[Experimental study on culturing Schwann cells of rats by single-enzyme digestion and explant-culture method].
OBJECTIVE
To establish an efficient method of isolating and culturing high activity and high purity of Schwann cells, and to identify the cells at the levels of transcription and translation.


METHODS
The sciatic nerves harvested from a 4-week-old Sprague Dawley rat were digested in the collagenase I for 15 minutes after dissecting, and then the explants were planted in culture flask directly. The cells were cultured and passaged in vitro, the growth state and morphological changes of the cells were observed under inverted phase contrast microscope. MTT assay was used to test the proliferation of cells and the cells growth curve was drawn. RT-PCR and immunohistochemistry staining were used to detect S100 and glial fibrillary acidic protein (GFAP) at the levels of transcription and translation, respectively. The purity of cells was caculated under microscope.


RESULTS
After the digestion of collagenase I, fibroblast-like cells appeared around explants within 24 hours, with slender cell body and weak refraction. After tissues were transferred to another culture flask, a large number of dipolar or tripolar cells were seen after 48 hours, with slender ecphyma, plump cell body, and strong refraction, and the cells formed colonies within 72 hours. The cells were covered with the bottom of culture flask within 48-72 hours after passaging at a ratio of 1 : 2, and spiral colonies appeared. Cells showed vigorous growth and full cytoplasm after many passages. MTT assay results showed that the cells at passage 3 entered the logarithmic growth phase on the 3rd day, reached the plateau phase on the 7th day with cell proliferation, and the growth curve was "S" shape. RT-PCR results showed that the cells expressed S100 gene and GFAP gene, and immunohistochemistry staining showed that most of the cells were positively stained, indicating that the majority of cells expressing S100 protein and GFAP protein. The purity of Schwann cells was 98.37% +/- 0.30%.


CONCLUSIONS
High activity and high purity of Schwann cells can be acquired rapidly by single-enzyme digestion and explant-culture method.
Department of Trauma Orthopedics and Hand Surgery, First Affiliated Hospital of Guangxi Medical University, Nanning Guangxi 530021, PR China.
Yiwu
Y
Qin
Jinmin
J
Zhao
Wei
W
Su
Xiaofeng
X
Li
Shixing
S
Luo
Junting
J
Liu
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D049109:Cell Proliferation; D002469:Cell Separation; D002478:Cells, Cultured; D003470:Culture Media; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D009418:S100 Proteins / Q000378:metabolism; D012583:Schwann Cells / Q000166:cytology* / Q000378:metabolism; D012584:Sciatic Nerve / Q000166:cytology*; D023822:Tissue Engineering / Q000379:methods
D003470:Culture Media; D005904:Glial Fibrillary Acidic Protein; D009418:S100 Proteins