heart, researchers have relied
on using natural scaffolds in the past. However, the advent
of decellularization has set a new path for cardiac tissue
engineering strategies. It is important to note that the process
of decellularization itself could potentially result in the
disruption to the ECM ultrastructure. Hence, the objective of
effective decellularization is to minimize undesirable effects
that are associated with cell removal techniques on the three-
dimensional (3D) architecture, while ensuring that all
immunogenic cells are removed (Crapo et al., 2011).
There is indeed a fine balance that needs to be struck, and
the current challenge is to optimize the amount of
Figure 1. Graphical representation decellularization research from 1994 to 2014. The bar chart shows the number of publications containing the word
‘‘decellularization’’ in the title–abstract–keyword published in the period of 19942014 (using Scopus database; date of search: 4 April 2014). The
prediction of the number of publications for the remaining time of 2014 full year is also shown by the white bar (long-term prediction using ANN
(artificial neural network)). The pie chart shows the distribution of different tissues and organs using decellularization technique published in the period
of 1994–2014 (using Scopus database; date of search: 4 April 2014).
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decellularization to the tissue type, sex and age of the original
tissue. Preserving the original architecture, mechanical
properties and vascular network of the scaffold are crucial
parameters that determine the eventual success of subsequent
recellularization and remodeling of the engineered heart.
Ultimately, research suggests that the most effective protocol
involves a combination of physical, chemical and biological
techniques (Gilbert et al., 2006).
Perfusion-based decellularization is one of the latest
approaches and has the ability to decellularize whole organs
(Remlinger et al., 2011). This process typically involves
passing a mild detergent through the intrinsic heart vascula-
ture, thereby removing cellular components (Gilpin et al.,
2014). Attempts at whole-heart decellularization protocol
integrating software-controlled automated coronary perfusion
with standard decellularization detergents have also been
successful (Aubin et al., 2013). The advent of numerous
promising techniques has shown that a lab-engineered
bioartificial heart could indeed be a clinical possibility
in the near future (Aubin et al., 2013). These various
methods or combinations thereof are summarized in
Table 1. Nevertheless, there are still various unresolved
issues (such as decellularization protocol optimization,
cell seeding techniques and bioreactor recellularization par-
ameters) and considerable challenges that need to be
addressed before whole-heart decellularization can be effect-
ively translated to become a clinical reality.
Physical methods
There is a plethora of physical decellularization methods,
including scraping, sonication (Azhim et al., 2011), agitation
(e.g. stirring and rocking) (Schenke-Layland et al., 2003b),
electroporation, pressure gradient (Sasaki et al., 2009), snap
freezing and supercritical fluid (Crapo et al., 2011; Eichhorn
et al., 2013; Sawada et al., 2008). Scraping is a simple
technique that allows for the removal of cells on the surface.
Other methods like sonication and supercritical fluids enable
cells to be removed more effectively and hence are more
commonly used. Azhim et al. (2014) demonstrated that
successful use of sonication to decellularize native aortic
tissues and reported minimal immune response. In another
example, Weymann et al. (2010) demonstrated effective use
of a pressure gradient system for decellularization of a whole-
heart model.
Snap freezing was adopted from the preservation tech-
nique, in which biological tissues are frozen rapidly at low
temperatures. Interestingly, during cryopreservation, the ice
crystals formed disrupts the cell membrane and in turn caused
cell lysis. However, it is important that the rate of temperature
decrease is carefully controlled in order to prevent disruption
to the ECM (Lehr et al., 2011). Sheridan et al. (2013) have
recently investigated the effects of various protocols on
decellularized porcine carotid arteries and determined the
optimum parameters for snap freezing. Results suggested that
slow cooling to the final temperature of 10 C produced a
stiffer and less distensible response than the nonfreeze-dried
scaffolds and also disrupted the collagen ultrastructure.
In addition, slow cooling to the final temperature of 40 C
demonstrated disruption to the elastin network. The results of
this study clearly showed the importance of optimizing the
nucleation and ice crystal growth, preventing ECM disruption
and subsequent inferior mechanical properties. Expert opin-
ion suggests that snap freezing in liquid nitrogen and
freeze drying to 40 C with a precooled shelf at 60 C
could produce scaffolds with adequate properties (Sheridan
et al., 2013).
