Methods 99 (2016) 112119

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Methods
journal homepage: www.elsevier.com/locate/ymeth

Kidney diseases and tissue engineering

Kyung Hyun Moon a,b, In Kap Ko a, James J. Yoo a, Anthony Atala a,
a
b

Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Medical Center Blvd, Winston-Salem, NC 27157, USA
Department of Urology, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Republic of Korea

a r t i c l e

i n f o

Article history:
Received 10 February 2015
Received in revised form 12 May 2015
Accepted 25 June 2015
Available online 29 June 2015
Keywords:
Kidney disease
Acute and chronic kidney disease
ESRD
Tissue engineering
3D kidney constructs
Whole kidney engineering

a b s t r a c t
Kidney disease is a worldwide public health problem. Renal failure follows several disease stages including acute and chronic kidney symptoms. Acute kidney injury (AKI) may lead to chronic kidney disease
(CKD), which can progress to end-stage renal disease (ESRD) with a mortality rate. Current treatment
options are limited to dialysis and kidney transplantation; however, problems such as donor organ shortage, graft failure and numerous complications remain a concern. To address this issue, cell-based
approaches using tissue engineering (TE) and regenerative medicine (RM) may provide attractive
approaches to replace the damaged kidney cells with functional renal specic cells, leading to restoration
of normal kidney functions. While development of renal tissue engineering is in a steady state due to the
complex composition and highly regulated functionality of the kidney, cell therapy using stem cells and
primary kidney cells has demonstrated promising therapeutic outcomes in terms of restoration of renal
functions in AKI and CKD. In this review, basic components needed for successful renal kidney engineering are discussed, and recent TE and RM approaches to treatment of specic kidney diseases will be
presented.
2015 Published by Elsevier Inc.

1. Introduction
The kidney is a highly complex organ, composed of more than
20 specialized cell types. When the kidney is injured, the damaged
renal tissue will undergo several disease states and stages such as
acute and chronic kidney symptoms. Acute renal injury (AKI) is
generally dened as a sudden increase in the serum creatinine concentration, often accompanied by decreased urine output [1,2]. In
pathological aspects, AKI shows tubular necrosis and apoptosis
[3]; changes of the ltration barrier; glomerular misltration;
vasoconstriction and tubular obstruction; as well as interstitial
swelling and activation of proteolytic enzymes [4]. The failure to
replace damaged renal cells with functional tubular cells results
in tubulo-interstitial brosis and scarring that lead to CKD. Current
treatment of AKI and CKD is limited to life-long dialysis, which
improved signicantly renal functions. While dialysis is capable
of replacing renal ltration function by removing certain toxins
from the blood, it is unable to restore many other kidney functions
such as production of erythropoietin (EPO) and activation of
Vitamin D. This limitation of dialysis frequently leads to a
suboptimal quality of life and is further associated with high morbidity and issues of long-term survival [5]. To address such

Corresponding author.
E-mail address: aatala@wakehealth.edu (A. Atala).
http://dx.doi.org/10.1016/j.ymeth.2015.06.020
1046-2023/ 2015 Published by Elsevier Inc.

complications, the injured kidney in AKI and CKD needs to be

regenerated with functional renal specic cells [6], suggesting that
cell-based approach to replace the damaged kidney cells may be
appropriate alternative to the current treatment.
When AKI and CKD develop into more severe states, that is,
end-stage renal disease (ESRD), the devastating conditions affect
multiple organs system. The only denitive treatment of ESRD is
kidney transplantation, but the scarcity of donor kidneys is perhaps the most important concern as the supply of donor kidneys
meets less than one-fth of the demand [7]. To address this issue,
tissue engineering (TE) and regenerative medicine (RM) have been
suggested as a promising solution to this problem through the
development of tissue engineered renal structures with normal
renal functions. Until now, the development of cell-based therapies
based on TE and RM strategies have provided a number of
alternative possibilities for the management of pathologic renal
conditions [8,9]. In this review, the basic components of TE and
RM-based kidney tissue engineering will be discussed and recent
advances in the treatments of kidney diseases will be reviewed.

