66 INDIRECT SOLID-SURFACE VITRIFICATION OF MURINE OVARIAN BIOPSIES AND ISOLATED FOLLICLES

Abstract

Vitrification is a quick and relatively easy cryopreservation technique, but requires high cryoprotectant concentrations and extremely high cooling rates. Possible disadvantages include contamination of specimens with liquid nitrogen (LN₂)-born pathogens and compromised post-thaw survival due to cryoprotectant toxicity and/or osmotic shock. The aims of the current research were to vitrify murine ovarian biopsies and isolated follicles using the Cryologic Vitrification Method 1 (CVM1^™, Linde Gas Cryoservices, Hedel, The Netherlands), and assess tissue viability after thawing. Ovaries from 5 mice were sectioned into small biopsies (≤0.5 mm³), and ovaries from 5 additional animals were enzymatically digested (collagenase 1 mg mL^–1) and filtered over a 100-µm sieve to isolate preantral follicles. Specimens were exposed to a series of vitrification solutions at 25°C, with the final solution consisting of sucrose (0.6 m), ethylene glycol (20%), and DMSO (20%). After 20 to 40 s of equilibration in the final medium, specimens were loaded into 3-µL droplets onto the hook of a plastic Fibreplug™. Droplets were vitrified by contact with the surface of the CVM1 metal block, and pre-cooled by partial submersion in LN₂. Fibreplugs were subsequently inserted into pre-chilled straws and plunged into LN₂ for 24 h. Specimens were warmed by removing the Fibreplug from the straw; the vitrified droplets were immersed in a series of sucrose solutions (0.3 m, 0.3 m, and 0.2 m) for 5 min at 25°C, and then transferred to PBS supplemented with 5 mg mL^–1 BSA. Post-warming viability of isolated follicles and biopsies was assessed by the fluorescent live/dead probe calcein-ethidium. Follicle survival was also appraised in vivo by autotransplantation of either devitrified isolated follicles or biopsies (6 to 12) to the left kidney capsule of the donor animals. The recipients were euthanized 7 days posttransplantation by CO₂ inhalation. Grafted tissue was fixed, paraffin-embedded, serially sectioned, and stained with hematoxylin-eosin for histological evaluation. Additional slides were immunohistochemically stained with proliferating cell nuclear antigen (PCNA) to differentiate between growing and non-growing follicles. In all digested samples, more than 80% of thawed isolated follicles were viable, as demonstrated by live/dead staining. In addition, fluorescent probes confirmed the presence of live follicles in 100% of thawed biopsies. In each of the 5 biopsy-transplanted animals, several biopsies survived, and in 3 mice grafted with isolated follicles, vital tissue could be retrieved. Histological examination of the 8 grafts revealed PCNA-positive follicles. These results indicate that indirect solid-surface vitrification of murine ovarian biopsies and isolated preantral follicles is a viable option to slow-rate freezing. The growing follicles in transplanted frozen–thawed tissue confirm the viability of surviving follicles. As specimens do not come into direct contact with LN₂ during the vitrification process, the contamination risk is greatly reduced.