Regenerative Engineering and Translational Medicine

https://doi.org/10.1007/s40883-020-00177-9

ORIGINAL RESEARCH

Decellularization and Recellularization of Rabbit Kidney Using

Adipose-Derived Mesenchymal Stem Cells for Renal Tissue
Engineering
Shabnam Sabetkish 1 & Nastaran Sabektish 1 & Masoumeh Ekhtiari 1 & Bahareh Mohammadi Jobani 1 &
Abdol-Mohammad Kajbafzadeh 1

Received: 24 March 2020 / Revised: 18 September 2020 / Accepted: 24 September 2020

# The Regenerative Engineering Society 2020

Abstract
Purpose The effectiveness of decellularization and recellularization of rabbit kidney scaffolds with autologous adipose-derived
mesenchymal stem cells (ADMSCs) was assessed. The static-based technique was judged against the perfusion-based method
both in decellularization and recellularization processes.
Methods Kidneys were obtained from 8 rabbits and divided into two groups. Two kidneys did not undergo any processing and
were served as the control group. Kidneys of group I (N = 7) were cannulated via the renal artery and decellularized with a
detergent-based method. We reseeded these constructs with ADMSCs using a peristaltic pump. The static method was applied to
decellularize and recellularize thin slices of the seven kidneys (group II, N = 7) using the L929 cell line. Several tests were
performed to evaluate the efficacy of the decellularization and recellularization processes.
Results All cellular and nuclear constituents were eliminated from the scaffolds of both groups confirmed with hematoxylin and
eosin (H&E), trichrome, 4′,6-diamidino-2-phenylindole (DAPI) staining, and DNA quantification. Cell viability was over 89%
as performed by MTT assay. Cells were effectively seeded in scaffolds of both groups, with a superior arrangement and a higher
concentration in the perfusion-based technique. Immunohistochemistry (IHC) staining demonstrated preservation of native
expression patterns of extracellular matrix proteins with better results in perfusion-based technique.
Conclusion The results suggest that organs with the distributed micro-vasculature system (perfusion method) may benefit from being
engineered with the dynamic procedure compared with organs with empty chambers and poor vasculature system (static system).
Lay Summary Previously, we evaluated the efficacy of the transplantation of decellularized kidneys in a rat model. In this study,
we try to assess the efficacy of two decellularization and recellularization protocols of kidney organs for further renal tissue
engineering. We also aim to produce renal scaffolds with structural, mechanical, and physiological characteristics that are crucial
for engineering main renal structures, using adipose-derived mesenchymal stem cells.
Future Studies Finding the most suitable and feasible decellularization and recellularization methods is important in several
medicinal therapeutics. In the future, we will emphasize on the long-term viability and functionality of obtained kidney scaffolds
in clinically translatable applications. We will also compare different cell lineages to obtain the most efficient scaffold for further
renal tissue engineering.

Keywords Kidney . Decellularization . Recellularization . Rabbit . Mesenchymal stem cells

Introduction with donor organ transplantation [1]. Moreover, the yearly

End-stage renal disease (ESRD) is a devastating problem that
may result in the progressive deterioration of renal function
* Abdol-Mohammad Kajbafzadeh
kajbafzd@sina.tums.ac.ir
1
Pediatric Urology and Regenerative Medicine Research Center,
Section of Tissue Engineering and Stem Cells Therapy, Tehran, Iran
Medicare expenses of these patients exceed $29 billion [2].
Due to decreased donor organ donation and the morbidity
related to immunosuppression, regenerative medicine tech-
niques have been considered as efficient therapeutic alterna-
tives [3].
State-of-the-art methods utilize tissue engineering tech-
niques to produce decellularized scaffolds that could prevent
allograft rejection and prevent the need for immunosuppres-
sive therapy [4]. Preservation of the ultrastructure, integrity,

