How to isolate urothelial cells? Comparison of four different methods and literature review

RESEARCH ARTICLE

How to isolate urothelial cells? Comparison of four differentmethods and literature review

T. Kloskowski • M. Uzarska • N. Gurtowska •

J. Olkowska • R. Joachimiak • A. Bajek • M. Gagat •

A. Grzanka • M. Bodnar • A. Marszałek • T. Drewa

Received: 8 April 2013 / Accepted: 28 June 2013

� Japan Human Cell Society and Springer Japan 2013

Abstract The aim of this study is to present the comparison

of four different methods for urothelial cell isolation and

culture and compare them to methods cited in the literature.

Four different techniques were examined for urothelium

isolation from rat bladders. Isolation effectiveness was cal-

culated using trypan blue assay. Confirmation of isolated cell

phenotype and comparison with native bladder tissue was

confirmed using immunohistochemical (IHC), immunocy-

tochemical (ICC) and immunofluorescence (IF) analysis.

The method with bladder inversion and collagenase P

digestion resulted in the highest number of isolated cells.

These cells showed positive expression of cytokeratin 7, 8,

18, a6-integrin and p63. Our results and the literature review

showed that the best method for urothelium bladder isolation

is dissection of the epithelium layer from other bladder parts

and digestion of mechanically prepared tissue in a collage-

nase solution.

Keywords Urothelium � Cell isolation � Cytokeratin �P63 � a6-integrin

Introduction

Urothelium covers the surface of genitourinary tracts from

the renal pelvis to the urethra. Normal urothelium consists

of basal, intermediate and superficial cell zones. Basal cells

form a single layer connected to the basement membrane.

Their maturation and differentiation lead to the formation

of an intermediate zone comprised of variable numbers of

cell layers. The superficial cell zone contains large, highly

specialized umbrella cells that exhibit unique characteris-

tics which enable them to form a barrier between urine and

the underlying tissues, including tight junctions, specific

membrane lipids, uroplakin plaques, and glycosaminogly-

cans on the apical membrane [1].

Recent developments in urinary tract tissue engineering

indicate that the application of multilayered urothelial cells

cultured on biocompatible scaffolds may be a promising

method for the treatment of urinary tract diseases, includ-

ing urothelial cancer [2]. However, due to a lack of com-

mercially available stable urothelial cell lines, the best way

to study normal urothelium in vitro is to isolate urothelial

cells from biopsy specimens.

Although normal urothelial cells are able to proliferate

under in vitro conditions, they require specific growth

conditions in comparison to other cell types. The first

attempts to initiate urothelial cell lines began 30 years ago.

Primary urothelial cultures were established from explants,

T. Kloskowski and M. Uzarska have contributed equally to this paper.

T. Kloskowski (&) � M. Uzarska � N. Gurtowska �J. Olkowska � R. Joachimiak � A. Bajek � T. Drewa

Department of Tissue Engineering, Ludwik Rydygier Collegium

Medicum in Bydgoszcz Nicolaus Copernicus University in

Torun, Karlowicza Str. 24, 85-092 Bydgoszcz, Poland

e-mail: tomaszkloskowski@op.pl

M. Gagat � A. Grzanka

Department of Histology and Embryology, Ludwik Rydygier

Collegium Medicum in Bydgoszcz Nicolaus Copernicus

University in Torun, Bydgoszcz, Poland

M. Bodnar � A. Marszałek

Department of Clinical Pathomorphology, Ludwik Rydygier

Collegium Medicum in Bydgoszcz Nicolaus Copernicus

University in Torun, Bydgoszcz, Poland

T. Drewa

Urology Department, Nicolaus Copernicus Hospital, Torun,

Poland

123

Human Cell

DOI 10.1007/s13577-013-0070-y

which were maintained in complete culture media [3, 4].

Methods using cell scraping and proteolytic enzymes for

predigestion of fragmented tissues have also been used for

epithelial cell isolation [5]. To increase urothelial cell

isolation efficiency, researchers often combine several

techniques. There are many existing methods of urothelial

cell culture, while the lack of a standard procedure for this

purpose leads to there being many combinations in isola-

tion and cell culture methods.

The aim of this study is to present a comparison of four

different methods for urothelial cell isolation and compare

them to methods cited in the literature. Each of them

includes the use of proteolytic enzymes to dissociate the

urothelium from the basement membrane. Two different

growth media and a 3T3 feeder layer were used to assess

the influence of specific growth factors on proliferation and

characteristics of isolated urothelial cells.

