Studies On The Spermatogonial Stem Cells And Leydig Cells Culture In Vitro Of Wuzhishan Porcine

The isolation and culturation of SSCs and Lc of different stage of WZSP were deaded in this study.The seminiferous cord fragment were cultured in vitro and examined under phase contrast microscopy,by this way,we have isolated and cultured SSCs and Lc of different age of WZSP successively and found a proper time of solation and culturation of SSCs and Lc.In the same time ,the cells of testes and Lc alone were be cryo-preserved as well.we found a simple and efficient way of solation and culturation of SSCs and Lc.It can be utilized as a references for the SSCs and Lc cells line.The experiment include three main part,that is:(l)The study of isolation and culturation of SSCs of WZSP in vitro. (2)The cryo-preservation of cells of porcine testes. (3) The study of isolation and culturation of Lc of WZSP in vitro.The results of the experiment described are as the following:The proper time of isolation and culturation of SSCs of WZSP is 1-20 old days of piglets.In this stage,the cells's type of testes were simple.including: SSCs Lc and Sc,different type of cells of testes can be differented easily under phase contrast microcopy; The treatment with 0.25% trypsin for 1-8 old days piglets and combination of enzymatic digestion for the two week old piglets show ideal cell dispersal .Using DMEM medium as a fundmental culture medium add different gradient at 34 °C in a water- saturated atmosphere of 95% air ,5%CO₂.The mulberry-shaped SSCs clusters appeared as original generation in 7-8 days culture,The SSCs clusters developed half-suspendedly in the culture medium; SSCs expressed AKP strong positive staining; By seminiferous cord fragment culturation can also appear SSCs clusters in 5 days; Mouse embryonic fibroblast was used as feeder layer for the SSCs passage cultured, SSCs show good attached attributes,but the number of SSCs decreased quickly.The result show SSCs can't be cultured longer time on the STO feeder layer; The Lc in testes appear about 8 old days piglets and two week old has a largest number Lc,this is the best stage of isolation and culturation of Lc of testes.Using excising and washing with PBS banlanced salt solution and colleagenase digestion ,both way can obtain higher concentration of Lc, that about 90% in all; Lc need to be cultured in the plate without Geletin, Geletin has a bad effect on Lc attach to the plate; After Lc cryo-preservation experiment,using PEF cryo-preservation mathod,the percentage of attach about 90%,but this way not for the cryo-preservation of cells of porcine testes. In addition, the testes placed in cold (4°C) PBS banlanced salt solution for 24h also can be used as a good materials for preparation of SSCs and Lc.