Application of Accutase enzymes in the separation of spermatogonial stem cells / 中国组织工程研究

ABSTRACT

BACKGROUND:It is reported that the cellsurface antigen may be damaged by the trypsin enzyme in some extent and the cellactivity may also be influenced, as a result of which, the subsequent separation and fol ow-up-test wil be affected. Accutase enzymes possess the activities of protease and col agen enzyme and need no special termination or cleaning at the end of digestion. Another special function of accutase enzyme is to protect cellsurface antigen and thus it has been widely applied in the stem celldigestion and culture. OBJECTIVE:To compare the digestive effect of accutase enzymes and trypsin enzyme in the separation and original culture of spermatogonial stem cells. METHODS:The testes of 5-7 days male Kunming mice (n=45) were col ected and primarily digested with col agenas. The suspension of digested tissue was divided into three parts with the same volume named accutases enzyme group, trypsin enzyme group and mixed enzyme group (trypsin enzyme and hyaluronidase). For the comparison of testis digestive status and the time required for the formation of single cells, the micrographs were taken at 1, 3 and 5 minutes respectively after enzyme digestion. The total number and the mortality of the single cells were estimated and compared. The leydig cells and sertoli cells were removed by differential adherent method and the remained spermatogenic cells were then treated with CD90.2 immune magnetic beads. The selected spermatogonial stem cells were subsequently labeled by glial cellline-derived neurotrophic factor receptor alpha-1 and sorted with flow cytometry. The number of spermatogonial stem cells positive for glial cellline-derived neurotrophic factor receptor alpha-1 in CD90.2+and CD90.2-cells was compared within different groups. RESULTS AND CONCLUSION:The digestive capacities of different enzyme were different. Accutases enzyme obtained the single cellsuspension more quickly than trypsin enzyme and mixed enzyme, and had the least cellmasses and broken cellmembrane than the other two groups. After the differential attached treatment, the highest total number of CD90+spermatogonial stem cells and lowest cellmortality could be found in the accutases group, when compared with the other groups. The results of cellsorting by flow cytometry showed a higher rate (72.24%) of GFRα1+/CD90+cells in the accutases group than the trypsin group (51.16%) and mixed enzyme group (71.27%). The GFRα1+/CD90-number in the accutases group was lower (15.03%) than that of the trypsin group (18.8%) and mixed enzyme group (24.23%), respectively. The results indicate a better effect of accutases enzyme on the primary separation of spermatogonial stem cells than that of trypsin or mixed enzyme.