Mechanical abrasion in combination with enzymes
can also effectively remove cells on the surface of tissue or
organ (Golberg & Yarmush, 2013; Hopkinson et al., 2008).
Mechanical agitation can be applied by using a magnetic stir
plate, an orbital shaker, or a low profile roller. In addition,
agitation can be applied for rinsing residual detergents, by
means of phosphate-buffered saline phosphate-buffered saline
(PBS) washing cycles. A novel tissue ablation method for
tissue decellularization, nonthermal irreversible electropor-
ation (NTIRE), has gained much attention (Golberg &
Yarmush, 2013). This technology usually provides ablation
within seconds without causing damage to ECM structures.
NTIRE has been used in in vivo rat models to decellularize
carotid arteries (Phillips et al., 2010). In this study, Phillips
et al. (2010) ensured that the electrical parameters did not
cause any thermal damage to the tissue scaffold by using a 2D
finite element solution of Laplace and heat conduction
equations. It was subsequently shown that after performing
NTIRE on a rat carotid artery, decellularization was mediated
by the immune system, leaving behind a functional scaffold.
After 7 days, recellularization occurred on the artery,
indicating that it was a viable platform on which cells could
adhere and proliferate on.
However, it must be noted that physical methods of
decellularization could potentially damage the ECM. For
instance, the process of snap freezing itself can also disrupt or
fracture the ECM during rapid freezing, and mechanical force
and agitation can also cause structural damage to the ECM, if
the decellularization process is not properly controlled. Thus,
care must be taken in using such methods by optimizing the
protocol specifically for the intended application.
Chemical methods
Acids and bases. Acids and bases are often used in combin-
ation with other methods such as detergents and alcohols.
Peracetic acid (also known as peroxyacetic acid, or PAA) is a
common disinfection agent that can remove residual nucleic
acids with minimal effects on the ECM composition and
structure (Gilbert et al., 2008; Hodde & Hiles, 2002). It has
also been proven that growth factors, cell membranes and
intracellular organelles can be completely eliminated from the
matrix by bases such as ammonium hydroxide (Reing et al.,
2010). One drawback of this technique is that acids and bases
could cause hydrolytic degradation of biomolecules (Li &
Vert, 1996). As they may adversely affect the mechanical
properties of the ECM, acids and bases are preferred where
preservation of mechanical strength is essential.
Hypotonic and hypertonic solutions. Dramatic alterations to
osmotic balance greatly affect the cells of tissues and organs
(Zhang & Sarras, 1994). This phenomenon has been exploited
as a decellularization technique by alternating the immersion
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Table 1. Summary of various decellularization techniques, their mode of action and cautions.
Decellularization technique Description Cautions References
Physical methods
Scraping Removes tissue and residue cells ECM can be affected by force Golberg and Yarmush (2013) and Hopkinson et al.
(2008)
Electroporation Induces significant ablation of biological tissues
with minimal heat generation
Pulsed electrical field can damage ECM Phillips et al. (2010)
Temperature (freezing and thawing) Used to remove cell contents from the ECM by
disrupting cell membranes
Ice crystal formation can disrupt or fracture ECM Sheridan et al. (2013)
Perfusion Preserves the underlying ECM, and provide an
acellular, perfusable vascular architecture, com-
petent acellular valves and intact chamber
geometry
Perfusion with pressure can disrupt ECM Remlinger et al. (2011) and Gilpin et al. (2014)
Supercritical fluid Removes cell residues when passed through tissues
at a controlled rate similar to critical point drying
Pressure necessary for supercritical phase can dis-
rupt ECM
Sawada et al. (2008)
Pressure gradient across tissue Used to supplement enzyme treatment, resulting in
superior preservation of ultrastructure
Pressure gradient can damage ECM Sasaki et al. (2009)
Agitation Used to lyse cells but more commonly applied to
removal of cellular material
Strong agitation can damage ECM Schenke-Layland et al. (2003b)
Sonication Used to lyse cells and resulted in significantly
cleaner matrices compared with the agitation
Aggressive sonication can disrupt ECM Azhim et al. (2014)
Chemical methods
Acids and bases Can cause hydrolytic degradation of biomolecules
such as nucleic acids and proteins
May damage collagen, GAG and growth factors and
harmfully affect the mechanical properties of the
ECM
Gilbert et al. (2008), Hodde et al. (2002), Reing
et al. (2010)
Hypotonic and hypertonic solutions Can readily cause cell lysis trough a simple osmotic
effect
Cannot completely remove the cellular debris in
some cases
Goissis et al. (2000), Vyavahare et al. (1997), Cox
et al. (2006)
Detergents Nonionic detergents disrupt DNA–protein inter-
actions, disrupt lipid–lipid and lipid–protein
interactions and to a lesser degree protein–protein
interactions. Ionic detergents solubilize cell and
nucleic membranes. Tend to denature proteins.