2. Fundamental components of kidney tissue engineering

The main components used to engineer functional kidney
constructs are living cells, scaffolding system based on biomaterials, bioactive factors, and appropriate microenvironments that

K.H. Moon et al. / Methods 99 (2016) 112119

facilitate cellular behaviors. A well-orchestrated combination of

these components is of critical signicance in creating engineered
tissues or organs for the development of functional substitutes
[10]. Engineering of kidney tissue constructs may involve scaffolding systems and/or bioactive factors alone, wherein the bodys natural healing ability to regenerate is utilized to guide or direct new
tissue growth. When cells are utilized, donor tissue is dissociated
into individual single cells that are either, implanted directly into
the host or expanded in culture or attached to a scaffolding system
and re-implanted after expansion.
Several TE and RM approaches have been introduced to improve
renal functions. Due to the difculty in regenerating damaged
renal structures, particularly glomerulus [11], early studies have
focused replacing pathological renal tissues with functional engineered constructs containing renal cells [12,13]. Implantation of
these constructs in vivo showed the formation of renal structures
that produce urine-like uid [13]. The results suggest the possibility of using engineered renal constructs for augmenting renal function. With the advances in stem cell biology and cell culture
techniques, additional fundamental studies have been performed
to identify specic cell types that contribute to the restoration of
renal function (e.g. ltration) [11]. For instance, podocyte, which
is a critical cell population in the glomerulus, plays a major role
in the glomerular ltration barrier among other cell types such
as mesangial and endothelial cells [14]. However, podocytes are
known to have limited proliferation capacity in the damaged kidney, and the depletion degree of podocyte up to 2040% and 60%
leads to scarring and glomerulosclerosis, respectively [15]. Due to
the low proliferation capacity of podocyte in the diseased kidney,
investigations have focused on developing methods to accelerate
podocyte regeneration, which include activation of podocytes by
exogenously administered bioactive factors [11] or infusion of cultured cells [16] into the damaged kidney. These strategies were
targeted to specically regenerate certain renal function (e.g. ltration). Likewise, many studies have aimed to achieve targeted specic renal functions using various cell sources, scaffolding system
and bioactive factors.
2.1. Cells sources
2.1.1. Primary renal cells
In most cases, approaches to renal regeneration are developed
using cell-based systems. The development of reliable kidney cell
sources is a prerequisite step for these approaches. Kidney tissue
is composed of more than 20 specialized cell types that are structurally organized into morphologically and functionally distinct
compartments. Primary renal cells can be harvested from normal
and diseased kidney tissue and grown in culture while maintaining
the phenotype and function of the kidney from which they were
derived. Proximal tubule cells (PTCs) play an important role in kidney physiological functions [17,18]. Therefore, it is essential to isolate functional human renal PTCs from kidney tissues. Primary PTC
cultures have many advantages and are more representative of
normal PTC physiology than immortalized cell lines; however, primary kidney cells, including PTCs, lose their expression of essential
genes during passaging and limit their use to only 25 passages
[19]. The optimal combination of high purity, consistently
well-preserved growth, and differentiation is observed at passage
23 [20].
Our group also established a cell culture method that enabled
expansion of primary renal cells from human kidney tissues [21].
Histological and immunohistochemical analysis demonstrate that
the majority of the cultured cells are able to retain proximal tubular cell phenotype while distal tubular cells and podocytes are present in a lower percentage of the cell population. Additionally,
when the cells were cultured under a 3D environment, the cultured

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cells form 3D tubule-like structures with functional properties.

