Stepped vitrification technique for human ovarian tissue cryopreservation

Abstract

The advantage of stepped vitrification (SV) is avoiding ice crystal nucleation, while decreasing the toxic effects of high cryoprotectant concentrations. We aimed to test this method for human ovarian tissue cryopreservation. Ovarian cortex was taken from 7 fertile adult women. Samples were subjected to an SV protocol performed in an automatic freezer, which allowed sample transfer to ever higher concentrations of dimethyl sulfoxide (DMSO) as the temperature was reduced. Histological evaluation of the vitrified-warmed tissue showed large numbers of degenerated follicles after 24 hours of in vitro culture. We therefore evaluated DMSO perfusion rates by X-ray computed tomography, ice crystal formation by freeze-substitution, and cell toxicity by transmission electron microscopy, seeking possible reasons why follicles degenerated. Although cryoprotectant perfusion was considered normal and no ice crystals were formed in the tissue, ultrastructural analysis detected typical signs of DMSO toxicity, such as mitochondria degeneration, alterations in chromatin condensation, cell vacuolization and extracellular matrix swelling in both stromal and follicular cells. The findings indicated that the method failed to preserve follicles due to the high concentrations of DMSO used. However, adaptations can be made to avoid toxicity to follicles caused by elevated levels of cryoprotectants.

Coaxial Alginate Hydrogels: From Self-Assembled 3D Cellular Constructs to Long-Term Storage

“…So far, cryopreservation remains the only clinically feasible option enabling long-term storage of native tissues and tissue-engineered constructs (TECs) using either slow freezing [38] or vitrification [39,40] approaches. Although being advantageous in the ice-free preserving of a wide range of biological objects, vitrification often fails to preserve relatively big and complex biological objects due to an increased cryoprotective agent (CPA) toxicity and requires complex handling [41]. Slow freezing is a prevailing strategy used in biobanks worldwide and is based on the application of lower concentrations of cryoprotective agents (CPAs), precise control over the cooling rate and gradual cell dehydration.…”

Research facilities and highlights at the Centro Nacional de Aceleradores (CNA)

“…Studies contributing to the standardization of clinical PET/CT with [ 18 F]FDG [33] and of radiotherapy planning [34] have been done. NanoCT was used for nanoparticle characterization in collaboration with ICMSE (Spain) [35], and also for cryopreservation studies in collaboration with UCL (Belgium) [36].…”

Yardimci Üreme Tekni̇kleri̇ Ve Ferti̇li̇te Prezervasyonunda Kri̇yoprezervason

“…As the primordial follicles are generally located 0.8 mm from the mesothelium, ovarian cortex grafts are recommended to be 1-1.5 mm in thickness. Cortical size is also important fort he permeation of CPAs as different types of cells and extracellular matrix compose the tissue (27). There is no consensus regarding the optimal procedure to cryopreserve ovarian tissue.…”