and bioactivity of the extracellular matrix (ECM) can be opti-
mized by using an effective decellularization methodology.
Decellularization of the kidney with intact ECM is crucial
for in vivo compatibility and targeted clinical application. In
addition, each tissue or organ requires specific processing be-
cause of the variability in function and biomechanical proper-
ties. As the kidney organ has complexity with more than thirty
different cell types, using tissue engineering and regenerative
medicine techniques, the unique microenvironment and pre-
served ECM may pave a road for the bioengineering of a
whole kidney which may be transplantable [5].
Cell-based therapeutic techniques have been considered
as a crucial step for the treatment of patients with early-
stage renal disease [6, 7]. However, these approaches may
not be effective in cases of ESRD, which may present with
severe fibrosis. Therefore, total organ regeneration using
seeded cells would be an interesting concept. In addition,
although organoids have shown the capacity to recapitu-
late native organ functions in animal models, they are typ-
ically much smaller compared with native human organs,
and implantation of several small organoids may not be
clinically practical [8].
Herein, we aimed to compare the efficiency of static- and
perfusion-based decellularization and recellularization tech-
niques in cell removal and biocompatibility of the obtained
scaffolds. Following our previous proof-of-concept study [9],
this study sought to determine the most effective method of
decellularization and recellularization with emphasis on bio-
logic composition and support of MSCs growth.
Material and Methods
Harvesting Renal Tissue
Eight female New Zealand rabbits weighing 2–2.3 kg were
included in this investigation under guidelines approved by
the local ethical committee of the Tehran University of
Medical Sciences. The animal experiment complies with the
National Institutes of Health guide for the care and use of lab-
oratory animals (NIH Publications No. 8023, revised 1978). An
intravenous dose of heparin was administered just before being
euthanized to decrease postmortem clotting. A midline abdom-
inal incision was made under the sterile condition to expose the
renal tissues. Two kidneys did not undergo any processing and
served as the control group. In group I, seven kidneys were
cannulated via the renal artery. After dissection of all the
connections, the kidney was resected attaching the cannula to
a peristaltic pump to undergo the perfusion-based
decellularization process. In group II, seven kidneys were sliced
into thin layers (0.5 mm of thickness) for static decellularization
techniques.
Regen. Eng. Transl. Med.
Perfusion-Based Decellularization Procedure
Seven complete kidneys weighing 8.2 ± 0.5 g were immedi-
ately perfused with 300 mL of distilled water and 0.01% hep-
arin to eliminate any remained clots. To dissolve cell mem-
branes, the organ was perfused with 300 mL of 2% Triton X-
100 for 2 h. Afterward, they have been washed again with
perfusion of 300 mL of distilled water for the subsequent
1 h. Renal perfusion of 1% sodium dodecyl sulfate (SDS)
was performed for the next 3 h to remove cytoplasmic proteins
and nuclear residues. The scaffolds were cleansed with 0.2 L
of distilled water to take the residual SDS out. All the previ-
ously mentioned steps were performed with a perfusion speed
of 10 mL/min under continuous shaking. All the previous
steps were repeated for 3 continuous cycles to obtain cell-
free scaffolds (Fig. 1a and b).
Static-Based Decellularization Procedure
After slicing the kidneys (n = 7) (5 mm of thickness), they
were washed with distilled water for a period of 1 h. Then,
they were immersed in 0.5% SDS for 12 h. The solution was
changed with 1% Triton X-100 for the next 12 h. The tissues
were placed again in 0.5% SDS for the following 24 h. The
