Abstract
Simple SummaryFreeze-drying (or lyophilization) is a method to preserve cells and tissues in which frozen material is dried by sublimation of ice. One of the main advantages is that nitrogen and dry ice are no longer required for the storage and shipment of biological material, which can be kept at room temperature or 4 °C, resulting in enormous reductions in costs. Although widely used to preserve biomolecules and macromolecular assemblies, freeze-drying of cells and tissues is currently experimental. Here, we lyophilized sheep ovarian tissue with a novel device named Darya and assessed effects on tissue integrity and gene expression. We show that ovarian tissue survives lyophilization procedures, maintaining its general structure and reacting to the different experimental steps by regulation of specific genes. Our results contribute to the optimization of protocols to freeze-dry ovarian tissues and may find application in programs of animal and human reproductive tissue preservation.Cryopreservation is routinely used to preserve cells and tissues; however, long time storage brings many inconveniences including the use of liquid nitrogen. Freeze-drying could enable higher shelf-life stability at ambient temperatures and facilitate transport and storage. Currently, the possibility to freeze-dry reproductive tissues maintaining vitality and functions is still under optimization. Here, we lyophilized sheep ovarian tissue with a novel device named Darya and a new vitrification and drying protocol and assessed effects on tissue integrity and gene expression. The evaluation was performed immediately after lyophilization (Lio), after rehydration (LR0h) or after two hours of in vitro culture (IVC; LR2h). The tissue survived lyophilization procedures and maintained its general structure, including intact follicles at different stages of development, however morphological and cytoplasmic modifications were noticed. Lyophilization, rehydration and further IVC increasingly affected RNA integrity and caused progressive morphological alterations. Nevertheless, analysis of a panel of eight genes showed tissue survival and reaction to the different procedures by regulation of specific gene expression. Results show that sheep ovarian tissue can tolerate the applied vitrification and drying protocol and constitute a valid basis for further improvements of the procedures, with the ultimate goal of optimizing tissue viability after rehydration.
Highlights
Long-term storage of biological samples in medical applications may be achieved by two different approaches: cryopreservation or lyophilization [1]
Despite various degrees of success reported in different species, which included morphological, cytological, and molecular alterations observed after cryopreservation with different approaches [31,32,33,34,35,36,37,38], ovarian tissue banking was recently accepted as a fertility-preservation technique by the American Society for Reproductive Medicine and is no longer considered experimental [39]
Relative expression of actin B (ACTB), SDHA, and ribosomal protein L19 (RPL19) in ovarian tissue subjected to lyophilization (Lio), lyophilization and rehydration (LR0h), lyophilization, rehydration and two-hour in vitro culture (LR2h), and in fresh tissue (CTR)
Summary
Long-term storage of biological samples in medical applications may be achieved by two different approaches: cryopreservation or lyophilization [1]. Cryopreservation allows preservation of cells, tissues, or any other biological construct by cooling samples to very low temperatures [2] It has become the most widely used and reliable approach to maintain cell or tissue structure and functional integrity during transportation and storage. Despite being currently considered the gold standard for the storage of active cells, it has some important limitations related to costs, as it requires a continuous and guaranteed supply of liquid nitrogen, regular maintenance, dedicated space, and trained staff; in addition, it greatly complicates sample transport and has a heavy carbon footprint due to the use of liquid nitrogen. Procedures to lyophilize ovarian tissue are still in progress and very limited data were reported to date; to our knowledge, only Lee et al described successful dry-preservation of cat ovarian tissue, with at least partial follicle survival after rehydration (2019) [40]
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