These results demonstrate that the established cell isolation and
culture method may ultimately be developed into an efcient
cell-based therapy for patients with CKD.
Whereas an abundance of data is at hand regarding PTC physiology, only limited information regarding cell culture systems of
other kidney cell types is available. Presnell et al. [22] developed
a culture method for primary cultures comprising autologous cells
from all major compartments of the kidney. In particular, two subpopulations, the rst corresponding to a tubular cell-enriched subpopulation (the B2 subpopulation) and the second corresponding
to an oxygen-responsive, erythropoietin (EPO)-producing subpopulation (the B4 subpopulation), were reproducibly established
from both healthy and diseased kidneys. Recent advances in
immunomagnetic cell isolation allowed the isolation of additional
subtypes of kidney-derived epithelial cells. Baer et al. [23] separated human renal epithelial cells from the human thick ascending
limb and the early distal tubule (TALDC) by using TammHorsfall
glycoprotein-coated magnetic beads. The results demonstrate that
the cultured TALDC can be utilized as in vitro system studies of the
human thick ascending limb of Henles loop and early distal tubule
as well as promising cell sources for treatment of renal failures.
2.1.2. Pluripotent stem cells
One type of pluripotent stem cells is embryonic stem cell (ESCs),
which is derived from the inner cell mass of the embryo. The ES cells
possess both self-renewal and multi-differentiation capability into
virtually any type of cell in the body [24]. Although ES cells have
great therapeutic potential, their use is currently limited by ethical
issues due to the destruction of embryos. Despite well-documented
ethical challenges, pluripotent ES Cs are considered as a viable
source of cells from which renal tissue can be derived. Several cell
culture and differentiation techniques [2528] have been developed to differentiate ESCs into renal-specic cells.
A type of genetic reprogramming technique has enabled
transformation of adult cells into pluripotent stem cells [29]. These
genetically modied cells, called induced pluripotent stem cells
(iPSCs), reportedly, possess the immortal growth characteristics
of self-renewing ESCs and express genes specic for ESCs that
can generate embryoid bodies in vitro and teratomas in vivo. Recent
progress in cell culture and differentiation methods has produced
iPS-derived renal cell lineages. Similar cell culture techniques, as
employed for ESCs, have been utilized in the production of tubular
cells and podocytes from the iPSCs [25,30]. In addition, iPSCs lines
from human mesangial cells [31], renal cells present in urine [32],
and broblasts from patients with polycystic kidney disease [33]
were established. These iPSCs can be considered as promising cell
sources for kidney tissue engineering. Despite the advantages of
these pluripotent stem cells for renal tissue engineering, their
use requires further safety studies prior to clinical translation [34].
2.1.3. Fetal and adult stem cells
Unlike ESCs and iPSCs, fetal and adult stem cells could be more
practically applied for renal engineering with fewer safety and
ethical issues. One type of fetal stem cells, amniotic uid-derived
stem cells (AFSCs), has been considered as a potential cell
source for tissue regeneration. AFSCs demonstrate extensive
self-renewal characteristics and differentiation capability to cells
from all three germ layers [35]. Since the AFSCs do not form teratomas in vivo, they could potentially be used for clinical applications. Differentiation of AFSCs into renal-specic lineage was
demonstrated in an in vitro culture system. Perin et al. [9] isolated
human AFSCs (hAFSCs) from male amniotic uid obtained at
1218 weeks gestation, transfected the hAFSCs with Lac-Z, and
microinjected the Lac-Z-transfected hAFSCs into the murine
embryonic kidney. The authors also utilized an in vitro culture

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K.H. Moon et al. / Methods 99 (2016) 112119

system to demonstrate the prospective capacity of hAFSCs to survive, proliferate, and integrate into the embryonic kidney during
organ development.
Adult stem cells, on the other hand, are usually isolated from
adult tissues and organs, or bone marrow and fat biopsies. As with
the stem cells described earlier, adult stem cells have the ability to
self-renew and they can differentiate into various types of cells
with limited tissue-specic lineages. Adult stem or progenitor cells
that have been used extensively for tissue repair and regeneration
include hematopoietic stem cells; neural stem cells from brain;
(MSC); liver stem cells from liver; muscle satellite cells from skeletal muscle; and epithelial stem cells from gut tissue; mesenchymal
stem cells (MSC) from bone marrow (BM) and fat tissue. In particular, BM- or adipose-derived stem cell is an attractive cell source,
since they are attainable via minimally invasive BM extraction or
liposuction surgery, respectively and they can be isolated and
expanded with high efciency in culture.
2.2. Scaffolding system
The principal function of a scaffolding system for tissue engineering is to provide a template to direct cellular behaviors,
which include cell migration, proliferation, and differentiation, as
well as to maintain cell type-specic phenotypes. For this reason,
it is desirable that a scaffolding system have the following features:
3D porous structures in order to provide enough space for the
seeded cells to reside with efcient nutrients and oxygen supply;
biodegradability to make account for tissue growth within the
scaffold; appropriate biological properties to stimulate and guide
tissue growth and formation of functional tissues and/or organs;
and mechanical properties to maintain the engineered tissue formation during in vitro culture and in vivo implantation [36].
Generally, three classes of biomaterials have been used for
tissue-engineering purposes: acellular tissue matrices, natural
and synthetic polymers [37]. The kidney possesses complex 3D
tubular structure with numerous types of cell populations.
Consequently, renal specic scaffolds for engineering kidney tissue
constructs require some allowance for the seeded cells to form normal kidney-like structures by permitting numerous cell growth
processes and cellcell/cellmatrix interactions during in vitro culture. Naturally derived polymers provide an alternative to decellularized tissues and include collagen, hyaluronic acid (HA), alginate,
agarose, chitosan, brin, and gelatin. These polymers all have the
ability to adequately support cell adhesion, migration, proliferation, and differentiation. Most naturally occurring polymers belong
to a class of highly hydrated polymer materials (water content P 30% by weight) [38] and are compatible with native tissue.
Synthetic polymers, including polyglycolic acid (PGA), polylactic acid (PLA), and poly lactic-co-glycolic acid (PLGA), are also
widely used in tissue engineering. The degradation products of
these synthetic polymers are nontoxic and correspond to natural
metabolites. Moreover, the polymer degradation rate can be controlled for specic purposes from several weeks to several years.
The disadvantages of synthetic polymers are that they are not
physiological and show less favorable interactions with the body
relative to biologically derived scaffolds. Biodegradation of synthetic polymers may also result in inammatory responses [39].
The use of decellularized tissue matrices as renal scaffolds is
very attractive, because naturally derived polymers and synthetic
polymers are unable to replicate the precise spatial organization
of complex structures like the kidney. In particular, acellular tissue
matrices are usually prepared by removing the cellular components from the tissues via mechanical and/or chemical manipulation to produce collagen-rich matrices [40]. These collagen-rich
matrices tend to slowly degrade upon implantation and replaced
with ECM proteins secreted by ingrowing cells. Therefore, much

