Annals of Diagnostic Paediatric Pathology

96
The aim of our study was to compare isolation efficiency of
various digestive mixtures and establish proper isolation methodology
of urothelial cells. Also different variants of growth
medium and its influence on proliferative capacity of growing
cells was assessed. In the future these cells can be used for urinary
tract reconstruction using tissue engineering methods.
Methods
Primary Rabbit Urothelial Cells (PRUC) culture was established
from 8 months old male rabbit urinary bladder wall.
After ketamine (25 mg/kg m.c. i.m.) and scoline (50 mg/kg)
overdosage bladder wall sample (5 x 5 cm) was obtained. Fragment
was immersed in sterile Phosphate Buffered Saline (PBS,
Biomed). In the next step urinary mucosa was mechanically
separated from muscle layer. At this stage samples were cut
into 12 equal sized parts (1 cm 2 each). Mucosa samples were
cut into 2 mm 3 pieces.
First of all 5 different digesting mixtures for isolation
of urothelial cells were tested. Incubation time was 4 hours.
Digesting solutions were as follow:
1. 0.125% trypsin and 0.01% EDTA
2. 0.08 % trypsin and 0.006% EDTA
3. 0.06 % trypsin and 0.005% EDTA
4. 0.05% trypsin and 0.02% EDTA
5. 0.1% collagenase-I
After incubation time the cell pellet was centrifuged
(800 g/5 min), resuspended in each of the tested culture media
and seeded on 25 cm 2 culture dishes (Greiner). Cells were
identified as epithelial cells by morphological criteria as well
as presence of cytokeratins (Anti-Cytokeratin, Clone: MNF
116, DAKO), after three passages.
In the next step 2 nd PRUC was established using the
most efficient digestive solution: 0.1% collagenase I. Digestion
efficiency was assessed using two incubation times (1
and 4 hours). Cells obtained from 0.1% collagenase I digestion
after 1 and 4 hours were checked for their growth potential
in 3 different media:
I- DMEM supplemented with 10% FBS and antibiotics: penicillin
(100 IU/ml) (Polfa) and streptomycin (100 ug/ml) (Polfa)
II- 3:1 DMEM and F-12 mixture supplemented with 5% FBS,
epidermal growth factor (EGF, Sigma), bovine pituary extract
(Sigma), cholera toxin (30 ng/ml) (Sigma), penicillin (100
IU/ml) (Polfa) and streptomycin (100 ug/ml) (Polfa)
III- 1:1 DMEM and F-12 mixture supplemented with 2.5%
FBS, epidermal growth factor (EGF, Sigma), bovine pituary
extract (Sigma), cholera toxin (30 ng/ml) (Sigma), penicillin
(100 IU/ml) (Polfa) and streptomycin (100 ug/ml) (Polfa).
Cells were seeded on 25 cm 2 culture dishes (Greiner)
and grown in 37 o C in a humidified atmosphere with 5% CO 2
.
Cells were counted using trypan blue exclusion test after four
days of cultivation. Neubauer«s cytologic chamber was used
for counting. Morphology was examined under inverted microscope.
Photographic documentation was done.
Mean values (+/-SD) from 10 counts for each culture were
compared to each other. The Student test was used for statistical
analysis. A p value