Materials and methods

For isolation of bladder epithelial cells, 20 male Wistar rats

were used. All experiments were approved by the local

bioethical commission.

Isolation of epithelial cells from bladder

Each rat was euthanized with pentobarbital (15 mg/kg),

and the bladder was exposed, surgically excised, and

washed in PBS supplemented with antibiotics (amphoteri-

cin B 5 lg/ml, streptomycin 100 lg/ml, penicillin

100 U/ml). Four methods were used in order to isolate the

highest number of urothelial cells (Table 1). Each method

was tested on five bladders.

Mechanical treatment and digestion of bladder tissue

Method 1 The dissected bladder was incubated in colla-

genase I (1 mg/ml; Sigma, Germany) for 4 h. Then, the

mucosal layer was carefully scraped, washed twice in PBS

and centrifuged. The obtained cell pellet was resuspended

in culture medium, seeded on a feeder layer (3T3 cells) and

cultured in standard conditions.

Method 2 The bladder was cut into small pieces

(1 9 1 mm) and incubated for 16 h in 0.05 % trypsin

(PAA, Austria) solution at 4 �C. After that, the trypsin was

inactivated by an equal volume of medium supplemented

with foetal bovine serum.The cell suspension was filtered

through a 100-lm membrane (BD Bioscience, USA). The

obtained cells were seeded on the feeder layer and cultured

in standard conditions.

Method 3 Bladder pieces (1 9 1 mm) were incubated for

4 h in 0.1 % collagenase I solution at 37 �C. The obtained

cell suspension was filtered through a 100-lm membrane,

and isolated cells were seeded on the feeder layer and

cultured in standard conditions.

Method 4 The epithelial site inverted bladder was tied

and digested with 1 mg/ml collagenase P solution (Roche,

France) in a spinner flask for 1 h at 37 �C. The urothelium

was carefully scraped, washed in PBS, centrifuged and

seeded on a feeder layer. Cells were cultured under stan-

dard conditions.

In vitro cultivation of bladder epithelial cells

Cells were cultured in standard conditions in plastic

T-flasks at 37 �C in 5 % CO2 and 98 % humidity.

Feeder layer preparation

Mouse fibroblast cell line 3T3 (ATCC, USA) was used for

feeder layer preparation. Cell growth was inhibited in

confluent phase by the addition of 10 lg/ml Mitomycin C

(Sigma). Cells were incubated with Mitomycin C for 3 h.

This prepared feeder layer was then seeded with isolated

urothelial cells.

Culture media

Cells obtained from each isolation were cultivated in two

different growth media. The most suitable medium was

Table 1 Methods used for epithelium isolation from rat bladders

Method

number

Method description

1 Bladder dissection

Incubation in collagenase I

Scraping of mucosal layer

Centrifugation and cultivation of obtained cells

2 Cutting bladder into small pieces

Incubation in trypsin

Filtration of cell suspension

Centrifugation and cultivation of obtained cells

3 Cutting bladder into small pieces

Incubation in collagenase I

Filtration of cell suspension

Centrifugation and cultivation of obtained cells

4 Bladder inversion to epithelial site

Incubation in collagenase P solution in spinner flask

Scraping of mucosal layer

Centrifugation and cultivation of obtained cells

T. Kloskowski et al.

123

selected on the basis of microscopic analysis of cell num-

ber and their morphology.

Medium 1 DMEM/Ham’s F12 (PAA) supplemented with

0.4 lg/ml hydrocortisone (Sigma), 10 ng/ml EGF (Sigma),

100U/ml penicillin, 100 lg/ml streptomycin and 5 lg/ml

amphotericin B (PAA).

Medium 2 Progenitor Cell Targeting medium, PCT (CnT-

16), with unknown trademarks supplements (CellnTec,

Switzerland), was additionally supplemented with 100 U/

ml penicillin and 100 lg/ml streptomycin (PAA).

