Purification And In Vitro Culture Of Spermatogenic Cells And Sertoli Cells On Adult Rabbits

In order to study the growth characteristic,cell morphologic and the relationship of spermatogenic cells and Sertoli cells, adult male rabbits` testis were used as material of experiment. The testis tissue was digested with different enzyme combinations, the production which contains spermatogenic cells and sertoli cells were treated with hypotonic shock and selective culture, through those methods, Sertoli cells were purified. Then Sertoli cells were cultured and identified. At the same time, the digesting production was treated with Percoll discontinue density gradient centrifugation and selective culture. Then, spermatogenic cells were purified. At last, the high purity spermatogenic cell was cultured single and with sertoli cell, respectively. The results of experiment were as follows:1 Both of two methods had successfully got single cell suspension. The total cells from single testis were 5.68×108 and 4.07×108, respectively, there was no significant difference between two methods (P>0.05). The round cell rate in the single cell suspension were 86.66 % and 79.21 % respectively, there was also no significant difference between two methods (P>0.05). But the live cell rate showed significant difference (95.28% VS 82.91%, P <0.05).2 The single cell suspension was treated with hypotonic shock, after that, the Sertoli cell rate was higher than before (23.31% VS 16.01%, P<0.05). But the live cell rate was lower than before (80.98% VS 95.96%,P<0.01).3 When cultured the two kinds of cell suspensions which were treated with hypotonic shock and no treated, as a result, the Sertoli cell rates in two kinds of cell suspensions were higher than before, both of which showed significant difference, (76.01% VS 23.31%, P<0.01 ) and (42.86% VS 16.01%,P<0.05), respectively. The live cell rate in the treated cell suspension was higher than before cultured, the difference was significant (88.02% VS 80.89%, P<0.05). In the untreated cell suspension, the live cell rate reduced, but the difference was not significant (90.48% VS 95.96%,P>0.05). Moreover, after cultured, the Sertoli cell rate in two kinds of cell suspensions showed significant difference (76.01% VS 42.86%,P<0.01).4 The single cell suspension was treated with Percoll discontinue density gradient centrifugation, and then, every cell line was checked, but spermatogenic cell almost existed in the cell line which was between the 25 % and 35 % Percoll. In that cell line, spermatogenic cell rate was 78.63 %. Besides spermatogenic cell, there was sertoli cell also, and the rate was 13.11 %. The total live cell rate in that cell line was 97.46 %. After selective cultured, that cell line contained spermatogonic cells and spermatid (94.37 % and 5.13 %, respectively).5 When the purified spermatogenic cells were cultured by single, they could not anchor and reproduce neither. Four days later, almost all of cells were dead. But the spermatogenic cells cultured with Sertoli cells begin reproduced after two days, they reached the highest multiplication at the seventh day. Two weeks later, all of cells were dead.