Isolation and recognition of spermatogenic cells lineages from mouse testis tissue
Introduction: This study was design to evaluate different method for separation and purification of spermatogenic cell and introduce a simple and cost-effective method. The main objective of this study is to compare the quality and effectiveness of Sedimentation Velocity at Unit Gravity (SVUG) with Purcell Density Gradient Centrifugation Method in separation of testis spermatogenic cells. Materials and Methods: In this study, the testes from male mice (NMRI strain) were obtained and transferred to Ham's F10 medium. By using of mechanical (scissors, forceps and needles) and enzymatic digestion method, (collagenase, trypsin and DNase) spermatogenic cells were isolated. Then the cell isolation done with Percoll gradient centrifugation method and sedimentation velocity at unit gravity (SVUG) method on the base of Human Serum Albumin (HSA) gradients and the May-Grunwald-Gimsa (MGG) staining method was used to identify isolated cells. Results: Our results show that enzymatic digestion after mechanical dissection is effective process for preparation of cell suspension, with this procedure most of spermatogenic cells were retrieved and protected the cell viability. Percoll gradients isolate the spermatogenic cells from sperms, tissue debris, dead cells and erythrocytes and aggregated cells. By SVUG method, the spermatogenic cells isolate as single cells and distribute in HSA gradients. MGG staining is a suitable method for spermatogenic cells identification and the different cells identified base on morphological criteria and staining properties. Conclusion: Applications of different methods are necessary for spermatogenic cells isolation and identification of best method in order to study of spermatogenesis process and its control pathways. Perhaps the combined method could be able to isolating more pure fraction of spermatogenic cells. It has potential to use as a simple and effective method to isolated spermatogenic cells as diagnostic or research propose.