1. Engineering a Bioartificial A paper was published in Nature
Medicine magazine that claimed to obtain a
Kidney Utilizing a functional bioartificial heart by
Decellularized Matrix recellularization of a decellularized
cadaveric heart [3]. Although the experiment
Christoph Neyer, Larry Liu, Regine Labog,
was conducted on murine and primarily on
Seyed Bozorgi
heart, it was claimed that the concept of the
experiment may be applied to a human heart
Introduction
and other organs. Thus by mimicking the
procedure of decellularizing a cadaveric
26 million Americans have chronic
kidney with no recorded medical history of
kidney disease with progression of disease
dysfunction and recellularizing the obtained
leading to kidney failure need for transplant
extra-cellular matrix (ECM) with the
to sustain life, and the number of new
patient's own cells, a functional bioartificial
patients with kidney failure has averaged
kidney may be obtained.
more than 90,000 annually [1]. Chronic
kidney disease is a progressive loss of renal
Background on human kidney physiology
function which may cause: blood wastes to
build to high levels and develop
Kidneys regulate body fluid volume
complications like high blood pressure,
and composition. As an endocrine organ,
anemia (low blood count), weak bones, poor
kidneys synthesize renin to regulate blood
nutritional health and nerve damage [1].
pressure, erythropoietin (the main factor for
Chronic kidney disease (CDK) may be
red blood formation in bone marrow), and
caused by diabetes, high blood pressure and
active vitamin D (enhances calcium
other disorders [1]. Current treatments for
absorption). Nephrons, as the functional unit
kidney's failure are dialysis and
of kidneys, filtrate through glomerular
transplantation. Dialysis does not cure
capillaries, reabsorb mostly through
kidney disease, costs a lot and patients will
proximal tubule and excrete the body fluid
need to have dialysis treatments for their
through collecting duct.
whole life unless they are able to get a
kidney transplant. Kidney transplants may
Design Ideas and Methods
come from living donors or a cadaver, and
require tissue typing and blood type
Decellularization of the Kidney
compatibility to reduce the risk of immune
rejection of the transplanted kidney.
Once a donor kidney has been
Furthermore, the treatment requires patients
obtained the kidney will then be
to wait for a match and take anti- rejection
decellularized using detergents. This can be
medications, which are expensive and cause
accomplished by first perfusing the matrix
more susceptibility to diseases. To reduce
with sodium doceyl sulfate (SDS), an ionic
the risk of post- transplantation immune
detergent commonly used in
rejection and provide patients relief from
decellularization that acts as a surfactant,
waiting for a tissue match, bone marrow
lysing the cells. The matrix is then perfused
stem cells, constructing a bioartificial kidney
with Triton X-100, a non-ionic detergent.
with the patient's own renal cells will be a
This process has been shown to remove over
potential treatment.
96% of all DNA traces and leaves no
detectable SDS residue. In the past intact

2. nuclei were detected using a DAPI stain and nutrient medium. The idea is that these cells
none were found [3]. will localize to the Bowman's capsule
because of it unique matrix composition.
Cell Source Next, mesangial cells will be added to the
perfusion solution. These cells will begin to
The next step in the process is to line the blood vessels present in the kidney.
obtain kidney cells to seed on the Lastly, endothelial cells will be added to line
matrix. Bone marrow-derived stem cells the interior of the blood vessels. The kidney
(BMSCs) have been reported to differentiate construct will then be cultured in a
into renal cells, specifically mesangial cells, bioreactor designed to mimic renal
endothelial cells, podocytes, and tubular physiology.
cells in the kidney [8]. Mesangial cells line
the vessels of the kidney, podocytes are Culturing of Bioartificial Kidney in
found in the Bowman's capsule, and tubular Bioreactor
cells are present in the renal tubule, which
connects the Bowman's capsule and the While the exact design of the
collecting duct. Using dual-wavelength flow bioreactor is beyond the scope of this
cytometric analysis [9], which is based on project, the bioreactor will need to perform a
the differential ability of cells to efflux few key functions. First, it must supply
Hoechst 33342 (a fluorescent DNA-binding oxygen and nutrients to the cells in the
dye), enriched populations of BMSCs can be kidney. Second, it must provide an artificial
obtained from adult murine bone marrow. outlet for waste products removed by the
Differentiation of the BMSCs into kidney. Lastly, it can recycle the isolated
mesangial cells is accomplished by seeding waste products back into the blood-like fluid
the BMSCs on a collagen I matrix. Non- being pumped through the blood vessels of
adherent cells were then transferred to a type the kidney to supply the kidney with waste
IV collagen matrix and subjected to a to remove. This set up also allows for the
differentiation medium containing retinoic easy measurement of waste removal
acid and platelet-derived growth factor-BB efficiency. Once the kidney exhibits
(PDGF-BB) for 24h [8]. Some of the physiological waste removal capabilities, it
resultant cells changed to a stellate shape can be transplanted into the patient. The
and expressed Thy1 and desmin, perfusion medium will contain nephrogenic
characteristic of mesangial cells. growth factors such as a combination of
retinoic acid, Activin-A, and Bmp7 in order
Reseeding of the Decellularized kidney to promote kidney growth and function [5].
The time frame for culturing the bioartificial
Our unique approach is in the kidney within the bioreactor is unknown
sequential perfusion of these different cell without actually doing the experiment, but
types into the decellularized kidney matrix. most likely after a period of weeks to
First, tubular cells will be perfused in a months the efficacy of the efficacy of the
retrograde fashion through the collecting kidney construct could be tested by adding
duct of the kidney. This will result in tubular waste to the blood-like solution being
cells lining the collecting duct and renal pumped through the construct and
tubule. Podocytes will then be perfused measuring the clearance. If the construct is
through the blood vessel system of the able to provide sufficient clearance, it can be
decellularized kidney, using an oxygenated