Confirmation of cells phenotype

Immunohistochemical/immunocytochemical analysis

(IHC/ICC)

Tissue specimens or single cell solutions were fixed with

7 % formaldehyde before analysis. Tissue slides pieces

(4 lm thick) were deparaffinized, rehydrated and washed

in distilled water. Antigenic determinants were exposed by

heating in citrate buffer (pH = 6) in microwave or EDTA

buffer (pH = 8) in a water bath. Incubation in 3 % H2O2

(RT) inhibited endogenous peroxidase activity. Non-spe-

cific binding of antibodies was blocked by the addition of

5 % BSA (Sigma). Tissue slides were then incubated with

primary monoclonal antibodies (Abcam, UK). Antibodies

were diluted by 1 % BSA solution in PBS in a ratio 1:50

for cytokeratines 7, 8, 18 and a6-integrin, as well as for p63

in the ratio 1:200. In the next stage, incubation with sec-

ondary antibodies was performed (DAKO EnVision

TM ? System Labelled Polymer HRP ? Anti Mouse;

DAKO, Denmark). Antigen–antibody complexes were

visualized using 3, 30-diaminobenzidine [DAB(?) Chro-

mogen, DAB(?) Substrate Buffer; DAKO]. Nucleus

visualization was performed using hematoxylin staining,

dehydration, radiography and closing in Canadian balsam.

Levels of analyzed markers expression was established on

the basis of the 12-point IRS scale (immunoreactive score)

by Remmele [6]. Cytokeratin 7, 8, 18 and p63 protein were

analyzed immunohistochemically, as were cytokeratin 7,

18, a6-integrin and p63 protein using immunocytochemical

analysis.

Immunofluorescence analysis (IF)

Cells cultured on glass slides were fixed with 4 % form-

aldehyde for 20 min and then washed twice in PBS. After

5 min of incubation with 0.1 % glycine, cells were washed

in PBS once again. Permeabilization was performed using

0.25 % Triton X-100 (10 min). After another washing,

non-specific binding sites were blocked by incubation with

1 % BSA (10 min) diluted in PBS. Then, cells were

incubated at room temperature for 1 h with primary anti-

body (Abcam). Antibodies were diluted by 1 % BSA

solution in PBS in a ratio 1:500 for cytokeratines 7, 8. 18,

as well as for p63 in the ratio 1:150. Antibody binding sites

were visualized using secondary antibody conjugated with

fluorophore (TRITC; Sigma). Cell nuclei were stained with

DAPI (Sigma) (10 min). Stained cells were examined

under a Nikon C1 (Japan) confocal microscope.

Results

Isolation of bladder epithelium cells

Adhesion of cell aggregates and epithelial-like cell growth

was observed after 2–3 days of culture using Method 1.

However, this method led to the isolation of a large number

of fibroblast-like cells, which after 6–7 days were domi-

nant in the culture (Fig. 1a). In consequence, the fibroblasts

displaced the epithelial cells. A small number of isolated

cells (150 ± 25 9 103) were achieved using Method 2.

After 5 days of culture, epithelial cells were not observed

despite the use of a feeder layer (Fig. 1b). Cell numbers

achieved after isolation with Method 3 were similar to

Method 1 (317 ± 34 and 275 ± 32 9 103, respectively).

In Method 3, small numbers of epithelial-like, spindle-

shaped cells were observed (Fig. 1c). The most effective

was Method 4 due to the large number of isolated cells

Fig. 1 Epithelial bladder cell culture: a Method 1, arrows show

epithelial-like cells surrounded by fibroblasts; b Method 2, lack of

epithelial cells, feeder layer is visible; c Method 3, arrows show

epithelial-like cells surrounded by fibroblasts; d Method 4, confluent

growth of epithelial-like cells. Inverted microscope, magnification

910

How to isolate urothelial cells?

123

(578 ± 56 9 103; Fig. 2). The morphology of cultured

cells indicates their epithelial character (Fig. 1d). For fur-

ther analysis, Method 4 was chosen.

Method 4 led to the isolation of single cells and cell

aggregates. Cell aggregates were often built from dozens of

cells and had an amber-gold color. On the first day of

culture, the migration of cells from aggregates and their

attachment to the culture flask surface was observed.

During the next days, cells were dividing intensively which

led to epithelial-like colony growth. Colonies were created

from small tightly packed polygonal cells. Cells which did

not adhere to culture flask surface were successfully

removed with every medium change. After 10–14 days of

culture, cells covered 80–100 % of the growth surface

(Fig. 1d).

Isolated cells were identified as normal bladder epithe-

lial cells and this type of cells could be passaged for a

maximum 5–8 times, similar to other normal cell lines.

The optimal growth medium

The results using the two different growth media were

similar. No differences in cell number and cell morphology

between the two tested media were observed.

