This study aimed to develop a process for engineering renal scaffolds using decellularized human kidneys and murine embryonic stem cells. Based on previous studies using different animal kidneys, the proposed method involves decellularizing a human kidney using 1% SDS for 21 days, then seeding it with 5x10^5 murine embryonic stem cells and circulating them through the kidney for 2.33 hours using a bioreactor. The seeded scaffold would be examined daily using fluorescence microscopy to validate sufficient cellularization throughout the kidney vasculature over 28 days. If successful, this could help address the shortage of kidneys available for transplant.

1. Engineering Renal Scaffolds for Humans Based on Trials of Increasingly Similar Anatomy
Abstract:
The goal of this study was to improve the process by which cells engraft and proliferate
onto a decellularized kidney scaffold. Reviewing various literatures, different kidneys were used
as models varying from pig to mouse. Decellularization will be performed using a detergent, 1%
sodium dodecyl sulfate (SDS). Then the scaffold will be seeded with murine Embryonic Stem
(mES) cells. The seeded scaffold is to then be transferred to a bioreactor so the cells can circulate
on the scaffold. After a few hours, the scaffold is then examined with an immunofluorescence
microscope to see if the scaffold is well cellularized.
Problem Statement:
End-stage renal disease is a major cause of death worldwide. This is caused by long-term
onset effects of chronic kidney diseases. A majority of these diseases lead to the nephrons of the
kidney being damaged, which inhibits their function [1]. Specific damage to the nephrons often
centralizes around glomerular damage. Crucial to the function of the kidney as a whole, the
podocytes are found in these glomeruli. The podocyte’s function in regards to general kidney
operation is to allow prevent macromolecules from travelling from the blood to the urine.
Damage to the nephrons compromises the structure of the podocyte, as illustrated in Figure 1 [2].
Figure 1: Illustration of how damage to the glomeruli influences podocyte activity within the nephron [2]. Loss of
slits in the diaphragm of the podocyte prevents certain molecules from flowing through,resulting in an inability of
the glomeruli to create ultrafiltrade used in producing urine.
Due to this inability to prevent macromolecules, such as proteins, from entering the blood, the
patient develops Nephrotic Syndrome. Nephrotic Syndrome is prevalent in most CKD’s, and is
characterized by elevated protein levels in urine. This, in turn, relates to a lack of protein in the
blood, causing fluids to pool in the body’s tissues instead of circulating [3].
An estimated 13% of the world’s population is struggling to live with these chronic
kidney diseases [4]. Over 500,000 of these people progress to end-stage renal disease, costing
upwards of $29 billion in Medicare spending [4]. Currently, treatment options are limited to
dialysis and transplantation. Dialysis can lead to a more difficult life, with increased risk of other

2. diseases. Transplantation allows for a better quality of life, but is often unsuccessful due to
rejection and lack of available donors. Bioengineered kidneys using donor cells can avoid these
problems, though the current technology to produce them is still in its infant stage.
Rationale:
The entire process of creating renal scaffolds from donor organs can be characterized into
several steps: decellularization, recellularization, and validation testing. Several additional
factors can be quantified for each step, including decellularization length, detergent and
concentration, recellularization length, the cell type being seeded to the scaffold, and amount of
cells to be seeded.
The decellularization methods observed in previous experiments have tested multiple
concentrations of both Triton X-100 and sodium dodecyl sulfate (SDS). One experiment
observed the use of SDS in 0.5% concentration to leave less than 50ng DNA per milligram of
dry tissue, however, this technique was used on a rat kidney, and higher concentration would be
needed in larger organisms. Additionally, in previous trials, prolonged exposure to Triton X-100
has lead to disruptions in the collagen structure of the kidneys [4].
Recellularization methods were also analyzed from previous experiments. The
recellularization protocol of both monkey and pig kidneys utilized 5x10^5 cells to be circulated
through the entire organ. This appears to be a standard in larger animals, based on multiple
research results [5], [6]. Fluorescent testing must be done on the organ in order to verify that the
cells proliferated through the entire kidney, and to verify that decellularization was successful in
clearing all the native cells. Due to the nature of the fluorescence staining, non-human cells must
be seeded into the scaffold. This is to make sure there are no residual human cells picked up on
fluorescence [7]. Additional testing for cells being fully seeded, and to test for cell growth days
after recellularization, are standard in similar experiments to those described below [4],
[5],[6],[7],[8],[9].
Method:
The methodology described below follows the idea that there are linear relationships
between blood volume and weight to decellularization and recellularization lengths, as shown in
Figure 2. Blood volume for each animal type was provided by Drexel University College of
Medicine [10]. Before any decellularization occurs, the native kidney and its cells must be
stained with Hematoxylin and eosin, so as to provide definitive proof whether the decellularized
scaffold does or does not still contain cells [8].SDS is needed for decellularization of the kidney
due to the higher cell density and fibrosity of the kidney as opposed to organs such as the heart,
lungs, and pancreas [9]. Due to the damaging nature of Triton, and the efficiency of SDS, it
seems obvious that perfusion of SDS in 1% concentration through a peristaltic pump connected
to the renal artery is the most effective combination to use in completely decellularizing a kidney