Zwitterionic detergents exhibit properties of
nonionic and ionic detergents
Efficacy dependent on tissue thickness Meyer et al. (2006), Grauss et al. (2005), Kasimir
et al. (2003), Pang et al. (2010), Cebotari et al.
(2010), Prasertsung et al. (2008)
Solvents
Alcohols Removes lipids from tissues, thus disrupting the
cellular membranes
Effectively removes cells from dense tissues and
inactivates pyrogens, but crosslinks and precipi-
tates proteins, including collagen
Dunmore-Buyze et al. (1995), Levy et al. (2003),
Flynn (2010) and Gorschewsky et al. (2005)
Acetone Can remove lipids from tissues and thus disrupt the
cellular membranes
Effectively removes cells from dense tissues and
inactivates pyrogens, but crosslinks and precipi-
tates proteins, including collagen
Montoya and McFetridge (2009)
TBP Can effect on retention of ECM constituent and
native mechanical properties and forms stable
complexes with metals, disrupts protein–protein
interactions
Mixed results with efficacy dependent on tissue,
dense tissues lost collagen
Deeken et al. (2011)
Biological methods
Enzymes Can provide high specificity for removal of cell
residues or undesirable ECM constituents
Difficult to remove from the tissue, could evoke an
immune response and enzyme residues may
impair recellularization
Wang et al. (2012)
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of the tissue in hypertonic and hypotonic solutions for several
cycles (Goissis et al., 2000; Vyavahare et al., 1997). While
hypertonic saline solution dissociates DNA from proteins
(Cox & Emili, 2006), in a hypotonic solution, the chemical
imbalance causes water to enter the cell by osmosis, causing
eventual cell lysis. It has been reported that treatment with
deionized water and sodium chloride solutions causes cell
lysis with minimal changes in matrix molecules and archi-
tecture (Gilbert et al., 2006). However, in some cases, this
technique does not completely remove the cellular debris.
Additional techniques are typically required to facilitate
the removal of the resultant cellular remnants from the
tissues. Remlinger et al. (2011) utilized a cocktail of
enzymes, detergents and acids combined with hypertonic
and hypotonic solutions to support the lysis and elimination
of cells. This protocol enabled successful detachment of all
nuclear materials from the native porcine cardiac tissue,
creating a site-specific cardiac ECM scaffold that can be
used in many applications.
Detergents. Ionic and nonionic detergents have been fre-
quently used in decellularization protocols to solubilize cell
membranes and dissociate DNA from proteins and many
reports have demonstrated effectiveness of this technique
(Crapo et al., 2011). Meyer et al. (2006) have demonstrated
that Triton X-100 (nonionic surfactant) can effectively
decellularize thicker tissues, such as valve conduits, where
enzymatic and osmotic methods are rendered insufficient.
There are mixed results concerning the use of Triton X-100,
and its efficacy is highly dependent on the type of tissue being
used. For instance, complete removal of nuclear material on
heart valves was observed, with the maintenance of the valve
architecture after 24 h. However, cellular material was found
in the adjacent aortic wall and myocardium. It was also shown
that Triton X-100 disrupted ECM components, and led to a
complete loss of glycosaminoglycans (GAGs) and decreases
the fibronectin and laminin content of heart valves (Grauss
et al., 2005). Sodium dodecyl sulfate (SDS) is an anionic
surfactant, which demonstrates a high efficacy for removing
cell residues from tissues (Kasimir et al., 2003; Pang et al.,
2010). However, there are some concerns about the disruptive
effects of SDS on the ECM structure (Kasimir et al., 2003).
