Urine Is a Highly Cytotoxic Agent: Does It Influence Stem Cell

Therapies in Urology?
J. Adamowicz, T. Kloskowski, J. Tworkiewicz, M. Pokrywczyńska, and T. Drewa

ABSTRACT
The state of art of stem cell therapies in urologic regenerative medicine is still under
development. There are still many issues before advances in tissue engineering can be
introduced for clinical application. The essential question is whether stem cells should be
seeded on the urinary tract lumen side. The present experiment, using Real-Time Cell
Analyzer (RTCA) DP (Dual Plate) of the xCellligence system (Roche Applied Science,
Mannheim, Germany), allowed us to monitor cellular events in real time. In this study we
examined the influence of urine on bone marrow– derived mesenchymal stem cells (MSC).
Cells were exposed to medium mixed with urine (1:1), medium mix with PBS (Phosphate
Buffered Saline) (1:1), only urine, and whole medium without cells as background. The cell
number was significantly lower in all groups exposed on medium mixed with urine and
urine alone. The results showed that urine is a highly cytotoxic agent whose role in urologic
regenerative medicine is underestimated.

UCCESS of the urinary bladder reconstruction based penicillin, and 10 ng/ml basic Fibroblast Growth Factor (bFGF).
S on tissue engineering methods depends on cellular
compartments within the neotissue. Stem cells are respon-
Cells were grown at 36°C in 5% CO2 with 95% humidity.

sible for tissue regeneration.1 However, only a small part of
transplanted cells survive during graft remodeling.2 The
survival of transplanted stem cells is important to promote
regeneration of the muscle layer and prevent scarring.3
Urine influences all structures used for tissue engineering
of the urinary tract. Stem cells are exposed to urine until the
urothelial layer regenerates. Regeneration of urothelium
takes about a week; by this time cell-seeded grafts have
absorbed urine. In this study we examined the influence of
urine on bone marrow– derived mesenchymal stem cells
(MSC), in a rat model.
METHODS
Cell Isolation and Culture
MSC were isolated from the bone marrow of rat femurs. The rats
were humanely killed with a ketamine overdose. The experiments
were performed with the approval of the local ethics commission.
The femurs were cut on both ends and the bone core was flushed
with culture medium. The cell suspension was centrifuged for 2
minutes at 400g. The cell pellet suspended in PBS was centrifuged
again for 10 minutes at 300g. After centrifugation the cell pellet
suspended in culture medium was seeded into culture bottles
(Nunc GmbH & Co. KG, Langenselbold, German; 25 cm2). MSC
were cultured in DMEM (Dulbecco’s Modified Eagle Medium)
medium containing 10% fetal bovine serum (FBS), supplemented
with 5 ␮g/mL amphotericin B, 100 ␮g/mL streptomycin, 100 U/mL
Cytotoxicity Assay
The experiment was performed using a Real-Time Cell Analyzer
(RTCA Roche, Mannheim, Germany) DP included in an xCelli-
gence system (Roche Applied Science, Mannheim, Germany) to
monitor cellular events in real time without the incorporation of
labels. We used E-Plates 16 (Roche Applied Science) with cells
(6.25 ⫻ 103 cells/cm2) cultured in DMEM and 10% ⫻ FBS (pH ⫽
7.4). Cells were exposed to medium mixed with urine (1:1),
medium mixed with PBS (1:1), only urine, and whole medium
without cells as a background. The experiment was planed for 105
hours. Urine was added 24 hours after the cells had been seeded in
E-Plates. In each group consisting of 5 cultures there were the
following exposures: 1 hour, 12 hours, and 24 hours. A 1-day
interval was used for culture recovery and cultures were exposed to
urine again.
The urine (10 mL fresh) was obtained by single catherization of
healthy Wistar rats. The technique of catherization has been
extensively described by Oliveira et al.4
From the Department of Tissue Engineering (J.A., T.K., J.T.,
M.P., T.D.), Nicolaus Copernicus University, Bydgoszcz, and
Department of Urology, City Hospital Toruń (T.D.), Poland.
Address reprint requests to Jan Adamowicz, MD, Department
of Tissue Engineering, Nicolaus Copernicus University, Karlow-
icza 24, str., 85-090 Bydgoszcz, Poland. E-mail: adamowicz.
jz@gmail.com