attention has focused on the development of kidney decellularization techniques for the production of decellularized kidney scaffold
that can maintain the native renal architecture and the integrity of
the ECM, bioactive factors, and vascular network. Several groups
have developed a kidney decellularization technique to produce
acellular kidney scaffolds for whole kidney engineering [41,42].
More details will be discussed in a later section.
2.3. Bioactive factors
Bioactive factors, such as growth factors and cytokines, are closely related with kidney regeneration from renal failure [43]. The
recovery process requires the replacement of damaged tubular
cells to restore the continuity of the renal epithelium. This process
involves a number of growth factors that are produced in renal tissues. The growth factors include epidermal growth factor (EGF), a
potent proximal tubule cell mitogen [44]; transforming growth
factor-a (TGF-a), a participant in the reconstruction of the injured
proximal tubule via the EGF receptor [45]; insulin-like growth factor 1 (IGF-1); broblast growth factor (FGF) [46]; and hepatocyte
growth factor (HGF). All of these factors have been utilized to
enhance renal recovery after AKI in experimental models [47].
EGF and TGF-a commit cells to the cell cycle via activation of extracellular regulated kinases to increase DNA synthesis after ischemic
and nephrotoxic renal failure [48,49]. The successful use of these
and other growth factors in animals has led to clinical trials of
IGF-1 actions in humans; however, IGF-1 failed to enhance recovery from AKI [50]. Nevertheless, Bach et al. [51] suggested that
IGF-1 may potentially enhance stem cell-mediated repair of kidney
injury, but further studies are required to determine the optimal
role of IGF-based therapies in kidney disease [51]. Additional studies are also required to identify the growth factors that are capable
of consistently increasing tissue proliferation, and for the
development of technologies to the delivery of these factors
either ex vivo or to transplanted cells. More promisingly, efcient
delivery systems of multiple growth factors at rates mimicking
the in vivo environment may have great potential in kidney tissue
engineering [52].
3. TE and RM approaches to therapeutic treatment of kidney
diseases (Fig. 1 and Table 1)
3.1. Acute and chronic kidney failure
3.1.1. Cell-based approach: cell therapy
Cell-based approaches have been considered as a promising
treatment option. Such treatments involve transplanting cells alone
or implantation of engineered kidney constructs. Current therapeutic strategies for treating AKI and CKD have been investigated using
primary renal cells and stem cells. In particular, BM-MSCs have
been used extensively as a cell source for this purpose in a wide
range of pre-clinical [5358] and clinical trials [59,60]. In early
studies, the mechanism by which exogenously injected BM-MSCs
facilitated the therapeutic effects in animals with AKI and CKD
has been suggested by trans-differentiation of the administered
BM-MSCs into renal specic cells such as renal tubular cells [61],
mesanglial cells [62], glomerular endothelia cells [63], and podocytes [16]. In addition, recent studies have demonstrated that the
therapeutic effects are also attributed to trophic factors released
from the injected BM-MSCs. Cytokines and growth factors that have
both paracrine and autocrine activities have been shown to be
secreted by BM-MSCs [64]. Furthermore, BM-MSCs secrete exosomes containing microRNAs that mediate some of the benecial
actions of these cells after injection [65,66]. Togel et al. [53] administered BM-MSCs to rats presenting with ischemiareperfusion