Cell phenotype

Native bladder epithelium and isolated bladder cells were

stained with antibodies against cytokeratin 7, 8, 18 and p63

protein using IHC and IF, respectively (Fig. 3). This ana-

lysis showed expression of cytokeratin 7 and 8, charac-

teristic for bladder epithelium. In all analyzed samples,Fig. 2 Number of cells isolated using four different methods

Fig. 3 Upper (IHC) Immunohistochemical staining of native rat

bladder specimens under light microscopy; the epithelial layer and

stroma can be easily distinguished; 910 magnification; middle; (IF)

immunofluorescence staining of in vitro cultures of rat urothelial cells

after 2 passages, by laser scanning confocal microscopy; nuclei

stained with DAPI, bar 50 lm; lower (ICC) immunocytochemical

staining under light microscopy, artificial tissue made from cultured

urothelial cells mixed with alginate which served as artificial stroma;

920 magnification, bar 100 lm. a Cytokeratin 7, b cytokeratin 8 (a6-

integrin for ICC), c cytokeratin 18, d p63. Because of the lack of a

cytokeratin 8 antibody for the IC method, a6-integrin staining was

used instead

T. Kloskowski et al.

123

strong expression of cytokeratin 18 and p63 was observed

(Table 2). Immunocytochemical analysis of isolated cells

confirmed this result (Fig. 3). Additionally, expression of

a6-integrin was observed in ICC analysis (Table 2).

Results presented in this paper were obtained only for

Method 4; the experiments were not performed for the

other methods. The obtained results indicate that we have

successful isolated and cultured urothelial cells. In Meth-

ods 1–3, fibroblast populations overgrew the bladder epi-

thelium cells (Fig. 1), which is why the cells obtained from

these methods were not used in cell phenotype analysis.

Discussion

Urinary tract epithelium possesses highly regenerative

properties in response to chemical or mechanical damage

[7]. Cultures of transitional epithelium of the urinary tract

are often used during in vitro studies, as a model for the

examination of urothelial cell properties during urinary

tract regeneration and reconstruction. Lack of commer-

cially available urinary epithelial cell lines makes experi-

ments with urothelial cells extremely difficult. Presently,

the best way to study urothelium in vitro is to use cells

from primary cultures. The use of autologous urothelial

cells is limited in the case of urothelial cancers [8].

Additionally, results obtained by Subramaniam et al. [9]

showed reduced capacity for proliferation and differentia-

tion of urothelial cells isolated from abnormal bladder,

compared to normal human urothelium. To resolve this

problem, cells from different origins, like mesenchymal

stem cells from bone marrow or adipose tissue, can be used

[10].

A literature review revealed a variety of methods used

for urothelium isolation (Tables 3, 4). Briefly, three types

of methods are generally used: tissue explants, digestion

with proteolytic enzymes and bladder washing. Establish-

ing culture from tissue explants is the oldest method, but

obtaining large numbers of cells requires long culture

periods [4, 11]. An advantage of bladder washing is min-

imal invasiveness, but culture establishment is not efficient,

with the success rate only about 55 % [12]. The most

frequently used method for urothelium isolation is enzy-

matic digestion. This technique requires invasive tissue

collection for cell isolation (bladder biopsy or bladder

excision in the case of small animals like rats). Methods

used for urothelial cells isolation differ in technique and the

enzyme used for digestion. From the literature review and

our own experience, an effective procedure requires sepa-

ration of the mucosal layer from the underlying stroma,

which minimizes contamination with other cell types. In

the case of bladders obtained from small animals, because

of tissue size, inversion of the bladder with mucosa enzy-

matic digestion, followed by gently scraping cells from the

epithelial layer, seems to be the most effective method.

This method also limits the risk of contamination. The

most frequently used enzymes for tissue digestion are

trypsin and collagenase type IV. Dispase and other colla-

genase types have also been used. Our experiment showed

that trypsin is less efficient in comparison to collagenase

(type I and P) in bladder epithelium cell isolation (Fig. 2).

After cell isolation, specific growth conditions should be

preserved. Collagen type I substratum or 3T3 feeder layer

were also used to improve urothelial cell attachment and

their growth [5, 13]. The use of a feeder layer provides

proper growth of epithelial cells and protects against ter-

minal differentiation. Keratinocyte Serum-free Medium

(KSFM) was the most frequently used medium for bladder

epithelium culture. This medium protects against differ-

entiation in fibroblast-like cells. The most commonly used

medium supplements were cholera toxin (CT), epidermal

growth factor (EGF), and bovine pituitary extract (BPE)

[11, 12, 14–23] (Table 5). However, the KSFM medium

for urothelial cell isolation is an expensive solution. From

our experience, the standard medium with EGF and

hydrocortisone addition is sufficient for bladder epithelium

growth when the proper isolation method is used. In such

cases, the use of a feeder layer is necessary to increase cell

numbers and cell growth.

