Validation of an automated technique for ovarian cortex dissociation: isolation of viable ovarian cells and their qualification by multicolor flow cytometry

BACKGROUND: Ovarian tissue cryopreservation is a technique for fertility preservation addressed to prepubertal girls or to patients for whom no ovarian stimulation is possible before initiation of gonadotoxic treatments. Autotransplantation of frozen-thawed ovarian tissue is the only available option for reuse but presents some limitations: ischemic tissue damages post-transplant and reintroduction of malignant cells in cases of cancer. It is therefore essential to qualify ovarian tissue before autograft on a functional and oncological point of view. Here, we aimed to isolate viable cells from human ovarian cortex in order to obtain an ovarian cell suspension analyzable by multicolor flow cytometry. METHODS: Ovarian tissue (fresh or frozen-thawed), from patients with polycystic ovarian syndrome (reference tissue) and from patients who underwent ovarian tissue cryopreservation, was used for dissociation with an automated device. Ovarian tissue-dissociated cells were analyzed by multicolor flow cytometry; the cell dissociation yield and viability were assessed. Two automated dissociation protocols (named laboratory and commercial protocols) were compared. RESULTS: The effectiveness of the dissociation was not significantly different between reference ovarian tissue (1.58 × 10(6) ± 0.94 × 10(6) viable ovarian cells per 100 mg of ovarian cortex, n = 60) and tissue from ovarian tissue cryopreservation (1.70 × 10(6) ± 1.35 × 10(6) viable ovarian cells, n = 18). However, the viability was slightly different for fresh ovarian cortex compared to frozen-thawed ovarian cortex whether we used reference tissue (p = 0.022) or tissue from ovarian cryopreservation (p = 0.018). Comparing laboratory and commercial protocols, it appeared that cell yield was similar but cell viability was significantly improved when using the commercial protocol (81.3% ± 12.3% vs 23.9% ± 12.5%). CONCLUSION: Both dissociation protocols allow us to isolate more than one million viable cells per 100 mg of ovarian cortex, but the viability is higher when using the commercial dissociation kit. Ovarian cortex dissociation is a promising tool for human ovarian cell qualification and for ovarian residual disease detection by multicolor flow cytometry.