Isolated culture method of functional Chinese softshell turtle ovarian granular cells

By using specific culture media and digestion methods, the problem of unsuitable culture media for Chinese soft-shelled turtle granule cells was solved, enabling efficient isolation and passage culture of Chinese soft-shelled turtle granule cells, thus improving reproductive efficiency and providing technical support for reproductive development research.

CN121896153APending Publication Date: 2026-04-21ZHEJIANG WANLI UNIV
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Patent Information

Application Number
CN202610057500.1
Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Filing Date
2026-01-16
Publication Date
2026-04-21

AI Technical Summary

Technical Problem

In the existing technology, chicken granule cell culture medium is not suitable for Chinese soft-shelled turtles, resulting in a high mortality rate of Chinese soft-shelled turtle granule cells. Furthermore, the isolation and in vitro culture of Chinese soft-shelled turtle granule cells are time-consuming and difficult to alleviate, thus affecting the reproductive efficiency of Chinese soft-shelled turtles.

Method used

A complete culture medium consisting of DMEM, 15% fetal bovine serum, 1% penicillin-streptomycin, and 1% insulin-transferrin-selenium additive, combined with 2 mg/ml type II collagenase digestion and trypsin digestion, was used to isolate and passage Chinese soft-shelled turtle ovarian granulosa cells, eliminating the need for pre-coating with rat tail collagen.

Benefits of technology

This study achieved efficient isolation and passage culture of Chinese soft-shelled turtle granule cells, maintaining cell functionality, reducing animal sacrifice, improving the reproductive efficiency of Chinese soft-shelled turtles, and providing an in vitro technology platform for reproductive development research.

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Abstract

The invention relates to an isolated culture method of functional Chinese softshell turtle ovarian granular cells. The isolated culture method comprises the following steps: S1, collecting ovarian tissues: collecting Chinese softshell turtle ovarian tissues; s2, separating and collecting a granular cell layer; s3, digesting and collecting granular cells; s4, primary culture of granulosa cells: transferring the collected cells into a cell culture bottle containing 4 ml of a complete culture medium for primary culture, wherein the complete culture medium comprises the following main components: a DMEM culture medium, 15% of fetal calf serum, 1% of penicillin streptomycin and 1% of an insulin-transferrin-selenium additive; and S5, subculturing the granular cells. The isolated culture method and the culture medium system provided by the invention are simpler and more time-saving and labor-saving; according to the method, the aromatase CYP19A1 can be expressed normally, the characteristics of the granular cells are kept, the aromatase CYP19A1 can be expressed normally, it is indicated that the in-vitro cultured cells can keep the functionality of the cells, and the in-vitro proliferation capacity of the granular cells is improved to a great extent.
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