Isolation And Cultivation Of Calf Sertoli Cells And Their Effection On Lymphocytes

The purpose of the research is to select an optimal method of the isolation and culture for calf sertoli cells in vitro.Observed the activity of the sertoli cells afer cryopreservation and resuscitation. Compared the effect of both sertoli cells and their conditioned medium on the calf PBMC and mice spleen cells transformation. The results as follows:1. Collagenase I, IV and trypsinase were sequentially used to separate sertoli cells. Demonstrated digest with 1 g/L collagenase for 10 min and then 2.5 g/L trypsin for 8 min was best. This digest method taked the shortest time and obtained more cell population, the percentage of living cells was over 95%.2. 3~4 h after incubation, sertoli cells stick to wall. After 5 h, majority of spermatogonias can be removed by changing the culture medium. After 24 h, treated with 20 mmol/L Tris-HCl (pH 7.0) for 3~5 min can remove the rest spermatogenic cells. Sertoli cells basically fuse into a monolayer after 48 h. Cryopreservation sertoli cells in liquid nitrogen for 21 d, after resuscitation, sertoli cells can grow normally.3. After culturing for 3~4 d, sertoli cells have a tight junction,they mix together and fuse into membrane monolayer, granulo- material or vacuolus can be seen in cytoplasm. Bipolar corpuscula in nucleus was clearly observed after Feulgen staining. SABC show the positive staining for FasL in sertoli cells.4. The depressant effect of calf sertoli cells and their conditioned medium on calf PBMC and mice spleen cells is conspicuous. This result shows that the sertoli cells can affect lymphocytes directly or by paracrine. The research established a new co-cultural method by paving agarose between two types of cells.This research established a simple and economical method to separate and purify calf sertoli cells. This result should be the foundation for the further research of sertoli cells. Through the determination of the depressant effect of calf sertoli cell and its conditioned medium on lymphocytes, provides the experimental basis for the effect of sertoli cells in local immune privilege. The research established a new co-cultural method which provided a new conception to further the co-culture research.