slices were washed distilled water for 1 week. All the solutions
were autoclaved and contained antibiotics and antimycotics to
obtain untainted scaffolds for the upcoming cell seeding pro-
cess. The decellularized renal tissues were accumulated in
phosphate-buffered saline (PBS) in 4 °C for further investiga-
tions (Fig. 1c and d).
Histological and Immunohistochemical Analyses
Kidney tissues obtained from both decellularization tech-
niques were fixed with 10% buffered formalin for 1 day at
room temperature for being prepared for histological evalua-
tion. After the fixation process, the scaffolds were embedded
in paraffin and serially sectioned (5 mm of thickness). The
slides were stained with hematoxylin and eosin (H&E),
trichrome, and 4′,6-diamidino-2-phenylindole (DAPI) to eval-
uate the efficacy of decellularization in cell removal and pres-
ervation of the extracellular matrix (ECM).
DNA Assay
Scaffolds obtained from perfusion- and static-based
decellularized techniques as well as normal matrices were
collected on dry ice and weighed. A Nanodrop 2000
(MaestroNano, USA) was used for the measurement of
DNA concentration (ng DNA/mg tissue) after DNA extrac-
tion according to the manufacturer’s protocol of the DNeasy
Blood & Tissue kit (QIAGEN, Netherlands).
Regen. Eng. Transl. Med.
Fig. 1 a–b Perfusion-based
decellularization and
recellularization procedure. c–d
Static-based decellularization and
recellularization procedure
Scanning Electron Microscopy
For evaluating the efficacy of the decellularization process in
cell removal and preservation of the ECM, several images with
different magnifications were taken from both natural and
decellularized scaffolds. Normal and decellularized rabbit kid-
ney samples (1 cm 3 for native- and perfusion-based
decellularized kidneys and 1 cm2 for static-based decellularized
kidney) were fixed in 2.5% glutaraldehyde and dehydrated in
ethanol series (30, 50, 70, and 90%). A critical point dryer was
applied for 15 min to process the samples. Electrical conduc-
tivity was obtained by coating all the samples with gold-
palladium (3.5 mm of thickness). Scanning electron microsco-
py (SEM) (S3500N; Hitachi High Technologies America) was
used to investigate the specimens (voltage, 5 kV).
Tensile Test
Natural and decellularized scaffolds obtained from perfusion-
based decellularization techniques were cut in 1 cm1 section
and evaluated with a tensile test device (Zwick/Roell, Model:
Hct 400/ 25, Germany) in room temperature to compare the
rigidity of kidney tissues. For this purpose, the specimens
were clenched in sample holders and subjected to mounting
uniaxial force with a constant elongation rate of 0.1 mm/s
(4 mm/min) at room temperature until the emergence of a tear
in the tissues. The maximal point of the final curve shows the
maximum pressure tolerated by the decellularized and natural
kidneys.
Isolation and Culture of ADMSCs from the Perivesical
Adipose Tissue
Biopsies were taken to obtain adipose tissue from the
perivesical adipose tissue of a rabbit in a sterile condition.
Adipose tissues were treated with 0.1 mg/ml type 1 collage-
nase (Sigma-Aldrich) for 1 h at 37 °C, following washing with
sterile PBS. In the next step, the cells were centrifuged and
Dulbecco’s Modified Eagle Medium (DMEM) (Sigma-
Aldrich), including L-glutamine (Sigma-Aldrich) and penicil-
lin/streptomycin, was used for cell culture. Subsequently, cells
underwent incubation in 10% AS, fetal bovine serum (FBS)
(Sigma-Aldrich), and 10 ng/mL bFGF (Sigma-Aldrich) at
37 °C. A humidified atmosphere with 5% CO2 and 95% air
was used for the process of cell culture. After 3 days, we
washed away the non-adherent cells and isolated the plastic-
adherent cells (adipose-derived mesenchymal stem cells
(ADMSCs)) for culture. A total of 0.025% Trypsin/EDTA
(Sigma-Aldrich) was applied for primary cultured cells
(2 min in the incubator) and the cell culture process continued
until the third passage.