K.H. Moon et al. / Methods 99 (2016) 112119

(I/R)-induced acute renal failure (ARF) via the carotid artery either
immediately or at 24 h after renal ischemia [53]. Provision of the
BM-MSCs to the I/R-ARF model rats proved signicantly renoprotective with favorable effects predominantly mediated by paracrine
rather than trans-differentiation-dependent mechanisms. Similarly, Wang et al. [67] showed that transplanted BM-MSCs
improved a wide variety of kidney disorders, from AKI to CKD,
which further indicates that these cells are renoprotective after
arterial delivery during the early stages of renal injury.
Adipose-derived stem cells (ADSCs) isolated from fat tissues
have been used for treatment of AKI and CKD in rodent [6870]
and large animal models [71,72]. Donizetti-Oliveira et al. [68] created a murine model of progressive renal brosis by unilaterally
clamping the renal pedicle for 1 h followed by administration of
ADSCs intraperitoneally at 4 h after surgery. Consequently, the systemic ADSC therapy reduced the progression of ischemia-induced
renal brosis through the downregulation of inammation and
hypoxia. In other studies, ADSC cultivation in tubular epithelial
cells-derived conditioned medium promoted ADSC differentiation
into epithelial cells [73], which may be relevant to mechanisms
of organ repair. The fundamental question of how BM-MSCs,
ADSCs, and other stem cells exert their actions in the in vivo
remains for debate, and investigation of long-term effects of stem
cells-based cell therapy continues. Regardless, the use of stem cells
as a therapeutic approach to renal disease holds great promise for
clinical application. Benecial therapeutic effects of other stem cell
sources such as iPS [74], AFSCs, and endothelial progenitor cells
(EPCs) [75,76] have been demonstrated in renal injury models
including glycerol [77] or cisplatin [9,78]-induced AKI, a murine
model of Alport syndrome [79], and unilateral ureteral obstruction
[80].
Recent studies in our group have demonstrated that primary
renal cells isolated from human kidneys also facilitated benecial
effects toward the recovery of renal functions. As previously
described, we developed a cell isolation and culture system to
obtain a number of human primary renal cells [21]. As renal injury
models, we have established two rodent models of kidney failure
by varying the length of the ischemic time [81]. In particular, we

115

demonstrated that the use of longer ischemic times (75 and

90 min) could be used to test new therapies for acute renal disease,
whereas shorter ischemic times (60 min) could be used to study
therapies for chronic renal insufciency [81]. To examine the therapeutic effects of the primary renal cells on the improvement of
renal functions, primary renal cells were administered into a CKD
rat model [81] and evaluated renal functions. In particular, several
reports focused on a cell population of EPO-positive renal cells.
EPO, a cytokine produced by broblast-like cells in the kidney
[82,83], has recently emerged as a renoprotective factor with
anti-inammatory and antioxidant activity. Our group developed
a cell purication method to enrich the EPO-positive renal cell population [83]. The results showed that intrarenal delivery of an EPO
positive-enriched cell population facilitated more benecial effects
in treatment of renal injury, inammation, and oxidative stress
compared to unsorted cell cultures in a chronic kidney injury
model, demonstrating that EPO-positive cells from primary renal
cells can be considered as potential cell population for degenerative kidney diseases. Another CKD model using two-step 5/6
nephrectomy in rats was used to demonstrate the positive effects
of primary renal cells on the recovery of renal functions [84]. In
this study, the authors showed that the tubular cells-enriched
population provided better therapeutic effects compared to
unfractionated heterogeneous renal cells by attenuating canonical
pathways of pro-brotic extracellular matrix production.
3.1.2. Cell-based approach: tissue-engineered 3D renal constructs
Another potential approach to treatment of AKI and CKD using
the cell-based system would be to engineer a 3D kidney-like tissue
capable of replacing the renal functions [85]. The general strategy
for engineering 3D renal structures is to seed cells into the desired
scaffolding systems and then to culture the cell-seeded construct
in vitro. Subsequently, the engineered renal construct can be
implanted in vivo for successful integration with host kidney tissues.
For this purpose, primary renal cells have been used mainly as
cell sources for seeding into several types of scaffolds.
Collagen-based hydrogels have been extensively used to generate
scaffolds for kidney tissue engineering. Wang et al. [12] isolated