In this study, four different isolation methods for bladder

epithelium cell culture establishment were used. In the case

of small laboratory animals, like mouse and rat urothelium,

separation from muscle layer is very difficult, which is why

in two methods the bladder was cut into small pieces

(Methods 2 and 3). In the two other methods, cells were

isolated through gently scraping (Methods 1 and 4). These

two methods differ in the type of collagenase and the use of

a spinner flask in Method 4. Both enzymes (collagenase I

and collagenase P) have additional enzymatic activities

(clostripain, tryptic and protease activity). Differences can

be observed in enzyme activity: 0.25–1 U/mg for colla-

genase I and 1.5 U/mg for collagenase P. Higher activity of

collagenase P could be one of the reasons for better iso-

lation results. Proteolytic enzymes were used in all the

Table 2 Immunohistochemical (IHC) evaluation of bladder transi-

tional epithelium, immunofluorescence (IF) and immunocytochemi-

cal (ICC) evaluation of isolated epithelial cells in IRS scale by

Remmele

Marker IHC IF/ICC

Cytokeratin 7 12 12/9

Cytokeratin 8 8 9/–

Cytokeratin 18 12 12/12

a6-integrin – –/3

p63 12 9/9

How to isolate urothelial cells?

123

Table 3 Enzymatic methods for urothelial cell isolation: literature review

References Source Method *

Trypsin

Rahman et al. [11] Human bladder Warm trypsinization mucosa was dissected from underlying

tissues. Explants were incubated in 0.25 % trypsin in HBSS

(30 min, 37 �C, stirred). Fresh trypsin solution was added

and the flask was incubated for an additional 30 min. Cell

suspension was centrifuged (5 min, 1100 rpm) and

resuspended in medium

Petzold et al. [12] Human renal pelvis,

ureter and bladder,

Explants were incubated in 0.25 % trypsin/0.02 % EDTA

(30 min, 37 �C). Cell suspension was filtered through a

stainless steel mesh, suspended in DMEM/10 % FCS,

centrifuged (5 min, 70 g) and resuspended in medium.

Irradiated 3T3 fibroblasts were used as a feeder layer

M2

Cheng et al. [21] Porcine bladder Explants were digested in 0.25 % trypsin/EDTA (10 min,

37 �C). Cells were scraped off the luminal surface,

centrifuged (5 min, 750 g) and resuspended in culture

medium

Magnan et al. [19] Porcine bladder Inner part of the bladder was cut into small pieces. Explants

were incubated overnight (4 �C, pH 7.4) in solution

containing thermolysin in HEPES 19 buffer with 1 mM

CaCl2. Urothelial cells were scraped off and incubated in

trypsin solution (37 �C, 30 min with agitation). Cell

suspension was filtered through 200 lm cell-strainer,

centrifuged (10 min, 1180 rpm) and resuspended in culture

medium. Irradiated fibroblasts were used as a feeder layer

Woodman et al. [22] Human bladder Mucosa was dissected from underlying tissues, washed and

incubated in 0.25 trypsin/EDTA solution (5–10 min, 37 �C).

Cells were suspended in medium, filtered through a 100-lm

nylon cell strainer and centrifuged (10 min, 4 �C, 428 g)

Shi et al. [23] Human ureter Inner part of ureter was separated from mucosa and connective

tissues and cut into small pieces. Explants were treated with

0.05 % trypsin (20 min, RT). Enzyme was inactivated and

cell suspension was filtered through 100-lm nylon mesh,

centrifuged (5 min, 1000 rpm) and resuspended in culture

medium

Trypsin and other enzymes

Rahman et al. [11] Human bladder Cold trypsinization. Explants were incubated in 3 % trypsin

and 1 % pancreatin in HBSS (2 h, 4 �C and 30 min, 37 �C).

