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Transport processes during cryopreservation of gametes and ovarian tissues

Subject Area Veterinary Medical Science
Term from 2014 to 2024
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 260387574
 
Final Report Year 2024

Final Report Abstract

Reproductive cells such as oocytes, sperm and embryos and reproductive tissues such as ovarian and testicular tissue used in reproductive medicine and animal breeding are only marginally stable. For long-term storage, biospecimens may be cryopreserved or freeze-dried. Such preservation processes, however, involve exposure to extreme temperatures, osmotic challenges, and dehydrating conditions. All these stress factors may result in mal-functioning of biomolecules and associated viability losses. The central aim of this project was to identify how freezing and drying affect cellular membrane properties and functioning. The following main specific aims were pursued. First, for the various steps involved in preservation of gametes, we identified the effect of the hydration level and role of membrane transport processes, i.e., movement of water and solutes across the membrane. Furthermore, we assessed permeation of protective solutes and related water fluxes involved in ovarian tissue preservation, and investigated physical properties of cryopreservation and freeze-drying formulations correlating with storage stability of biospecimens. The following important major findings were made: (1) Ice formation triggers a membrane phase transition, dependent on the nucleation temperature and cooling rate as well as presence of cryoprotective agents. (2) Freezing alters the rate of water movement across membranes as compared to suprazero conditions. (3) Cryoprotective agents modulate the rate of water transport and do not prevent freezing-induced membrane phase changes. (4) Freezing makes membranes permeable for molecules for which they are normally impermeable. (5) The latter can be used for intracellular delivery of protective disaccharides for preservation in the dried state. (6) Furthermore, we found that permeation behavior of protective agents differs in cells and tissues, and solute movement in mixtures is altered. We determined cell-specific water and solute membrane permeability parameters, for simulating cell volume responses, (un)loading of cryoprotective agents, and predicting equilibration times and optimal cooling rates. Furthermore, we developed a real-time spectroscopic approach to study permeation of solutes in mixtures and related water fluxes, and translated findings to the tissue level. We found that freezing-facilitated uptake of sucrose and trehalose effectively stabilizes sperm chromatin structure in the dried state, even under accelerated aging conditions. Storage stability of biospecimens largely depends on the glass transition temperature and related physical properties of the preserved state. Both for cryogenic and dried storage, using compounds that strengthen the viscous state and hydrogen bonding interactions increase stability.

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