Cryopreservation and xenografting of human ovarian fragments: medulla decreases the phosphatidylserine translocation rate

Abstract

BackgroundPhosphatidylserine is the phospholipid component which plays a key role in cell cycle signaling, specifically in regards to necrosis and apoptosis. When a cell affected by some negative factors, phosphatidylserine is no longer restricted to the intracellular side of membrane and can be translocated to the extracellular surface of the cell. Cryopreservation can induce translocation of phosphatidylserine in response to hypoxia, increasing intracellular Ca2+, osmotic disruption of cellular membranes, generation of reactive oxygen species and lipid peroxidation. As such the aim of this study was to test the level of phosphatidylserine translocation in frozen human medulla-contained and medulla-free ovarian tissue fragments.MethodsOvarian fragments from twelve patients were divided into small pieces of two types, medulla-free cortex (Group 1, n = 42, 1.5–3.0 × 1.5–3.0 × 0.5–0.8 mm) and cortex with medulla (Group 2, n = 42, 1.5–3.0 × 1.5–3.0 × 1.5–2.0 mm), pre-cooled after operative removal to 5 °C for 24 h and then conventionally frozen with 6 % dimethyl sulfoxide, 6 % ethylene glycol and 0.15 M sucrose in standard 5-ml cryo-vials. After thawing at +100 °C and step-wise removal of cryoprotectants in 0.5 M sucrose, ovarian pieces were xenografted to SCID mice for 45 days. The efficacy of tissues cryopreservation, taking into account the presence or absence of medulla, was evaluated by the development of follicles (histology with hematoxylin-eosin) and through the intensity of translocation of phosphatidylserine (FACS with FITC-Annexin V and Propidium Iodide).ResultsFor Groups 1 and 2, the mean densities of follicles per 1 mm3 were 9.8, and 9.0, respectively. In these groups, 90 and 90 % preantral follicles appeared morphologically normal. However, FACS analysis showed a significantly decreased intensity of translocation of phosphatidylserine (FITC-Annexin V positive) after cryopreservation of tissue with medulla (Group 2, 59.6 %), in contrast with tissue frozen without medulla (Group 1, 78.0 %, P < 0.05). In Groups 1 and 2 it was detected that 21.6 and 40.0 % cells were viable (FITC-Annexin V negative, Propidium Iodide negative).ConclusionThe presence of medulla in ovarian pieces is beneficial for post-thaw development of cryopreserved human ovarian tissue.

Comparison of the enzymatic efficiency of Liberase TM and tumor dissociation enzyme: effect on the viability of cells digested from fresh and cryopreserved human ovarian cortex

“…This procedure was performed as published previously [ 48 – 58 ]. In our protocol, we used DMSO and ethylene glycol as cryoprotective cocktail to support a multi-cellular structure of ovarian tissues to protect all types of cells [ 59 ].…”

“…Rehydration of the tissue by stepwise rehydration followed. This was also performed using the same, previously published [ 5 , 48 – 58 ] ‘dropping’ methodology: slow addition of basal medium to the solution of sucrose with ovarian pieces. For ‘dropping’, we used 50-ml of basal medium in a 50-ml tube (Greiner Bio-One GmbH, Frickenhausen, Germany).…”

“…For ‘dropping’, we used 50-ml of basal medium in a 50-ml tube (Greiner Bio-One GmbH, Frickenhausen, Germany). The final sucrose concentration was 0.083-M, resulting in almost isotonic conditions [ 5 , 48 – 58 ]. The last step involved three washes in a basal medium for 10 min immediately prior to preparation.…”

Preserving fertility in female patients with hematological malignancies: a multidisciplinary oncofertility approach

“…According to several studies, systematic reviews and metaanalyses assessing the risk of reintroducing malignant cells, autotransplantation of frozen-thawed ovarian tissue should be absolutely contraindicated in all types of ovarian carcinomas and leukemia (high risk) while it could be carried out in HL, breast, bone, and connective tissue malignancies (mild risk) and in NHL and gastrointestinal cancers (moderate risk) [134,[172][173][174][175]. To assess the risk of reintroducing malignant cells, several tests can be carried out including histological examination, in vitro culture, immunohistochemistry, PCR, multicolor flow cytometry, and long-term xenografting of ovarian tissue portion into immunodeficient mice [176][177][178][179][180][181][182][183][184][185]. Recently, the first delivery in a leukemia survivor after autotransplantation of cryopreserved ovarian tissue, evaluated for leukemia cells contamination before reimplantation, has been reported [186].…”

Increasing of malignancy of breast cancer cells after cryopreservation: molecular detection and activation of angiogenesis after CAM-Xenotransplantation.

“…Cryopreservation (freezing and thawing) of the model tissues Cryopreservation of compacted fragments of cancer cells was implemented based on the protocols for cryopreservation of human ovarian tissue [13] with modi cations and peculiarities as described below.…”