title: Development of a cryopreservation protocol for type A spermatogonia.
abstract: The aim of this study was to develop a cryopreservation protocol for type A spermatogonia. Testes from 5- to 7-month-old calves were collected, and type A spermatogonia were isolated using two-step enzymatic digestion and Percoll separation. Cells were resuspended in minimum essential medium (MEM) supplemented with 1% bovine serum albumin (BSA) in a final concentration of 6 x 10(6) per mL, and the effects of different cryoprotectants and freezing protocols were tested. Cells frozen/thawed in medium containing 10% fetal calf serum (FCS) and 1.4 M glycerol or dimethyl sulfoxide (DMSO) had a significantly (P <.05) higher percentage of living cells compared to medium with only FCS, whereas DMSO gave a significantly better cell survival rate than glycerol did. An increase in the concentration of FCS in the DMSO-based medium to 20% had no effect on survival after freezing and thawing. Furthermore, inclusion of 0.07, 0.14, or 0.21 M sucrose in DMSO-based medium resulted in a significant improvement of cell survival, cell proliferation in culture, and colonization efficiency in recipient testes. A controlled slow-freezing rate (1 degrees C/min) resulted in significantly (P <.05) more viable cells than fast (5 degrees C/min) freezing. However, noncontrolled-rate freezing, with a comparably low cooling rate, gave even better results than the controlled-rate slow freezing. Cryopreservation in MEM-based medium containing 10% FCS, 10% DMSO, and 0.07 M sucrose using a non-controlled-rate freezing protocol appeared to be a simple and effective way to preserve type A spermatogonia, with a high yield of almost 70% living cells after thawing. Frozen/thawed spermatogonia survived in culture and retained the ability to proliferate as determined by colorimetric and bromodeoxyuridine incorporation assays. To test whether the stem cells among the A spermatogonia retained their ability to colonize the testis of a recipient mouse, bovine spermatogonia were transplanted. This resulted in colonization 2-3 months after transplantation. In conclusion, for the first time, a method specific for cryopreservation of type A spermatogonia, including spermatogonial stem cells was developed, which allows long-term preservation of these cells without apparent harmful effects to their function.
authors.affiliation: Department of Endocrinology, Faculty of Biology, University Medical Center, Utrecht, The Netherlands. fizadyar@lab.azu.nl
authors.forename: Fariborz
authors.initials: F
authors.lastname: Izadyar
authors.forename: J J
authors.initials: JJ
authors.lastname: Matthijs-Rijsenbilt
authors.forename: Krista
authors.initials: K
authors.lastname: den Ouden
authors.forename: Laura B
authors.initials: LB
authors.lastname: Creemers
authors.forename: Henri
authors.initials: H
authors.lastname: Woelders
authors.forename: Dirk G
authors.initials: DG
authors.lastname: de Rooij
mesh_terms: D000818:Animals; D002417:Cattle; D002455:Cell Division; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D007124:Immunoenzyme Techniques; D008297:Male; D051379:Mice; D008819:Mice, Nude; D013093:Spermatogonia / Q000166:cytology* / Q000187:drug effects / Q000637:transplantation
chemical_list: D003451:Cryoprotective Agents