In vitro Recellularization of Perfusion-Based
Decellularized Kidneys
Whole decellularized rabbit kidneys were connected to a
closed system bioreactor that was placed in a cell culture
incubator with 5% CO2 at 37 °C for ADMSCs)seeding. A
100-mL glass bottle that had two holds (the connectors
that tubing attaches to) on its cap was connected to a
pump. One of the holds was connected to the renal artery,
while the other cannula was designed for perfusion of the
medium. The kidney was preserved in a hanging situation
in the container and the medium was circulated with
57 min intervals. The resting time was considered to let
the trapped cells adhere to the scaffold. During the first
3 days, 70 mL of culture medium having a total of 1 × 107
million cells were circulated into the scaffold via the renal
artery cannula with a flow rate of 5 mL/min. In the next
12 uninterrupted days, the medium was changed every
3 days. To avoid cell adhesion to the bottle wall and
decrease of ADMSC concentration, the recellularization
process was performed on a shaker.
In vitro Recellularization of Static-Based
Decellularized Kidneys
For in vitro cell seeding of sliced decellularized kidney tissues,
1 × 104 ADMSCs extracted from the rabbit’s perivesical adi-
pose tissue were seeded on each specimen after completion of
the rinsing step and removal of detergents. The scaffolds and
cells were placed in an incubator at 37 °C and 5% Co2 for 3 h
until the adherence of ADMSCs to the scaffold surface.
Afterward, 10% FBS) and 3 mL of DMEM were added to
the sliced scaffold and kept in the incubator. After 48 h of
incubation, the specimens were transferred to new plates and
kept for 10% DMEM for another 1 week. The environment
was changed with 2 days intervals. After the last step of
recellularization, the samples were washed with PBS and im-
mersed in 10% formalin for further histopathological
evaluations.
MTT Assays
The oxidative activity and survival rate of cultured ADMSCs
was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) assay, before recellularization.
After adding 80 μL of MTT into each well, it was incubated
at 37 °C for 4 h in the next step; 800 μL of dimethylsulfoxide
was replaced with 10% DMEM. After shaking for 10 min,
150 μL of the solution was extracted to 96-well. A multi-
well spectrophotometer was applied to determine the absor-
bance value at 490 nm.
Regen. Eng. Transl. Med.
Immunohistochemical Examinations
Recellularized kidney tissues obtained from both techniques
were fixed in 4% paraformaldehyde and immersed in Triton
X-100 diluted 1:100 in PBS. Paraffin-embedded samples were
sectioned and incubated with certain antibodies including
vimentin, cytokeratin, and PAX8.
Results
Both scaffolds obtained from perfusion- and static-based
decellularization technique turned whitish and translucent in
appearance with maintained gross appearance and size. H&E,
trichrome, and DAPI staining of the decellularized rabbit kid-
neys revealed that both techniques were successful in cell
removal and preservation of ECM (Fig. 2). Investigation of
DNA content after decellularization treatment revealed a sta-
tistically significant reduction in DNA versus the natural renal
tissues (p < 0.001). Therefore, the DNA content was signifi-
cantly decreased in renal matrix decellularized with perfusion-
based technique (0.32 ± 0.12 ng DNA/mg tissue) compared
with renal matrix decellularized with perfusion-based tech-
niques (0.35 ± 0.15 ng DNA/mg tissue) and native tissue
(83.74 ± 23.12 ng/mg wet dermal tissue). Biomechanical
properties of the decellularized renal matrix (maximal force,
4.12 N) and normal kidney tissue (maximal force, 4 N)
showed no significant changes, suggesting no detectable col-
lagen loss during the decellularization process (Fig. 3). SEM
analysis of the decellularized scaffolds of both techniques
showed a well-preserved ECM comparable with the native
kidney. Nonetheless, some extent of deterioration was found
in the scaffolds obtained from the static-based
decellularization technique (Fig. 4).
The results of static cell seeding are shown in Fig. 5 that
were obtained from the light microscope, showing the cells
were seeded on decellularized scaffolds after 1 and 2 weeks of
the recellularization process.
The results of both H&E and immunohistochemistry (IHC)
staining revealed the superiority of the perfusion-based tech-
nique in the recellularization of renal tissue compared with the
static-based process. Accordingly, IHC staining with vimentin
as a cytoskeletal protein, tubular injury marker, and a good
marker of epithelial-mesenchymal transition showed that
specimen of perfusion-based methods were significantly
stained with this marker (78.25 ± 0.5 vs. 23.75 ± 0.75, p =
0.001). Similar results were obtained in the comparison of
the cytokeratin marker. The anti-cytokeratin antibody was sig-
nificantly stained in all tubular cross-sections of specimens in
the perfusion-based method. PAX8 is considered as a nephric
lineage transcription factor that is essential for the organiza-
tion of renal lineage cells and the development of the kidney.
Statistical analysis showed that PAX8 marker was
Regen. Eng. Transl. Med.

