METHOD OF PREPARATION OF TISSUE ENGINEERING AND CELL CULTIVATION
COLLAGEN BY ACID EXTRACTION OF CALF SKIN
L. V. Kukhareva,1 I. I. Shamolina,2 E. V. Polevaya 1
1 Institute of Cytology RAS and 2 Laboratory of Ecological Chemistry and Biotechnology,
State University of Technology and Design, St. Petersburg;
e-mail: kochka-0734@mail.ru
Seven methods of preparation of intact native collagen with telopeptides by acid extraction of calf skin have
been compared; the hide was first dehaired by original mild enzymatical method using Bacillus licheniformis
protease. The noncollagenous proteins and proteoglycans were previously removed by different ways and collagen
was extracted by acid solvents and purified by salt precipitation. The dynamic of noncollagen impurities removing
was followed by noncollagen proteins and hexuronic acids analysis in extracts. The purity of the resulting
collagen was determined by polyacrilamide gel electrophoresis. The gel-forming capacity of the collagen
was determined and the suitability of the product for tissue engineering was estimated by cultivation of fibroblasts
and cardyomyocytes of new-born rats on collagen gells. All collagens obtained were not cytotoxic and had
good gel-forming capacity. The preparation method with noncollagen impurities removing with 0.02 M K2HPO4
and collagen solution with 0.5 M acetic acid and 5 mM EDTA proved to be the best by final yield of the product
and cell reaction to it. Hence, the optimal variant of collagen preparation method has been developed. The Russian
Federation patent on this method was taken out.
Key words: tissue engineering, collagen, collagen preparation, collagen fibril structure, cells in collagen gels
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