3. implanted into the patient and the patient’s To characterize the contribution of
blood will be monitored for waste levels. bone marrow cells to the turnover rate of
renal cells, male BMSCs can be transplanted
Product Characterization and Validation into a female murine kidney and tested for
Y-chromosome kidney cells.
In Vitro
In Vivo
The BMSCs were immunostained
with CD34, CD133, c-Kit, and GATA-4 to To assess kidney function,
make sure they were bone marrow derived immunofluorescence will be used to
stem cells. After the BMSCs are determine whether the decellularized kidney
differentiated in a tissue culture, the renal is composed of differentiated renal cells.
cells can be isolated using immunoselection Glomerular flow rate in and out of the
with monoclonal antibodies (mABs) such as kidney within the bioreactor can be
ST.12, which intercalated and principal cells measured by injecting inulin into the
in the renal collecting duct react to or by bioreactor. Inulin is neither reabsorbed or
conjugating renal cells with cell markers and secreted by the kidney after glomerular
separating with FACS [12]. filtration so its rate of excretion is directly
Cells with slow cycling time can be proportional to the rate of filtration of water
distinguished by retention of a nucleotide and solutes across the glomerular filter. The
label such as bromodeoxyuridine (BrdU), normal kidney functions with a glomerural
which is incorporated into the DNA of cells flow rate of 90mL/min/1.73m2.
during DNA synthesis [7]. If, after
administration of a pulse of BrdU the cells Renal Blood Flow tests could also be
are monitored for long periods of chase, done by determining the level of creatinine,
only the slowly cycling cells retain a BUA (Blood Uric Acid), and BUN (Blood
concentration of label sufficiently high to Urea Nitrogen) in the blood, which is
allow their staining and thus adult organ- normally above levels during kidney
specific stem cells are often called “label- disfunction.
retaining cells”
Discussion
Flow cytometry of the acutely
dispersed cells from the papillae of 8 rats Key Techniques and Improvements
revealed that 3% of the cells were positive
for CD45, indicating that a small fraction of The most important technique
the isolated cells were blood cells. However, introduced in our project is the sequential
less than 0.5% of the total cells were perfusion of the different cells types.
positive for CD34, CD44, or c-Kit, Perfusion is done by pumping medium
indicating that if hematopoietic stem cells or through the decellularized blood vessels of
bone marrow mesenchymal stem cells were the kidney or the collecting ducts. The
isolated with the renal papillary cells, their importance of this method is that it controls
contribution to the population of the BrdU- where cells end up, but eliminates the need
retaining cells (about 40% of the total cells) for a tedious manual seeding process. Since
was small the matrix content and geometry is
preserved during decellularization, the
differentiated cells are likely to localize to

4. the correct locations within the kidney allogeneic donor is available, a xenogenic
construct and sequential perfusion further kidney could theoretically be used.
controls where the cells end up. If all the However, this brings with it issues of
cells were perfused at the same time, the immunogenic matrix components. There are
endothelial cells, which will tend to line the some important drawbacks to consider in
interior of the blood vessels, will prevent the this case. First, the decellularized kidney
other cells from migrating further into the with implanted stem cells need to be
kidney matrix. cultured in a bioreactor. However, such a
bioreactor that can grow an entire kidney
Feasibility has not yet been invented and its parameters
are complicated. Second, during the kidney
The feasibility of this project can be growth inside the bioreactor, it will be
separated into three parts. First, it is difficult to observe and monitor cell
definitely feasible to decellularize the growth/allocation, taking into the account
kidney, as the decellularization of the that there are going to be different cell types
murine heart was successfully conducted and layers interacting at same time. The
recently in literature. Second, it is also third drawback as well as a major challenge
feasible to generate the renal cells from bone is the time that's required to complete the
marrow-derived stem cells (BM-dSC) and entire process, which includes finding the
other forms of stem cells. However, the part best kidney match, decellularize the selected
that's not quite feasible is the perfusion of kidney, implant the stem cells, grow kidney
the bioartificial kidney because there might inside bioreactor, implantation and potential
be various variables not known and post-implantation treatments.
parameters not quantified.
Alternatives
Works Cited
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