title: Cryopreservation of mouse testicular tissue: prospect for harvesting spermatogonial stem cells for fertility preservation.
abstract: OBJECTIVE
  To investigate the efficacy of vitrification, rapid freezing, and slow freezing in preserving testicular tissue for subsequent isolation of spermatogonial stem cells.
  
  
  METHODS
  Experimental study.
  
  
  METHODS
  University-based laboratory.
  
  
  METHODS
  Immature mouse testicular tissue.
  
  
  METHODS
  The tunica of the testis was manipulated before cryopreservation. The tunica was either breached with a fine needle or completely removed, or the testis was sectioned longitudinally into halves.
  
  
  METHODS
  Cell viability by Trypan blue exclusion test and flow cytometry analysis of live-dead cytotoxicity test, measurement of hormonal production, enrichment of spermatogonial stem cells with use of magnetic-activated cell sorting technology.
  
  
  RESULTS
  Samples with tunica minimally penetrated with a needle point gave the highest cell viability after freezing and thawing. Vitrification protocol with use of an ethylene glycol-sucrose-based vitrification solution (40% vol/vol ethylene glycol-0.6 mol/L sucrose) was able to maintain postwarming cell viability and functions similar to those of noncryopreserved controls and significantly better than both conventional slow and rapid freezing protocols. Primitive spermatogonial stem cells were enriched successfully from vitrified tissue via magnetic-activated cell sorting.
  
  
  CONCLUSIONS
  Vitrification of testicular tissue is a time- and cost-efficient strategy to preserve spermatogonial stem cells for potential transplantation procedure.
authors.affiliation: National University Medical Institutes, Yong Loo Lin School of Medicine, Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore.
authors.forename: Sok Siam
authors.initials: SS
authors.lastname: Gouk
authors.forename: Yu Feng Jason
authors.initials: YF
authors.lastname: Loh
authors.forename: Srinivasan D
authors.initials: SD
authors.lastname: Kumar
authors.forename: Paul F
authors.initials: PF
authors.lastname: Watson
authors.forename: Lilia L
authors.initials: LL
authors.lastname: Kuleshova
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D002469:Cell Separation / Q000379:methods; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D019855:Ethylene Glycol / Q000494:pharmacology; D005298:Fertility; D005434:Flow Cytometry; D007248:Infertility, Male / Q000503:physiopathology / Q000628:therapy*; D007265:Inhibins / Q000378:metabolism; D008280:Magnetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D053625:Sperm Retrieval; D013093:Spermatogonia / Q000187:drug effects* / Q000378:metabolism; D013395:Sucrose / Q000494:pharmacology; D013737:Testis / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D013739:Testosterone / Q000378:metabolism; D013997:Time Factors; D046509:Tissue Culture Techniques; D058989:Vitrification
chemical_list: D003451:Cryoprotective Agents; C422364:inhibin B; D013739:Testosterone; D013395:Sucrose; D007265:Inhibins; D019855:Ethylene Glycol