Fig. 1. Schematic diagram of possible cell-based therapies for kidney diseases.

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K.H. Moon et al. / Methods 99 (2016) 112119

Table 1
Tissue engineering and regenerative medicine approaches to treatment of kidney diseases.
Cell or tissue sources

Scaffolds

Type of
disease

Model creation

BM-MSC

N/A

Acute

I/R (rat and mouse), cisplatin (mouse), gentamycin (rat), [5356,59]

adriamycin (rat), acute renal failure (clinical trial)
Remnant kidney model (rat), kidney allograft model (rat), [57,58,60]
chronic allograft nephropathy (clinical trial)

Chronic

Refs.

ADSC

N/A

Acute
Chronic

I/R (mouse), I/R (rat), cisplatin (rat)

Atherosclerotic renal artery stenosis (pig)

[6870]
[71,72]

iPSC
AFSC

N/A
N/A

Acute
Acute

[74]
[7780,103]

Primary renal cells

N/A

Chronic

I/R (rat)
Glycerol (mouse), cisplatin (mouse), Alport
syndrome (mouse), unilateral ureteral
obstruction (mouse)
Two step 5/6 nephrectomy (rat)
I/R and gentamycin (rat)

EPC
N/A
ESC-derived renal cells
N/A
Renal cells and embryonic renal tissue Collagen/vitrigel, Collagen/Matrigel, HA
Kidney segments
Biodegradable synthetic polymer (PGA)
Primary renal cells
Polycarbonate
Renal cells from broblast
PGA
N/A
Decellularized kidney scaffold
Renal cell and EC
Decellularized kidney scaffold
EC

Decellularized kidney scaffold

Chronic
Non-disease
N/A
Non-disease
Non-disease
Non-disease
Non-disease
Non-disease

Renal artery stenosis (pig)

Injection to kidney rudiments (Rat)
In vitro characterization of 3D kidney-like construct
Subcutaneous implantation (athymic mouse)
Subcutaneous implantation (athymic mouse)
Subcutaneous implantation (athymic mouse)
Partial graft into nephrectomized animal (rat)
Experimental orthotopic implantation using
whole kidney construct (rat)
Non-disease Experimental orthotopic implantation using
whole kidney construct (pig)

[84]
[81,83]
[75,76]
[26]
[12,86,87]
[89]
[13]
[90]
[102]
[42]
[100]

Bone marrow-derived mesenchymal stem cells (BM-MSC), adipose-derived stem cells (ADSC), embroynic stem cell (ESC), induced pluripotent stem cells (iPSC), amniotic
uid-derived stem cells (AFSC), endothelial progenitor cells (EPC), ischemia/re-perfusion (I/R), hyaluronic acid (HA), polyglycolic acid (PGA), endothelial cells (EC), not
available (N/A).

