[An experimental study of using chitinous membrane as the culture scaffold for epidermal stem cells].
OBJECTIVE
To investigate the feasibility of constructing a skin tissue engineering covering on chitinous membrane using rat epidermal stem cells (ESCs).


METHODS
Rat ESCs were isolated and cultured by cold digestive method and collagen type IV adherent method. Cell colonies were observed with inverted microscope. Expressions of DNA and RNA of ESCs were detected with laser scanning confocal microscope. Growth curves of cells were determined with Alamar BlueTM colorimetric method. Expressions of surface markers of ESCs (CD29, CD71, CD49d, and CD34) were detected with flow cytometer. Positive expressions of CK15, CK19, and P63 of ESCs were determined by immunohistochemistry. Influence of original chitinous membrane leachate in different dilutions on ESCs was observed. Condition of growth of ESCs on the vehicle was observed.


RESULTS
Isolated cultured cells were verified as ESCs, of which the doubling generation time was 48 hs. CD29 and CD49d were positive; CD71 and CD34 were negative; CK19, CK15, and P63 were positive. Compared with that of control group, ESCs cultured in chitinous membrane leachate showed slight cell proliferation when diluted to 1:8-1:512 dilutions, but there was no statistically significant difference (P > 0.05). The checkerboard-form cell colonies of ESCs could be visualized with naked eyes on the chitinous membrane in 2-4 weeks of culture. A multitude of ESCs were seen to grow on fibres under microscope.


CONCLUSIONS
Chitinous membrane may be used as ESCs culture vehicle, and biological compatibility is good.
Institute of Traumatic Surgery, Guangzhou Red Cross Hospital Affiliated to Jinan University, Guangzhou 510220, China.
Yan
Y
Shen
Xiao-Jian
XJ
Li
Rong
R
Liang
Yan-Cang
YC
Li
Yan
Y
Zhang
Pei-Hong
PH
Liang
Li-Bing
LB
Dai
Xiao-Hong
XH
Yang
Jian-Rong
JR
Tan
Ye-Yang
YY
Li
Yue
Y
Huang
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D022082:Cellular Structures; D002686:Chitin; D004847:Epithelial Cells / Q000166:cytology; D005260:Female; D008297:Male; D051381:Rats; D013234:Stem Cells / Q000166:cytology*; D023822:Tissue Engineering / Q000379:methods; D054457:Tissue Scaffolds
D002686:Chitin