Caution should be advised when using SDS due to cytotoxic
concerns. In this regard, even thin tissues such as valve
leaflets require multiple (more than six rounds) agitated
washes to completely remove residual amounts of detergent
(Cebotari et al., 2010; Prasertsung et al., 2008).
Solvents. This simple, yet elegant, technique involves
treating tissues with alcohol, removing lipids and thus
disrupting the cellular membrane. For instance, glycerol
aids in tissue decellularization by dehydrating and lysing cells
(Prasertsung et al., 2008). Phospholipids found in valve
leaflets and conduits have also been effectively extracted by
alcohols (Dunmore-Buyze et al., 1995; Levy et al., 2003).
Alcohols such as isopropanol, ethanol and methanol have
been demonstrated to efficiently remove lipids from tissues in
a relatively short period of time (Flynn, 2010). Combinational
strategies such as methanol with chloroform have also been
successfully used to delipidate and hence decellularize tissues
(Prasertsung et al., 2008). Despite its efficacy, caution needs
to be taken while treating tissues with alcohols, as they can
damage ECM ultrastructure and/or precipitate proteins
(Gorschewsky et al., 2005). Alternative solvents include
tributyl phosphate (TBP) (Deeken et al., 2011) and acetone
(Montoya & McFetridge, 2009). TBP could potentially have a
protective effect on preservation of ECM fundamental
and native mechanical properties. Acetone can also remove
lipids from tissues and thus disrupts the cellular membranes,
and can also be used as a tissue fixative.
Biological methods
Enzymatic treatment (e.g. trypsin, nucleases, collagenase,
lipase, dispase) is perceived as the most well-known bio-
logical method for decellularization. Although enzymes
possess the advantage of specific cell removal, residual
enzyme activity can impair recellularization and/or elicit
unwanted immune reactions (Crapo et al., 2011). Trypsin has
been extensively used as an enzymatic agent for decellular-
ization of tissues including arteries and human amniotic
membrane (Patnaik et al., 2013). However, it may damage
the ECM at high concentrations. Nonetheless, lower concen-
trations of trypsin can be used adjunctively with other
detergents such as SDS can be highly effective to remove
cellular contents (Wang et al., 2012).
Characterization of decellularized cardiovascular
tissues
The effectiveness of a given decellularization protocol is
highly dependent upon the tissue of interest. In determining
the success of decellularization, one should take into account
parameters such as the degree of preservation of vasculature,
whether the chemical compositions is ideal, and the extent of
preservation of the inherent mechanical properties and the
shape of the 3D framework.
The validity of decellularized cardiovascular tissues can be
evaluated by techniques such as genomic DNA extraction,
DNA content analysis, polymer chain reaction (PCR), stained
histological sections and surface structure analysis by
scanning electron microscopy (SEM). It is important to note
that each decellularization protocol has differing effects on
different tissues, depending on the tissue type, gender, age and
various environmental effects. For instance, Grauss et al.
(2003) demonstrated that a trypsin-based decellularization
protocol was ineffective at removing the cellular materials
from a rat aortic heart valve, while Schenke-Layland et al.
(2003a) reported complete decellularization of a porcine
pulmonary valve using a similar protocol. In another study,
Zou and Zhang (2012) reported that all three protocols for
decellularization – using enzymatic detergent, anionic deter-
gent and nonionic detergent, were effectively implemented to
decellularize the thoracic aorta in a porcine model. However,
the structure of ECM in the model treated with Trypsin was
severely disrupted, while treatments with SDS and Triton
X-100 had similar elastic properties with intact aortic tissues
(Zou & Zhang, 2012).
These studies again attest the effectiveness of decellular-
ization. Admittedly, the numerous decellularization strategies,
protocols and combinational approaches have made it
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difficult to settle on the optimal decellularization formula.