Fig. 2 H&E staining of

perfusion- (a) and static-based (b)
decellularized kidneys. DAPI
staining of perfusion- (c) and
static-based (d) decellularized
kidneys. Trichrome staining of
perfusion- (e) and static-based (f)
decellularized kidneys

significantly higher expressed in the perfusion-based
recellularization manner (72.5 ± 0.75) than the specimen ob-
tained from the static-based technique (31.5 ± 0.25) (p = 0.03).
CD34, S100, and SMA markers were also extensively higher
expressed in perfusion-based specimens compared with sam-
ples obtained from the static-based technique (p ≤ 0.05)
(Fig. 6).
Discussion
In this study, we demonstrated that the perfusion-based
recellularization technique would be able to produce kidneys
with structural, mechanical, and physiological characteristics
essential for engineering main renal structures as compared
with the static-based method.
Decellularization techniques are different from shared con-
cepts of the use of detergents to remove all cellular and nuclear
components as well as the preservation of the ECM [10, 11].
Recently, 0.1% and 0.5% SDS were applied to achieve
decellularized mouse and canine kidney scaffolds, respective-
ly [12, 13]. In the current study, we identified a favorable
protocol of decellularization that generates the finest scaffold
for kidney engineering to examine our second principle of
recellularization. Histologically, the currently applied protocol
resulted in no harm to native vascular construction.

Fig. 3 Force/displacement (kN/mm) curves comparing the maximum
force. Assessment of natural and decellularized kidney organs
Significantly, we have produced scaffolds with an undamaged
vascular system, which could be the preliminary starting ma-
terial for the perfusion and static-based delivery of target cells.
In one study in 2018, a comparative analysis of two differ-
ent kidney decellularization techniques was demonstrated in
the porcine model for the maintenance of functional vascular
architectures. The outcomes showed that the perfusion of a
combination of 1% Triton X-100 and 0.5% SDS was more
efficient in cell removal and ECM components and architec-
ture preservation compared with 0.5% SDS and DNase treat-
ment [14]. A standardized immersion protocol was recently
applied to decellularize kidney tissue in the porcine model
with Triton X-100, SDS, and sodium deoxycholate (SDC) at
changeable temperatures. A scoring system was developed
that allows intra- and inter-study comparison of
decellularization techniques. The results verified that
decellularization with 1% SDS at 4 °C presented the highest
structural and composition scores [15]. Successful detergent-
based decellularization technique has been used to the murine
kidney, supporting murine embryonic stem cells [16].
Fig. 4 Scanning electron microscopy of (a) perfusion-based, (b) static-based, and (c) normal kidney
Regen. Eng. Transl. Med.
Though, this method may not be appropriate for clinically
relevant sized kidney and will bring new challenges which
require the development of clinically effectual methods.
With this in mind, we tried to develop a decellularization
and recellularization system to guarantee the reproducibility
of rabbit kidneys to develop an engineered whole kidney for
further transplantation.
MSCs are easily accessible and abundant with multilineage
differentiation ability, which is the major advantage of these
cell types. The application of MSCs can cause little discomfort
without any ethical considerations compared with embryo stem
cells [17]. Although there are successful protocols for the pro-
duction of several scaffolds, the recellularization of organs is
challenging [18]. Cell-scaffold technology is an interesting con-
cept developed in the field of tissue engineering and regenera-
tive medicine. In recent years, several studies have
decellularized animals’ small solid tissues, with the aptitude to
re-seed these scaffolds to obtain architectural resemblance to
the native organ [16, 19, 20]. In this study, we aimed to apply
these methods to the rabbit’s kidney and developed a
recellularization method that can be scaled up for application
in other organs. This study represented a step toward the devel-
opment of kidney organs using tissue engineering techniques.
It has been mentioned that the optimal recellularization
technique would be perfusion of cells via the combined renal
artery and ureter [3]. Additionally, perfusion seeding will be
able to enhance cell engraftment when compared with the
manual injection of the cell suspension [21]. The results of
our study revealed that the cell seeding process was more
effective in scaffolds of the perfusion-based method compared
with the static-based technique. Furthermore, we used a bio-
reactor that allowed perfusion through sterile ports with vari-
ables such as used pressure and the circulated media and the
culture time. We used a method to deliver MSCs to the scaf-
folds through the renal artery at high pressure with improved
results compared with low pressure arterial infusion [22]. The
Regen. Eng. Transl. Med.