Cells were separated from the tissue by gentle pipetting. Cell

suspension was centrifuged (5 min, 1100 rpm) and

resuspended in medium

Truschel et al. [14] Rabbit bladder Mucosa was incubated in MEM (overnight, 4 �C) containing

2.5 mg/ml dispase. Scraped cells were incubated in 0.25 %

trypsin/1 mM EDTA solution (15–30 min, 37 �C),

resuspended in MEM and centrifuged (5 min, 1000 rpm).

Cells were washed two times in MEM and then resuspended

in KM

Kurzrock et al. [25] Rat bladder Inverted bladder was placed in 0.05 % trypsin/0.53 EDTA or

1 % collagenase IV solution in a spinner flask and stirred for

1 h (37 �C, 5 %CO2). Bladders were gently scraped to

remove urothelial cells. Cell suspension was centrifuged

(5 min, 1000 rpm), resuspended in culture medium and

seeded on the 3T3 feeder layer

M4

Collagenase

Sugasi et al. [15] Human bladder,

ureter

Urothelium was separated from the stroma and cut into small

pieces. Explants were digested in collagenase IV (200 U/ml)

for 2 h in shaking water bath (37 �C). Cell suspension was

centrifuged (5 min, 1000 rpm), rinsed in HBSS and

centrifuged for the second time. Finally, cells were

resuspended in culture medium

M3

T. Kloskowski et al.

123

methods. Drewa et al. [24] showed that collagenase is an

effective enzyme for bladder epithelium isolation. These

findings are consistent with our results showing the

advantage of collagenase over trypsin (Fig. 2). Enzymatic

digestion with scraping after bladder inversion turned out

to be the most effective method. The use of a spinner flask

Table 4 Non-enzymatic methods for urothelial cell isolation: literature review

Study Cells origin Method for cells isolation

Explants

Kirk [33] Human bladder, ureter Explants were seeded on culture dishes (20 per dish)

Dubeau and Jones [34] Human bladder Explants were placed on culture dishes coated with R22CIF

Kreft et al. [4] Mice bladder Urothelium and lamina propria were separated from muscle

layer. Mucosa was transferred on culture inserts with porous

membranes placed in culture plates

Bladder washing

Fossum et al. [37] Human bladder Bladder irrigation fluids were centrifuged (10 min, 1500 rpm).

Cells were resuspended in culture medium, centrifuged

second time (5 min, 1500 rpm) resuspended in culture

medium and seeded on J2 feeder layer

Nagele et al. [2] Human bladder Bladder irrigation fluids were centrifuged (5 min, 250 g) and

resuspended in culture medium

R22CIF rat smooth muscle matrix, J2 mouse fibroblast cell line

Table 3 continued

References Source Method *

Zhang et al. [16] Rat bladder Inverted bladders were incubated in 1 % collagenase IV

(60 min, 30 �C, shaker) or in 0.1 % EDTA (4 h, 4 �C).

Urothelial cells were scraped, washed, and resuspended in

KSFM on Primaria cell culture flasks. Irradiated 3T3

fibroblasts were used as a feeder layer

M4

Southgate et al. [17] Human renal pelvis,

ureter and bladder

Explants were placed in stripping solution (1 M HEPES buffer

pH 7.6, 20 KIU aprotinin, 50 ml of 1 % EDTA, 500 ml

HBSS without Ca2?and Mg2?) overnight at 4 �C or 4 h in

37 �C. Urothelium was separated from underlying stroma,

centrifuged (4 min, 250 g) and incubated in collagenase

(100 U/ml) for 20 min in 37 �C. After addition of KSFM cell

sheets were disaggregated by gentle pipetting, centrifuged

and resuspended in culture medium

M1

Collagenase and other enzymes

Adelow and Frey [20] Human bladder Explants were digested in Liberase Blendzyme�-mixture of

collagenase and protease enzymes (1–2 h, 37 �C). Cell

suspension was filtered through 70-lm cell strainer,

centrifuged (5 min, 174 g) and suspended in culture medium.

Urothelial cells were isolated using epithelial beads (EpBer4

conjugated) and magnetic separator

Liao et al. [35, 36] Rabbit bladder Urothelium was gently scraped from explants. Obtained tissue

was digested using collagenase I and elastase

Dispase

Fraser [18] Porcine bladder Excess stroma was removed from full-thickness section.