Nonetheless, a decellularization protocol for a specific
cardiovascular should be one that places emphasis on
maintaining mechanical properties of the native tissue,
given the high-pressure environment these tissues will be
subjected to in vivo. Ultimately, even within cardiovascular
tissue engineering, decellularization protocols should be
tailored to specific tissues of interest.
Techniques for verification of cell removal
There are a number of methods available to assess the
efficiency of removal of cell material. Histological and
biochemical assays can effectively examine the rate of cell
removal after decellularization. Before implantation in vivo of
any decellularized tissue, a series of verification techniques
are required to certify successful removal of cells (Schenke-
Layland et al., 2003a).
Confirmation at a genomic level: DNA extraction and
content analysis
DNA purification, also known as DNA isolation, is an
effective technique to identify the efficiency of decellulariza-
tion. Carvalho et al. (2012) demonstrated that decellulariza-
tion of rat heart model was assessed using genomic DNA
extraction, which subsequent DNA content analysis using
DNAzol reagent (Figure 2). Briefly, after lysing the sample,
DNA is then precipitated, washed in ethanol and finally
diluted in sodium hydroxide (NaOH). The isolated genomic
DNA was quantified using a spectrophotometer in order to
assess genomic DNA content of the decellularized matrices
(Carvalho et al., 2012).
Confirmation at a Genomic Level: PCR
PCR is a popular biochemical technology that allows the
amplification of DNA, to generate millions copies of a
specific DNA sequence. The technique is extremely useful as
it enables amplification of small amounts of DNA, and hence
enabling the detection of trace amounts of DNA present on
decellularized tissue. For instance, Lehr et al. (2011) have
demonstrated using real time polymerase chain reaction
(RT-PCR) that decellularized allograft vascular tissues does
indeed reduce the host allogeneic immune responses. By
means of RT-PCR analysis, they proved that decellularization
reduced the number of grafts expressing various cytokines
such as interferon gamma and interleukin-10, but there were
no changes in the number of grafts expressing transforming
growth factor beta 1. It was suggested that the rejection of the
allograft vascular tissues was mediated by both cellular and
humoral immune responses and that decellularization attenu-
ates both of these response mechanisms (Lehr et al., 2011).
Providing visual confirmation: histological analysis
Histology is the study of the cellular organization of tissues
and organs, usually with the aid of a histological stain in order
to enhance visualization. Hematoxylin and eosin (H&E) is a
common stain used as the primary examination techniques.
Other histological stains such as Masson’s Trichome, Movat’s
Pentachrome, or Safrin O can be also used for the detection of
nucleic acid structures and complex organic compounds in the
decellularized tissues (Sasaki et al., 2009). In one commonly
performed technique for histological analysis, decellularized
heart tissues are fixed using paraformaldehyde and embedded
in paraffin. Samples are then cut into micrometer-sized
Figure 2. (A) Schematic illustrating decellularization protocol, showing the initial dense and red rat heart tissue transitioned to a white-translucent
colored heart while maintaining its overall three-dimensional structure. (B) and (C) DNA content analysis of the decellularized matrix, indicating that
in contrast to original heart tissue, the decellularized matrix presented virtually no DNA according to DNA quantification in spectrophotometer, and to
PCR amplification, using primers for genomic DNA. Adapted with permission from Carvalho et al. (2012).
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sections and stained using H&E and Masson’s Trichrome
stains. This technique is capable of confirming an acellular
structure postdecellularization, with a well-preserved vascular
structure (Carvalho et al., 2012).
In another investigation, Lu et al. (2013) showed that
histological analysis using H&E staining could accurately
detect the absence of nuclei in decellularized hearts.
Similarly, Ott et al. (2008) carried out antegrade coronary
perfusion of cadaveric rat hearts on a modified
Langendorff apparatus and subsequently used histological
evaluation of nuclei or contractile elements to compare
the decellularization efficacy amongst three chemicals
were present. Histological stains clearly revealed that the
use of SDS yielded a fully decellularized construct and
gave better results than polyethylene glycol (PEG), Triton-
X100 or enzyme-based protocols (Ott et al., 2008)
(Figure 3).