© 2012 by Elsevier Inc. All rights reserved. 0041-1345/–see front matter

360 Park Avenue South, New York, NY 10010-1710 http://dx.doi.org/10.1016/j.transproceed.2012.01.128

Transplantation Proceedings, 44, 1439 –1441 (2012) 1439

1440 ADAMOWICZ, KLOSKOWSKI, TWORKIEWICZ ET AL

RESULTS
The cell number was significantly lower among all groups
exposed to medium mixed with urine or urine alone (Fig 1A,
1B, 1C, and 1D). Twenty-four– hour incubation in medium
with urine was deadly for all cells in culture (Fig 1A). The
replacement of medium with a fresh one did not produced
recovery of the cultures. The number of cells decreased
quickly after the addition of urine to the medium; The stem
cells managed to survive for 1 hour and 12 hours of incubation
with urine mixed with medium (Fig 1B and 1C). The first
exposure to pure urine that lasted 1 hour was deadly for
almost all cells in culture. Nevertheless stem cells started to
regrow in fresh medium. All stem cells did not survive a
second exposure to pure urine (Fig 1D). Stem cell culture
recovery was observed after 1 hour and 12 hours exposure to
medium with urine (Fig 1B and 1C). In these cases stem cell
growth rates in fresh medium depended on the prior exposure
time. The culture recovery was slowest after 12 hours of
incubation with medium containing urine. In the last recovery
period the number of cells was approximately 50% lower than
at the beginning of the study.

DISCUSSION
Urinary tracts are characterized by poor self-regeneration
properties of the urothelium. Scarring is a dominant pro-
cess during urinary tract healing, even after slight trauma.5
Scarring of the reconstructed hollow organs of the urinary
tract, such as the ureter and urethra, is not acceptable from
the clinical point of view. The scar closes the lumen,
reducing urine passage leading to obstructive nephropathy.
Tissue engineering offers a technology that enables com-
plete regeneration of the ureter, urinary bladder, and urethra.6
Regenerative templates, composed of biodegradable material
and stem cells, have been proven to induce urinary tract
regeneration after implantation.7 Cells play a key role to
induce urinary tract regeneration.8 Severe fibrosis always
occurs after implantation of a decellularized graft, conse-
quently leading to graft contraction in a few weeks. MSC were
chosen to be the stem cell model in this study due to their
widespread use in experimental approaches of regenerative
medicine.9 Bioengineered grafts composed of biomaterials
and MSC have shown reliable, long-lasting regenerative ca-
pacities for porcine and canine bladder augmentation com-
pared with deceleularized matrices. MSC have been shown to
be effective to prevent graft contraction and to induce smooth
muscle regeneration. MSC stimulate urinary tract regenera-
tion by several mechanisms, including release of paracrine
signals and stimulation of endogenous regeneration by recruit-