Urothelium was separated by incubation in 2 % dispase II in

HBSS (16 h, 4 �C). Urothelial sheets were incubated in 100

U/ml collagenase type IV (20 min, 37 �C) and disaggregated

by pipetting. Cells were suspended in KSFM and seeded into

Primaria� tissue culture flasks

HBSS Hank’s balanced salt solution, FCS fetal calf serum, RT room temperature, MEM minimal essential medium, KM keratinocyte medium,

3T3-mouse fibroblast cell line, KSFM keratinocyte serum-free medium

* Methods similar to those used in our study (M1 similar to Method 1)

How to isolate urothelial cells?

123

enhances the isolation effectiveness by a more gentle and

efficient method of epithelial cell detachment from the

basal membrane and reducing contamination by fibroblasts.

A similar method of cell isolation was presented by Ku-

rzrock et al. [25]. Isolated cells were seeded on a 3T3

feeder layer what is a popular method during primary

epithelial culture establishment [26, 27]. The use of a

feeder layer allows for the adoption of epithelial mor-

phology by isolated cells. It can be assumed that the 3T3

feeder layer was this factor, which increased cell adhesion

and was a source of signals determining proper morphol-

ogy of the bladder epithelium [25, 28, 29]. A medium

without serum was chosen for selective proliferation of

epithelial cells [4] to avoid culture contamination with

fibroblast-like cells [28, 30]. Unfortunately, no percentage

of urothelial and fibroblasts cells was estimated just after

each isolation. However, in Method 4, the percentage of

fibroblasts was the lowest in in vitro culture. In other

methods, the number of fibroblasts exceeded the number of

epithelial cells (Fig. 1). The presence of other cell types

was not observed in any method during long-term in vitro

culture.

The epithelial nature of isolated cells was confirmed

using immunofluorescence and immunocytochemical

analysis. The examined cells expressed cytokeratin 7, 8,

18, p63 protein and a6-integrin, which are characteristic

markers for bladder transitional epithelium. The presence

of cytokeratin 7 in the urothelial basal layer and cytoker-

atin 8 and 18 in the umbrella cells have been previously

proved and were confirmed in our study [3, 31, 32].

Conclusion

The development of an effective method for urothelial

cell isolation provides a safe and rich source of cells for

in vitro experiments. Stable cell lines can be used to study

normal urothelium and the processes of pathogenesis and

cell responses to exogenous signals. Isolation of urothelial

cells is also the first step that opens the way for the

regeneration and reconstruction of urinary tracts by tissue

engineering.

Acknowledgment This work was supported by research task within

framework of the statutory activities no. 585 from Nicolaus Coper-

nicus University.

Conflict of interest The authors declare that they have no conflict

of interest.

Table 5 Comparison of media used for urothelial cell culture: literature review

Medium type Supplements References

KSFM EGF, FGF, Insulin [23]

BPE, EGF, CT [2, 15–18, 20]

BPE, EGF [35, 36]

DMEM/Ham’s F12 FCS, EGF, CT, Hydrocortisone, Transferrin, Liothyronine,

Adenine

[12]

FBS, EGF, CT, Insulin, Hydrocortisone [19]

FBS, CT, Insulin, Hydrocortisone, Transferrin, Adenine,

Triiodothyronine

[37]

RPMI 1640 FBS, EGF [21]

FBS, Fungazone, Glutamine, HEPES buffer [22]

HMRI-2 BPE, EGF, Insulin, Hydrocortisone, Transferring,

Ethanolamine, Phosphoethanolamine

[33]

DMEM/MCDB 153 (1:1) EGF, CT, Insulin, Hydrocortisone, Ethanolamine,

Phosphoethanolamine, Adenine, MCDB vitamins, MCDB

trace elements, CaCl2

[34]

MCDB153/advanced-DMEM (1:1) Insulin, Hydrocortisone, Adenine, Ethanolamine,

Phosphoethanoamine, Ca2?[4]

M-199 FBS [11]

FGM and KGM ? (1:1) resuspension

and first medium change

FGM: FBS, DMEM, L-glutamine [25]

KGM ? second or third medium change KGM?: FBS, BPE, EGF, CT, Insulin, Hydrocortisone

AMEM BGS [20]

EGF epidermal growth factor, FGF fibroblast growth factor, CT cholera toxin, BPE bovine pituitary extract, FCS fetal calf serum, FBS fetal

bovine serum, BGS bovine growth serum, KSFM keratinocyte serum free medium, AMEM alpha minimal essential medium, FGM fibroblast

conditioned growth medium, KGM? augmented keratinocyte growth medium

T. Kloskowski et al.

123

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How to isolate urothelial cells?

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