3. [8]. The trend, based on graphical analysis, has led to a decellularization length of 21 days for a
human kidney.
Figure 2: Trends relating both weight and blood volume to the length of decellularization and recellularization of
kidney scaffolds.Points on the line designate the rates used for rats [8], pigs [5], and this proposal’s derived human
approximation. “*” denotes an erroneous plot derived from an experiment conducted on Rhesus Monkey kidneys
[6]. The methodology differed from the experiments that yielded the rest of the trends. However, the monkey
represents the most human-like specimen, and is worth considering for future works.
Recellularization begins after these 21 days, and after checking for residual staining and
verifying that the kidney is absent of parent cells. The protocol to be followed for
recellularization is the same as that of decellularization: perfusion through a peristaltic pump
over the duration chosen, in order to assure that all of the cells have seeded throughout the entire
kidney vasculature. As mentioned previously, non-human cells must be seeded into the scaffold
in the amount of 5x10^5 cells per kidney. Embryonic stem cells are pluripotent, and can
differentiate into many cell types. With this in mind, murine Embryonic Stem (mES) cells are to
be treated with anti-green fluorescent protein (GFP), which will show up in later tests to verify
that cells have proliferated throughout the entire kidney [7]. Based on the trend of
recellularization length in relation to blood volume, a length of 2.33 hours was chosen. The
recellularized scaffolds are to be stored connected to the peristaltic pump, which will continue to
circulate cell medium throughout the kidney. Kidneys will be stored for up to 28 days, with
fluorescence testing being conducted daily.
Validation testing for the proposed methodology will need to be conducted through
fluorescence microscopy. In order to detect whether or not the newly implanted murine cells

4. have seeded throughout the entire scaffold, kidneys are analyzed under a fluorescence
microscope with the goal of observing fully occupied vasculature, as depicted in Figure 3. In the
event that fluorescence micrographs indicate the cells were unable to perfuse throughout the
entire kidney, an additional method may be employed to “pull” the cells through. The scaffolds
should be mounted in a seeding chamber under a vacuum to create a pressure between 40 and
60cm H2O [11]. After the fluorescence has verified full organ proliferation, Hematoxylin and
eosin can be applied to the newly seeded scaffold, see Figure 4. This technique allows for
recordings to be taken after certain periods of time, to see whether or not the cells are continuing
their growth deeper into the tissue [7].
Figure 3: Various fluorescence micrographs, showing various
sections of kidney vasculature and the amount of cells seeded
into these regions. This is a positive result when conducting
validation testing [7]. Figure 4: Cells stained with Hematoxylin
and eosin,allowing for easy observation of their proliferation
throughout the architecture of the kidney.
Conclusion:
This amalgam of different individual techniques seeks to take what has been most
effective in previous works, and to scale it to hopefully provide a basis for more successful
human works. It is quite clear that there is a shortage of kidney donors, and to establish a
standard for which kidneys can be decellularized and re-used would greatly impact this crisis.
Looking forward, this technique seeks nothing more than to improve upon current methods.
Realistically, this will not be the perfect method for kidney decellularization and
recellularization, but it should prove to be a reference point and a step in the right direction.

5. References
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[2] V. Dumont and Pauliina Saurus. “Regulation of Podocyte Apoptosis.” Internet:
http://www.hi.helsinki.fi/sannalehtonen/projects/podocyte_apoptosis.html, [14 March, 2014].
[3] D. Mangusan. “Nephrotic Syndrome – What is Nephrotic Syndrome?” Internet:
http://www.kidneyhealthcare.com/2010/07/nephrotic-syndrome.html, [10 March, 2014].
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[8] Barbara Bonandrini, et. al. “Recellularization of Well-Preserved Acellular Kidney Scaffold
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[9] Marcus Salvatori, et. al. “Regeneration and Bioengineering of the Kidney: Current Status and
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[10] Drexel University College of Medicine. “A Compendium of Drugs Used for Laboratory
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