glomerular epithelial and mesangial cells from kidney tissues and

cultured the cells on a stable, thin, transparent collagenous gel
membrane termed a collagen vitrigel. The collagen vitrigel
provided an optimal environment that mimicked the native
glomerular basement membrane, which formed a reconstructed
renal glomerular tissue when co-cultured with renal cells [12]. In
a similar approach, Lu et al. [86] reconstructed 3D renal tissues
by using mixed neonatal rat renal cells in a 3D collagen/Matrigel
scaffold in vitro. Subsequently, the seeded cells within the 3D
hydrogel scaffold self-assembled into engineered-kidney tissues
containing both tubules and glomeruli-like structures. Using the
same collagen scaffold system, our group established a 3D
kidney-like construct for in vivo study. Our results demonstrated
that human primary renal cells are effectively expanded in a 3D
collagen-based culture system [21], and the 3D-cultured cells
retained their phenotypes, migration ability, and albumin uptake
functions. Also, the human renal cells formed tubular structures
in 3D culture that stained positively for proximal tubule, distal
tubule, and collecting duct markers [21]. When the 3D kidney construct was subcutaneously implanted into mice, the cells within
the implant maintained their survival and renal phenotypes for
6 weeks. These ndings demonstrate that human renal cells grown
under 3D culture using a collagen gel system are able to generate
kidney-like structures in vitro and can be used for treatment of
renal failures.
Another type of hydrogels, hyaluronic acid (HA) is a
glycosaminoglycan of great importance to tissue engineering as it
plays a vital role in mammalian development. Rosines et al. developed a 3D cell culture method to construct kidney-like tissues from
fetal kidney tissues [87]. In the study that was based on the kidney
organ development programs, the authors utilized HA scaffold to
support ureteric bud (UB) branching; promote mesenchymal
to-epithelial transformation; and stimulate differentiation of the
metanephric mesenchyme (MM) and the UB. They suggested that
HA might serve as an ideal scaffolding material for kidney tissue
engineering. Nevertheless, use of hydrogel-based scaffolds such

as collagen, HA, and other hydrogel polymer scaffolds are

limited by their low mechanical strength and high rate of
shape-retention failure [88].
In an attempt to address issues of low mechanical strength by
using hydrogel-based scaffolds, synthetic polymer-based biomaterials have been used as an alternative source for cell seeding. Our
group developed an initial approach to construct renal structures
using a synthetic biodegradable polymer with rabbit renal cells.
The renal cells from distal tubules, glomeruli, and proximal tubules
were expanded separately and then seeded onto PGA scaffolds. The
cell-seeded constructs were implanted subcutaneously into athymic mice. Histological analysis showed that nephron segments
had formed within the implant structures, and the seeded cells
were able to proliferate, as evidenced by BrdU staining. These
results demonstrated that the cultured primary renal cells are capable of reconstituting tubular structures with homogeneous cell
types within each tubule. Other synthetic polymers have been used
as scaffolds in renal tissue engineering as well. A tubular device
constructed from polycarbonate material was utilized as a scaffold
for supporting mouse-derived renal cells [13]. The tubular device
was connected at one end to a silicone catheter that terminated
in a reservoir, and this construct was implanted subcutaneously
in host athymic mice. Histological analysis demonstrated that the
implanted device possessed highly organized tubule-like structures and formation of glomeruli and integrated with extensive
vascularization. Furthermore, the tubular-like structures contained
both proximal and distal tubular cells as well as cells of the thin
ascending loop of Henle, as conrmed by immunohistochemical
staining. Interestingly, the newly formed structures exhibited a
renal function by excreting high levels of solute through a yellow
urine-like uid. A similar study using kidney segments was
performed with the same PGA scaffold. Kim et al. [89] created renal
units in vivo by transplanting isolated renal segments onto 3D
biodegradable PGA polymer scaffolds. Three-dimensional renal
reconstructs were formed that contained glomeruli and tubules,
suggesting that renal structures can be reconstituted by