Fig. 5 Recellularized kidney

tissue obtained by static-based
technique after (a) 1 and (b)
2 weeks

limited size of thin slices of tissues and the lack of a large
circulatory system limits the clinical translation, which is in-
compatible with the results of the current study.
Decellularized whole kidney scaffolds with preserved micro-
structure and ECM can be effectively employed for kidney
bioengineering using different cell lines.
In a recent study, concomitant bilateral kidney transplanta-
tion was performed; the result of which demonstrated that this
has the potential to serve as a feasible and practical approach for
further clinical approaches [23]. In one study in 2013, rat kid-
ney scaffolds were seeded with epithelial and endothelial cells,
perfused in a bioreactor, and transplanted in the rat. The results

Fig. 6 Histopathological evaluations of perfusion- and static-based recellularized kidneys: H&E, trichrome, and IHC staining with vimentin, anti-
cytokeratin, PAX8, CD34, S100, and SMA markers

showed that the grafts produced urine through the ureteral con-
duit [20].
In the study of Osele et al., human induced pluripotent stem
cell–derived endothelial cells were infused through the renal
artery for engineering the vasculature of the decellularized rat
kidney. The results showed that endothelial cells reached all
vascular sections, allowing the repopulation of the glomeruli
and peritubular capillaries [24]. In this study, the capability of
the kidney scaffolds to manipulate the self-organization of
M S C s i n t o r en a l co m p o s i t i o n w a s i n ve s t i g a t e d .
Immunohistochemical staining of the matrix showed that the
cells in renal scaffolds had differentiation toward endothelial
cells and tubular cells, with preservation of proteins within the
scaffolds especially after the perfusion decellularization meth-
od. According to the study of Bonandrini et al. [22],
decellularized kidney scaffolds were recellularized with murine
embryonic stem cells using infusion technique via the renal
artery. However, seeded cells were only reached peritubular
capillaries without cells in the tubular lumen. We established
two different kidney scaffold recellularization methods using
MSCs due to their capacity to proliferate and differentiate when
in intimate contact with ECM. Although whole organ scaffolds
were effectively implanted and connected to the vasculature,
considerable thrombosis developed after implantation
[25–27]. In another study, tighter control of blood coagulation
was performed. However, inflammatory cells increasingly
destroyed the ECM and infiltrated in the scaffold [28]. The first
study for the recellularization of rat kidney organs, which was
infused into the renal artery, demonstrated that cell attachment
was low and mainly at the glomerular capillary level [16]. Our
attempt in this direction with the recellularization of rabbit kid-
ney scaffolds using MSCs showed cell attachment to the scaf-
folds and proliferation that was, however, rather higher in group
I, without significant inflammation. Several organs such as the
kidney, liver, and lung have been recently decellularized and
recellularized using the perfusion-based method with promising
outcomes [9, 29, 30]. The current outcomes confirmed that the
recellularization of the rabbit kidney with the perfusion tech-
nique can result in uniform cell seeding throughout the whole
volume of the decellularized scaffolds. Fundamental limitations
that remain are that detergent and antibiotic traces were not
quantified, and we also did not evaluate the possible immune
response issues. Additionally, the longevity of MSCs was not
evaluated during a long period. Furthermore, the application of
IHC markers was limited to general markers, and kidney-
specific markers were not widely applied. In this preliminary
study, the main focus was on evaluating the static- and
perfusion-based techniques in the recellularization of the renal
scaffold with ADMSCs that were obtained from an adjacent
anatomical area and repopulation of different anatomical kid-
ney components were not studied specifically.
Taken together, further studies with more recellularization
experiments and different cell lineages are necessary to
Regen. Eng. Transl. Med.
evaluate the full potential of these scaffolds. Furthermore,
we will emphasize on the long-term viability and functionality
of obtained kidney scaffolds in vivo in the next steps.
Conclusion
In this study, both static- and perfusion-based techniques were
applied for both decellularization and recellularization of the
renal tissue, and the ADMSCs were obtained from the
perivesical adipose tissue. Obtaining the adipose tissue from
the adjacent part to the targeted tissue may result in more
favorable outcomes that need to be addressed in future studies.
The perfusion-based recellularization method of the whole
rabbit kidney may be successful in better cellular repopulation
regardless of the specific type of seeded cell compared with
the static-based process.
Funding The authors thank the Tehran University of Medical Sciences
for funding this study (Grant Number 32213).
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict of
interest.
Ethical Approval All procedures performed in this study were in accor-
dance with the ethical standards and under guidelines approved by the
local ethical committee of the Tehran University of Medical Sciences.
The animal experiment complies with the National Institutes of Health
guide for the care and use of laboratory animals (NIH Publications No.
8023, revised 1978).
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