Method for separation and purification, long-term passage cultivation, cryopreservation and recovery of sheep spermatogonia stem cells

Abstract

The invention relates to a method for separation and purification, long-term passage cultivation, cryopreservation and recovery of sheep spermatogonia stem cells. The method includes the steps that various solutions are prepared, 2-3 g of tissue blocks of the seminiferous tubule are taken out from the testis of a sheep of 3-4 mouths of age, single-cell suspension is prepared through enzymolysis and digestion of two steps, purified spermatogonia stem cells are separated according to the difference adherence method, the long-term passage cultivation of the sheep spermatogonia stem cells is achieved through an SSCM culture solution, and the sheep spermatogonia stem cells are cryopreserved and recovered after the long-term passage cultivation. By the technical scheme, the sheep spermatogonia stem cells with high purity can be obtained without using specific sheep spermatogonia stem cell antibodies, a long-term passage cultivation system of the sheep spermatogonia stem cells is built, and the method for preserving the sheep spermatogonia stem cells for a long term through a liquid nitrogen freezing method is successfully provided. The sheep spermatogonia stem cells achieving separation, purification and passage according to the method are cultivated at least for more than 40 generations, and the longest cultivation period reaches four years if the cryopreservation time is included.

Description

technical field [0001] The invention relates to the field of biotechnology such as preservation of livestock germplasm resources, breeding of fine varieties, regeneration of tissues and organs, and in particular to a method for separation and purification of sheep spermatogonial stem cells, long-term subculture, cryopreservation and recovery. Background technique [0002] Spermatogonia stem cells (SSCs) are one of the most important adult stem cells. Spermatogonial stem cells not only have the genetic function of procreation, recent studies have shown that spermatogonial stem cells can easily produce pluripotent stem cells through dedifferentiation, which makes spermatogonial stem cell manipulation techniques favored by experts in life sciences, agricultural sciences, and medicine. Since the establishment of spermatogonial stem cell transplantation technology, the research on spermatogonial stem cells began to accelerate. After 2000, the long-term in vitro culture method of ...

AI Technical Summary

Problems solved by technology

In the current domestic and foreign scientific literature on the separation and purification of spermatogonial stem cells using enzymatic hydrolysis + differential attachment method, only the mechanism of action of enzymatic hydrolysis and differential attachment method is emphasized, while the details of each operation are ignored, but spermatogonial stem cells Stem cells are biologically active cells, and any external factors will have a direct impact on the separation and release, maintenance of activity, or death of cells. Therefore, referring to these scientific and technological documents, many experiments are often carried out, and it is difficult to successfully separate and purify spermatogenesis. stem cell

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