K.H. Moon et al. / Methods 99 (2016) 112119

transplanting renal segments. Other cell-sourced renal structures

have been created using a nuclear transfer technique. Lanza et al.
[90] created bioengineered renal tissues by using a nuclear transfer
technique, where renal cells were isolated from early-staged
cloned bovine broblasts and seeded onto polymer scaffolds incorporating PGA scaffolds. The secretion of urine-like uid and the formation of organized glomeruli- and tubular renal specic
structures were demonstrated after transplantation of the grafts
into the subcutaneous space of the same cow from which the
broblasts were derived.
3.2. Whole kidney engineering for end-stage renal failure (ESRD)
When AKI and CKD develop into end-stage renal failure (ESRD),
it is a devastating disorder involving multiple organs in affected
individuals. Currently, available renal therapies are not optimal
for most patients. The only denitive treatment of ESRD is kidney
transplantation. Due to a critical shortage of available donor kidneys, physicians and scientists have sought novel solutions to this
problem.
Recent progress in whole organ engineering involving the
decellularization of organs followed by recellularization of the
resulting collagen matrix has provided a promising approach for
the production of transplantable organ constructs [9195]. The
decellularization process involves the removal of all the cellular
components from an organ with the structural architecture left
intact. During this process, primary immunogenic factors such as
cellular proteins and DNA are removed, largely negating reactions
to the transplanted scaffold by the recipient immune system. Many
decellularization methods are based on perfusion of an organ with
detergents and other chemical agents through the vasculature.
These perfusion-based decellularization protocols are effectively
designed to remove all native cells and DNA, while maintaining
the structural integrity of the organs extracellular matrix. Subsequently, the resulting acellular matrix is repopulated with
organ-specic cells (recellularization) that may undergo some
degree of self-organization within the matrix, which often results
in pockets of functional tissue.
Current decellularization/recellularization techniques have
enabled researchers to successfully create a number of bioengineered whole organs such as heart [91], lung [95], liver [92,94],
and recently, the kidney [42] in vitro, which has led to pre-clinical
in vivo studies using small animal models. These in vivo studies
have been limited to short-term evaluations as these engineered
organs function for only a few hours following transplantation.
Recently, the implantation of a bioengineered rat kidney demonstrated the feasibility of the decellularization/recellularization tissue [42]. In the recellularization process of acellular kidney
matrices, previous studies utilized embryonic stem cells [96,97]
and viable kidney explants [98] to re-create engineered kidney
structures in vitro [42]. Although these preliminary approaches
demonstrated the feasibility of using recellularization of a renal
matrix to bioengineer a kidney, the eld of whole-organ tissue engineering is still in its infancy and numerous challenges must be
addressed before these techniques can be used clinically.
One major challenge for long-term in vivo success of bioengineered organs is vascular patency. In the absence of complete
and functional endothelial reseeding of vascular matrices, signicant thrombosis is likely to occur within the vasculature of the
scaffold, which renders the recellularized construct nonfunctional
[92,99]. To address this issue, we have developed an endothelial
cell seeding method that permits effective endothelial cell coating
of the vascular walls of decellularized porcine kidney scaffolds
[100]. Acellular renal matrices were processed from normal pig
kidneys using our previously described decellularization technique
[41]. Furthermore, conjugation of CD31 antibodies to the vascular

117

matrix improved endothelial cell retention on the vasculatures

that enhanced vascular patency of the implanted scaffold.
These results demonstrate that our endothelial cell seeding
method, in combination with antibody conjugation, improves
endothelial cell attachment and retention leading to vascular
patency of tissue-engineered whole kidney in vivo. For this technology feasible toward clinical translation, other critical challenges
should be addressed. Such challenges include: fabrication of acellular renal scaffold of a clinical scale [41,101]; efcient recellularization of scaffolds using clinically relevant cell sources to
recreate fully functional kidney construct [21,31,32]; long-term
implantation without severe thrombosis [100].
4. Concluding remarks and future perspective
Recent advances in established cell culture methods,
biomaterial-based scaffolding systems, and understanding of biological, molecular, and physiological aspects of renal regeneration
have allowed the eld of cell-based therapy and kidney tissue engineering to make promising advances in repairing and restoring
kidney functions from renal failures. Many experimental studies
of cell-based therapy using transplantation of primary renal and
stem cells alone have been performed to test new strategies for
the repair of the damaged kidney structure and functions, and
some of them are now clinically applicable.
When the kidney disease progresses to devastating conditions
such as CKD and ESRD, the degenerating kidney tissue needs to
be partially and fully replaced with functional normal renal tissues.
For this purpose, current progress in 3D cell culture methods has
enabled the production of engineered kidney constructs in vitro.
The resulting 3D functional renal structures have been successfully
validated in non-diseased renal models; however, the pre-clinical
and clinical application of the fully and whole functional renal
structures to the kidney specic disease remains challenging.
Indeed, the recapitulation of the normal kidney in terms of its
structural and functional complexity will ultimately require a master system capable of successfully mimicking all key aspects of kidney physiology. To achieve this goal, multidisciplinary researches
in this eld should be conducted in a well-balanced manner. Such
studies should include a number of pre-clinical studies on model
creation of kidney diseases and application of tissue-engineered
kidney constructs [102]; more sophisticated approaches on partial
and whole kidney implantation dealing with immunological
issues; innervation of the implant; and renal physiology.
Transparency Document
The Transparency document associated with this article can be
found in the online version.

Acknowledgement
We wish to thank Dr. Heather Hatcher for editorial assistance
with this manuscript.
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