The aim of our study was to design and validate a high throughput vitrification approach for human ovarian tissue based on the successful protocol of Suzuki and colleagues reporting two deliveries.40
The technical advantage of the vitrification procedure is avoidance of ice crystal formation contrary to conventional freezing approaches.41,42,43 The avoidance of ice crystal formation prevents the cellular damage, which can otherwise impair tissue inegrity or even follicular survival.
Our results indicate that follicular viability rates are compareable to our standard slow freezing approach, suitable for a clinical application. As a carrier system, we used metal made cell meshes linked with caps of 1.8 ml cryovials, suitable for fast loading of tissue fragments prior rapid vertical vitrification in grid embedded cryovials resulting in a high troughput process. This is important, because in a clinical laboratory application, the amount of tissue and the numbers of tissue fragments for cryopreservation may variate substantially per patient. Processing time per each fragment is critical in order to minimize prolonged exposition to a high ratio of cryoprotectants in the final step of equilibration solution – ressembling the vitrification process of oocytes, pronuclear (PN) stages or embryos. Additionally, the use of metal made meshes may provide faster cooling rates compared to plastic based systems and can be sterilized by autoclavation.
The protocol of Suzuki includes a carrier system consisting of four fine stainless needles linked with the cap of a cryogenic vial, before immersion into liquid nitrogen and insertion into cryovials after vitrification.44
Partially, our handling approach ressembles the protocol of Nikiforov, using unlinked metal meshes for carrying ovarian tissue samples with 0.28 mm wire diameter and mesh aperture of 1.31 mm for vitrification and insertion into cryovials after vitrification.45
Our approach included metal meshes with wire diameters of 0.25 mm and opening sizes of 0.38 mm. This ensures a large contact area of the cortex tissue on a metal carrier thin as possible, potentially influencing on the thermal conductivity while the meshes were pre linked with the caps of the cryovials. This enables the use of grid embedded cryovials, prefilled with liquid nitrogen ready for fast vertical insertion of loaded meshes into the cryovials directly during the process of vitrification. This results in a standardized, high throughput vitrification process of ovarian cortex tissue.
One major key aspect to assess the quality of ovarian cortex tissue is follicular viability that can be evaluated with calcein, a well described fluorescence based live assay.46,47,48 The incubation period of 24 h prior viability measurements of fresh, slow frozen/thawed and vitrified/rapid warmed tissue enables the tissue to express potential damage that could potentially occur with these procedures and may potentially not be observed when analysed at an earlier stage. The different types of cell death can take minutes to hours.49 Thus differences caused by the different methods might become visible later than directly after thawing/rapid warming.
Using 2x2 mm biopsy samples for evaluation of follicular viability provide valueable, but limited insights due to the fact that follicular density is unevenly50,51,52 distributed in the human ovary potentially influencing the results. To minimize these effects, a cohort of 30 patients with individual determination of follicular viability prior cryopreservation, post slow freezing and vitrification was evaluated by two experienced embryologists to mitigate researcher specific bias. In a clinical setting, preparation of at least 2x2 mm biospy samples for individual quality control assessment prior and post freezing is recommended.
The design of our experimental setup is limited by the fact that the amount of tissue per patient variates substantially and our ethical vote restricts research to 10% of the amount of tissue, reflecting the limited access of researchers to ovarian tissue in a routine setting.53 It will be interesting to investigate other tissue specific quality markers with potential impact on implantation success like angiopoietic factors after tissue culture in a future study with a larger sample number and selected patients with larger amounts of ovarian tissue available for research purposes.
In terms of economical parameters, our results implicate that vitrification is less cost demanding regarding cryopreservation time that can have a major impact on personell deployment planning - keeping in mind that in a busy cryobank ovarian tissue from external referrer centers scheduled for e.g. overnight transportation54 may occasionally arrive on the next day late in the afternoon during the week, or on saturdays, prior e.g. a slow freezing process lasting for several hours.
Contrary to vitrification, the time consuming slow freezing cooling process must not be interrupted due to potential technical errors related with the programmable freezer unit, computer or software malfunctions and continuous nitrogen supply that could have a major impact on tissue integrity.
Additionally, slow freezing ist cost intensive regarding purchasing and maintenance of equipment like controlled freezing systems with an attached nitrogen supply tank, personal computer, freezing software and requires in contrast to vitrification security of electricity during the cooling procedure.
In summary, our results indicate that rapid vertical vitrification of ovarian tissue may be equivalent to slow freezing in terms of follicular viability while offering a cost efficient alternative to conventional slow freezing procedures. We follow the argumentation of Suzuki55, Keros56, Sugishita57, Silver58 and Nikiforov58 that vitrification of ovarian tissue is a promising alternate approach to conventional slow freezing systems for ovarian tissue.