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

Abstract

BackgroundOvarian 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.MethodsOvarian 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.ResultsThe effectiveness of the dissociation was not significantly different between reference ovarian tissue (1.58 × 106 ± 0.94 × 106 viable ovarian cells per 100 mg of ovarian cortex, n = 60) and tissue from ovarian tissue cryopreservation (1.70 × 106 ± 1.35 × 106 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%).ConclusionBoth 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.

A protocol to isolate and qualify purified human preantral follicles in cases of acute leukemia, for future clinical applications

“…The most common cancer in prepubertal girls is leukemia, with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) accounting for 85 and 15% of cases, respectively [ 2 ]. Real time quantitative polymerase chain reaction (RT-qPCR) [ 10 , 14 ], multicolor flow cytometry (MFC) [ 15 – 18 ] and ovarian tissue xenografts in immunodeficient mice [ 11 , 19 ] are techniques proposed for the detection of minimal residual disease (MRD) in cryopreserved ovarian tissue.…”

New method of FACS analyzing and sorting of intact whole ovarian fragments (COPAS) after long time (24 h) cooling to 5 °C before cryopreservation

“…Analysis of single cells by flow cytometry has become a common tool for both clinicians and basic science researchers (Theunissen et al 2017 ; Wagner et al 2020 ; Zver et al 2017 ). To help improve the grafting technique, it was investigated whether short-term xenografting of a suspension containing ovarian stromal and endothelial cells without follicles could enhance graft survival and revascularization (Dath et al 2011 ).…”

Minimal residual disease detection by multicolor flow cytometry in cryopreserved ovarian tissue from leukemia patients

“…Ovarian cortex was cut into small pieces of 1-2 mm 3 . Depending on the timing of the MRD test, a socalled "laboratory" protocol (as previously described) [44] or a commercial protocol was used for ovarian cell isolation. Brie y, the laboratory protocol is based on mechanical and enzymatic dissociation using a cell dissociator (GentleMACS; Miltenyi Biotec) and collagenase Ia (1.6 U/ml; Sigma) with DNase I (Roche) in 5 ml of RPMI (ThermoFisher Scienti c) in combination with C Tubes (Miltenyi Biotec).…”

“…Brie y, the laboratory protocol is based on mechanical and enzymatic dissociation using a cell dissociator (GentleMACS; Miltenyi Biotec) and collagenase Ia (1.6 U/ml; Sigma) with DNase I (Roche) in 5 ml of RPMI (ThermoFisher Scienti c) in combination with C Tubes (Miltenyi Biotec). For the commercial protocol, a Tumor Dissociation Kit was used according to the manufacturer's instructions (Miltenyi Biotec); it was previously validated in our laboratory for ovarian tissue dissociation [44]. After ovarian tissue dissociation, we performed cell suspension ltration with a 70 µm cell strainer (Dutscher) and washed the suspension with 5 mL of RPMI.…”