Isolation, Purification And Culture Of Spermatogonia In Human

Object:1.To research the effect methods of Separation and purification of human spermatogonia.2.To study the effects of growth factors on the survival and proliferation of human spermatogonium stem cell (SSCs) in vitro.Methods:1.Using two-step enzymatic digestion to prepare germ cells suspension,then the germ cells suspension was centrifuged by Percoll discontinuous density gradient centrifugation. Finally,purificated human spermatogonia was obtained according to different time and times of cellular adhesiveness (per2h,3h,4h and one time or times) .2.Different concentrations of growth factors were added to culture. Each factor was divided into four groups, repeated three times and observed the survival time and proliferation of cells in every eight to twelve hours. Light microscope and electron microscope were applied to observe the appearance and morphological features of SSCs.Results:1.The percentage of spermatogonia only after separated by percoll was 48.5%,and the percentage of spermatogonia of purificated one time by interval 2,3,4h was 51.3%,59.0%,56.4% respectively;2.The percentage of spermatogonia was 57.7%,67.8%,57.8% respectively through 2,3,4h interval and continuous 2 times ;The highest of percentage of spermatogonia were interval of 3h;3.Compared with purificated one time,the percentage of spermatogonia interval of 2,3h and purificated two times were higher than that of one time;But as far as interval of 4h,there was no different between purificated two times and one time.4.After treatment with different concentrations of SCF,LIF and bFGF,the survival time of cells were significantly longer than the control group (P<0.05). Conclusions:1.Percoll centrifugation combined with different time of cellular adhesiveness can be effectively used to separate and purify the human spermatogonia.2.The percentage of spermatogonia of separation of 2,3h and purificated two times were higher than that of one time;3.If purificated two times,interval 3 h was most effective.4.The growth factors such as SCF, LIF and bFGF could promote the survival and proliferation of cells in vitro.