Fig 1. The influence of urine on MSC proliferation rate depend-

ing on exposition time. (A, B, C) The cultures were exposed on
urine mixed with medium (1:1). (D) The cultures were exposed on
pure urine. The yellow arrow points to the time of addition of
urine. The black arrow points to the time of replacing the medium
with fresh one. The cell number is expressed as the cell index.
STEM CELL THERAPIES IN UROLOGY
ing local resident stem cells. MSC may also modulate the
concentrations of metaloproteinases that play important roles
in remodeling of extracellular matrix within the neo-tissue.10
Fibrosis processes need to be limited during graft remodeling
to guide ingrowth of regenerating smooth muscles cells into
the implanted scaffolds. The survival of MSC within a graft
after implantation is a major demand for successful regener-
ation.11 Urinary tracts are particularly unfavorable for trans-
planted cells, because they are exposed to damaging agents
like urine.
This study reports urine to be a highly cytotoxic agent whose
role in urologic tissue engineering has been underestimated.
The observed cytotoxic effect was not specific for MSC. A
study demonstrating urine cytotoxic effects on human urothe-
lial cells was published by Davis et al.12 These results provide
evidence for a crucial role of urine in the etippathogenesis of
interstistial cystitis (IC), a disease that manifests as recurrent
pain or discomfort in the bladder and the pelvic region that
surrounds it. The exact cause of IC remains unclear, however,
its development inhibits bladder cell proliferation and renders
healing of cell layers much more difficult.
The challenging question is whether cells that have contact
with urine would be able to proliferate and support urinary
tract regeneration. Taking the results of this study into ac-
count, cells seeded on the side of the urinary tract lumen have
no chance to survive 24 hours after transplantation. It is worth
noting that the pH of cultivation medium mixed with urine was
adjusted to 7, 4, the cytotoxic properties of urine might be
greater in physiological conditions when there is an acidic pH
(it can be even 5.0). The results of the present study provide
important information when seeking to repair the urinary tract
using in vitro constructed cellular grafts. We should also ask
what is the most optimal pH for urinary tract regeneration?
Despite cytotoxic effects of urine on stem cells, urine may
also impact stem cell differentiation. Wlodarski et al observed
an interesting phenomenon following urine leakage into the
ileo-lumbalis muscle, namely, heterotorpic bone formation.
Analyzing this study, we can suppose that urine may have an
impact on the differentiation of urothelial or MSC located in
multiple niches within the urinary tract and the muscles.13
Unlike stem cells that are particularly sensitive to an
unfavorable environment in the urinary bladder, oral epi-
thelial cells survive in the urinary bladder up to 7–10
months.14 This is a consequence of the lack of protective
barrier against adverse environmental conditions. Mature
urothelial cells form uroplakin complexes as the fundamen-
tal part of the cell protection barrier separating cells from
the urinary tract lumen. Epithelialization of implanted
grafts occurrs up to 7 days after urinary bladder wall
augmentation. Complete coverage of the luminal surface of
the graft by urothelium begins as a monolayer, gradually
becoming a normal appearing, multilayered urothelium
indistinguishable from the normal bladder.
There is no need to transplant stem cells on the luminal
surface of the urinary tract. The only acceptable reason to
seed stem cells on the urinary tract lumen side is the
intention to restore the urothelial layer in patients who
1441
undergo regeneration of a bladder previously removed due
to cancer. In all other cases stem cells should be seeded only
on the external side of grafts used for urinary tract regen-
eration to reduce contact with urine during the first days
and weeks after transplantation.
Patients with invasive urothelial cancer should undergo
radical treatment that includes resection of most structures
covered with urothelium. The urotheliual layer of artificial
bladder wall replacements cannot be restored from autolo-
gus cells harvested from the host urinary tracts before
treatment. The theory of urothelial cancer development
assumes an overall change in the urothelium with many
transformed cells. The survival, proliferation, differentia-
tion, and phenotypic stability of these cells is affected by the
disease. It may be improper after transplantation leading
even to carcinogenesis in the host.
Although stem cells provide successful regeneration with-
out any special protection, we must pay more attention to
the survival of the transplanted stem cells to optimize
cell-based therapies for clinical application to reorder costs
reasonable for public health care.
REFERENCES
1. Drewa T: Using hair-follicle stem cells for urinary bladder-
wall regeneration. Regen Med 3:939, 2008
2. Burst VR, Gillis M, Pütsch F, et al: Poor cell survival limits
the beneficial impact of mesenchymal stem cell transplantation on
acute kidney injury. Nephron Exp Nephrol 11:4107, 2010
3. Kinebuchi Y, Aizawa N, Imamura T, et al: Autologous
bone-marrow-derived mesenchymal stem cell transplantation into
injured rat urethral sphincter. Int J Urol 17:359, 2010
4. Oliveira PA, Pires MJ, Nóbrega C, et al: Technique of bladder
catheterization in female mice and rats for intravesical instillation
in models of bladder cancer. Scand J Lab Anim Sci 36:5, 2009
5. Ferguson MWJ, O’Kane S: Scar free healing: from embryonic
mechanisms to adult therapeutic intervention. Phil Trans R Soc
Lond B 359:839, 2004
6. Matoka DJ, Cheng EY: Tissue engineering in urology. Can
Urol Assoc J 3:403, 2009
7. Drewa T, Adamowicz J, Lysik J, et al: Chitosan scaffold
enhances nerve regeneration within the in vitro reconstructed
bladder wall: an animal study. Urol Int 81:330, 2008
8. Drewa T, Sir J, Czajkowski R, et al: Scaffold seeded with cells
is essential in urothelium regeneration and tissue remodeling in
vivo after bladder augmentation using in vitro engineered graft.
Transplant Proc 38:133, 2006
9. Drzewiecki BA, Thomas JC, Tanaka ST: Bone marrow-
derived mesenchymal stem cells: current and future applications in
the urinary bladder. Stem Cells Int 765167, 2011
10. Ortiz LA, Gambelli F, McBride C, et al: Mesenchymal stem
cell engraftment in lung is enhanced in response to bleomycin
exposure and ameliorates its fibrotic effects. Proc Natl Acad Sci U
S A 100:8407, 2003
11. Nishijima S, Sugaya K, Miyazato M, et al: Restoration of
bladder contraction by bone marrow transplantation in rats with
underactive bladder. Biomed Res 28:275, 2007
12. Davis NF, Callanan A, McGuire BB, et al: Evaluation of viability
and proliferative activity of human urothelial cells cultured onto xeno-
genic tissue-engineered extracellular matrices. Urology 77:1007, 2011
13. Wlodarski K, Kuzaka B, Wlodarski P, et al: Is heterotopic
bone formation, occurring near a cut ureter, a sign of osteoinduc-
tive potency of human urothelium? Urol Int 55:115, 1995
14. Lu M, Zhou G, Liu W, et al: Remodeling of buccal mucosa
by bladder microenvironment. Urology 75:7, 2010