Abstract
Spermatogonial stem cell (SSC)-based technologies may have application for preserving and/or restoring male fertility. We evaluated the recovery of stem and progenitor spermatogonia from frozen/thawed testicular cell suspensions versus testicular tissue fragments frozen at a controlled rate (CR) or by vitrification. Laboratory study using human tissue. Human testicular tissue was obtained through the Center for Organ Recovery and Education with approval from the University of Pittsburgh Institutional review board (#0506140). Testicular tissue was digested with enzymes to produce a cell suspension and frozen at CR or minced into large (6-10 mm3) or small (3-5 mm3) testicular tissue fragments and frozen at CR or by vitrification. The efficiency of each technique was analyzed by immunocytochemistry (ICC) for the spermatogonia marker UTF1 and human to nude mouse xenotransplantation. Cryopreservation method had a significant effect on the recovery of UTF1 positive spermatogonia per gram of tissue (p<0.001). Large testicular tissue fragments frozen at CR had the greatest number of UTF1 cells/g tissue (14.9±2.2), followed by CR small (10.4±1.3), vitrified small (5.4±0.9), vitrified large (4.4±0.5), and frozen/thawed cell suspension (1.5±0.2). The difference in UTF1 positive cell recovery from CR large tissue fragments versus CR cell suspension was statistically significant (14.9 vs. 1.5, p=0.027). Human to nude mouse xenotransplants confirmed that recovery of transplantable SSCs (colonies/g tissue) from CR small (1045.9±240.6) and CR large (570.0±147.1) testicular tissue fragments was greater than the CR cell suspension (62.6±12.3, p<0.05). Recovery of transplantable stem cells from vitrified small (562.4±186.0) and vitrified large (75.4±33.2) testicular tissue fragments was not significantly greater than the CR cell suspension (p>0.05). CR freezing of testicular tissue fragments (large or small) leads to the greatest recovery of UTF1 positive spermatogonia and transplantable SSCs. Recovery of SSCs from vitrified small testicular tissue fragments was also very good. Freezing and thawing of intact testicular tissue is compatible with several downstream applications, including testicular tissue grafting, testicular tissue organ culture or digestion with enzymes to produce a cell suspension for SSC transplantation.
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