Providing visual confirmation: SEM
As a simple method for high-resolution imaging of surfaces,
SEM is frequently used for observing the microstructure of
decellularized tissues. Prior to using SEM, a fixing procedure
needs to be carried out on the targeted tissue. In order to
examine the structure of decellularized tissues, glutaraldehyde
is always applied to fix the tissues in sodium cacodylate
buffer. Thereafter, osmium tetroxide is applied to the fixed
tissue and subsequently, different increasing concentrations of
ethanol ranging from 30% to 100% to cause dehydration and
drying. The dried samples are then sputter-coated with an
ultrathin coating of electro-conductive materials such as
gold or gold/palladium alloy before microscopic examination.
An alternative to coating is increasing the conductivity
of the bulk material by impregnation with osmium (Malick
et al., 1975).
Figure 3. Perfusion decellularization of whole rat hearts. (A)–(C): Photographs of cadaveric rat hearts mounted on a Langendorff apparatus.
Retrograde perfusion of cadaveric rat heart over 12h using, (A) PEG, (B) Triton-X-100 and (C) SDS. The heart becomes more translucent as cellular
material is washed out from the right ventricle, then the atria and finally the left ventricle. (D)–(F) Corresponding H&E staining of thin sections from
LV, showing complete decellularization in (F) and incomplete decellularization in (D) and (E). (D) and (E) Hearts treated with PEG or Triton-X-100
retained nuclei and myofibers. Scale bars, 200 mm. (F) H&E staining of SDS-treated hear t showing no intact cells or nuclei. Scale bar, 200 mm. All
three protocols maintain large vasculature conduits (black asterisks). Note: Ao, aorta; LA, left atrium; LV, left ventricle; RA, right atrium; RV, right
ventricle. Source: Adapted with permission from Ott et al. (2008).
DOI: 10.3109/07388551.2015.1007495 Hearts beating through decellularized scaffolds 7
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Providing visual confirmation: immunostaining
Immunostaining refers to the use of antibody-based staining
techniques for the detection of specific proteins in tissue
sections. In a typical procedure, targeted tissues are fixed
using paraformaldehyde and passed through serial baths of
saccharose. Following treatment in saccharose baths, the
samples are embedded in a compound, which functions as a
matrix for cryostat sectioning at freezing temperatures. Then,
the sections are dehydrated and rehydrated by passing through
decreasing and increasing concentrations of ethanol. The
sections are then incubated with antibodies based on the
targeted proteins for the detection, and observed under
fluorescence microscopes (Carvalho et al., 2012).
In a study by Lu et al. (2013), mouse cadaveric hearts
were decellularize using Trypsin and detergents, including
SDS and Triton X-100, to lyse the cellular content and obtain
decellularized heart with the yield of a transparent ECM
scaffold. Immunostaining was used to visually compare the
decellularization efficacy of the various agents used. Results
showed that the typical ECM components such as fibronectin,
laminin and collagen type II remained postdecellularization
and 40,6-diamidino-2-phenylindole (DAPI) positive nuclei
were detected, indicating the complete removal of intact heart
cells (Lu et al., 2013) (Figure 4).
Recellularization of acellular scaffolds
There have been several attempts toward using decellular-
ized and recellularized cardiovascular tissues to successfully
create functional organs (Ga
´lvez-Monto
´n et al., 2013;
Ketchedjian et al., 2005). Recellularization involves seeding
cells onto the decellularized scaffold and placing the
construct in a bioreactor, where the growth and proliferation
of the tissue is supported (Ga
´lvez-Monto
´n et al., 2013).
Prior to recellularization, the decellularized heart has
been sterilized to remove any pathogens. Once sterile, the
recellularization process commences by first repopulating
the scaffold with multipotent cardiovascular progenitor cells,
supplementing them with additional exogenous growth
factors which stimulates the differentiation of the precursor
cells into specific cell types (Laflamme & Murry, 2005).
It has been reported that the recellularized cardiovascular
tissues carry a significantly lower risk of rejection in
comparison to traditional transplants (Song & Ott, 2011;
Traphagen & Yelick, 2009).
