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• W1985133035 abstract "Cryopreservation of spermatozoa is routinely performed as a standardized procedure in many laboratories. Because of its typical structural character, the spermatozoon requires only a mild cryoprotectant such as egg yolk and simple freezing procedure utilizing nitrogen vapors. With the refinement of treatment for azoospermia that involves isolation of testicular tissue, this also has been subjected to such cryopreservation. However, the different character of testicular sperm membranes, a larger cytoplasmic component, and the coexistence of germ cells at different maturational stages may make them more prone to cryo damage. Vitrification has been proposed as a method to minimize this, specifically crystallization, and at the same time reducing the length of the procedure. In this work, we examined the utility of vitrification as a way of cryopreserving mammalian testicular tissue. Minced mouse testicular tissue was processed by vitrification or standard egg yolk freezing. In order to assess whether the integral structures of seminiferous tubules, testicular samples were also vitrified as micro-dices. End term yardsticks were cell survival, integrity, morphology, and motility. A cell surface marker was used to confirm germ cell membrane integrity. Testes were retrieved from a 19 week old B6D2F1 mice in HTF medium divided into three sections: minced and processed for egg yolk cryopreservation, minced and processed by vitrification, or first shaped in micro-dices of 3 mm3 before vitrification. To establish a baseline, all standard parameters were confirmed in all three groups prior to cryopreservation. Standard cryopreservation was performed by adding v/v in egg yolk solution, exposure to vapors and plunging in liquid nitrogen, with this procedure taking 60 min. Thawing was performed by gradually warming to room temperature in 30 min. Vitrification of minced tissue and tissue squares was carried out in increasing concentrations of ethylene glycol over a period of 7 min. Thawing of vitrified tissue was performed by adding decreasing concentrations of sucrose for a total duration of 5 min. Post-thaw analysis included cell concentration, cell integrity by direct visualization under oil and TestSimplets™ staining and eosin-nigrosin test. Thawed micro-diced tissue was finally minced prior to evaluation. To confirm membrane integrity, germ cells were stained with a monoclonal antibody against integrin-α-6. Three male mice were sacrificed and their six testes assigned to standard, vitrification, and diced-vitrification groups. Vitrification proved to be the most expeditious as a method for cryopreserving testicular tissue, this resulting in a higher degree of germ cell integrity (P < 0.05). However, when the vitrified micro-dices were assessed, the concentration, viability, and number of intact germ cells were higher (P < 0.05) than the minced tissues independently from the cryopreservation methods. Vitrification can improve the cryopreservation of germ cells in mouse testicular tissue and provides a higher level of cell integrity. Independently of the method used, the cryostorage of testicular tissue as micro-dices appeared to favor both the cell integrity and viability of the immature germ cells." @default.
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• W1985133035 date "2004-09-01" @default.
• W1985133035 modified "2023-09-28" @default.
• W1985133035 title "Vitrification of testicular tissue is more gentle on germ cells" @default.
• W1985133035 doi "https://doi.org/10.1016/j.fertnstert.2004.07.483" @default.
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