Primary cultures from rat vibrissae as a potential cell source for in vitro construction of urinary bladder wall grafts.
BACKGROUND
In vitro-constructed grafts can be used for human bladder augmentation. There are many diseases in which autologous cells cannot be used for this purpose. The aim of the present study was to examine the potential of rat vibrissae hair follicle cells to form cultures, which could serve as a source for in vitro creation of urinary bladder wall grafts.


METHODS
Two hundred vibrissae were excised from young Wistar male rats. Two different digestions were performed, in dispase and in collagenase. All follicles were additionally incubated in trypsin and ethylenediamine tetraacetic acid. Two different culture media based on DMEM (Dulbecco's Modified Eagle's Medium) were used: the first was supplemented with keratinocyte growth factor (KGF) and the second with epidermal growth factor. Immunocytochemical detection of cytokeratin, CD34, p63, Ki-67 (proliferation index), and HMB45 (Human Melanoma Black 45) was performed.


RESULTS
Forty-eight primary cultures of rat follicle vibrissae cells were established from 200 hair follicles (24% successful rate). Twenty-four primary cultures were obtained after dispase digestion and 24 after collagenase treatment. Each group was cultured in 2 different media. A heterogeneity of primary cultures was observed. Cells formed a monolayer within a period of 2 to 4 weeks. The 24 primary cultures established after dispase treatment exhibited monolayers of small cuboid cells expressing cytokeratin and CD34. In the 40th passage 20%-40% of cells expressed p63; 85% of these cells from late passages were positive for Ki-67, indicating preserved mitotic potential. Epithelial-like phenotype was observed after dispase digestion and cultivation in KGF-supplemented medium. After 3 weeks, the morphology of these cells changed into fibroblast-like. These cultures were negative for CD34. Fibroblast-like cell growth was observed after collagenase treatment in both KGF- and EGF-supplemented media. These cells were positive for the melanocyte cell marker (HMB45).


CONCLUSIONS
Culture media and isolation conditions influence hair follicle stem cell differentiation. The stem cell niche within the hair follicles is a reservoir of cells, which can be potentially used for in vitro creation of urinary bladder wall grafts.
Drewa
T
T
Department of Tissue Engineering, CM UMK, Bydgoszcz, Poland. tomaszdrewa@wp.pl
Joachimiak
R
R
Kaznica
A
A
Sarafian
V
V
Sir
J
J
D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism; D018929:Cell Culture Techniques; D004815:Epidermal Growth Factor / Q000494:pharmacology; D006801:Humans; D007633:Keratins / Q000378:metabolism; D008297:Male; D008938:Mitosis; D051381:Rats; D017208:Rats, Wistar; D001743:Urinary Bladder / Q000601:surgery*; D014738:Vibrissae / Q000166:cytology* / Q000187:drug effects / Q000637:transplantation*
D018952:Antigens, CD34; D004815:Epidermal Growth Factor; D007633:Keratins