Recellularization can be divided into two components:
seeding cells onto the scaffolds to achieve a similar spatial
configuration and cell distribution as in the native organ, and
a perfection culture to ensure cells are functional and ready
for implantation. In terms of cell seeding, endothelial cells are
known for its antithrombogenic effects and also ensure that
blood flow is restricted to the vascular network. Ensuring that
the cell numbers are appropriate for the organ type is an
important consideration. For a tissue-engineered heart, it must
be fully functional at time of implantation. Indeed, the heart
has high cellular density, with around 10
8
cardiomyocytes/
cm
3
, and its 3D scaffold has to be fully recellularized at the
time of implantation in order to be fully functional (Vunjak-
Novakovic et al., 2009). With regards to the cell type, there is
still no general consensus on whether it would be more
advantageous to use a large number of fully mature cells or a
small number of stem/progenitor cells. However, experts
believe that a combination of the two might be the answer.
Nevertheless, seeding such large numbers of cells would
require longer periods (i.e. weeks) of organ culture in a
bioreactor before it is suitable for implantation. Seeding
strategies can include intramural injection or infusion of cells,
followed by continuous perfusion. It has been reported (Ott
et al., 2008) that recellularization occurred with 50–75 10
6
neonatal cardiac cells that were delivered in five 200 ml
injections into the anterior left ventricle in a murine model,
with a seeding efficiency of 50%. Bioreactors are often
needed for whole-organ engineering, and it has been reported
that it takes 4 weeks for a heart to be recellularized (Ott et al.,
2008). Oxygen delivery is also an important aspect in
perfusion bioreactors, and it is known that the metabolic
demand for oxygen is 27.6 nmol/[mg protein min] for cells of
the heart. Biophysical stimuli are also needed to render cells
functional, and are especially true for cardiomyocytes. It has
been shown that cardiac cells engineered under mechanical
stretch and biophysical stimuli displayed similar propagation
velocities to native cardiac cells, and also responded to
external stimuli by synchronized contractions (Bursac et al.,
2007; Radisic et al., 2008).
Immunogenicity of implanted scaffolds
One of the important criterions for clinical use of decellular-
ized natural scaffolds is that they should be relatively
nonimmunogenic. It is a concern that the residual proteins
on the decellularized scaffolds could potentially elicit an
immune response (Wagner et al., 2013). Nevertheless,
Hawkins et al. (2003) demonstrated that decellularized
grafts elicited lower levels of class I and class II human
leukocyte antigen formation compared to standard cryopre-
served allografts, suggesting that decellularized grafts are less
immunogenic. Further optimization studies should be under-
taken to assess the ideal decellularization levels depending on
various parameters such as tissue type and age. This is
important, as it is crucial to strike a balance between
removing immunogenic cellular components, while maintain-
ing the shape and mechanical properties of the scaffold.
Prior to recellularization, it is necessary to ensure that the
decellularized organ scaffolds are adequately sterilized to
prevent cross-contamination and the risk of infection.
Sterilization may involve simple treatments such as incuba-
tion in solvents and acids, but these methods may not be
sufficiently effective. Sterilization techniques used in con-
ventional medical implants can also be used, such as ethylene
oxide, gamma irradiation and electron beam irradiation.
However, these techniques may alter the mechanical proper-
ties of the construct, and may even cause undesirable host-
immune response and may even render residual lipids
cytotoxic (Crapo et al., 2011).
Conclusion and future perspectives
The challenges in developing complex 3D functional tissues
will be mimicking the normal dynamic integrated network of
natural tissues and organs. Although the translation to clinical
care is admittedly a few decades away, there have been great
8S. Zia et al. Crit Rev Biotechnol, Early Online: 1–11
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For personal use only.

advances in the field of cardiac regeneration. The use of
decellularization and recellularization processes has led to the
production of constructs with an architecture similar to that of
a native organ that promote the repopulation of cells, cellular
growth and possess remodeling capabilities. This area shows
immense promise, and achieving this objective will mark a
new era in regenerative medicine.
Declaration of interest
The authors report no declaration of interest.
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