Alginate beads as a tool to handle, cryopreserve and culture isolated human primordial/primary follicles

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• 作者：A. Camboni ; A. Van Langendonckt ; J. Donnez ; J. Vanacker ; M.M. Dolmans ; C.A. Amorim
• 关键词：Follicle isolation ; Follicle cryopreservation ; In vitro culture ; Human ovary ; Alginate hydrogel ; Primordial follicles
• 刊名：Cryobiology
• 出版年：2013
• 出版时间：August, 2013
• 年：2013
• 卷：67
• 期：1
• 页码：64-69
• 全文大小：602 K

文摘

Background

One major concern of grafting cryopreserved ovarian tissue to restore fertility in cancer patients is the possibility of reintroducing tumor cells. Cryopreservation of isolated primordial/primary follicles (PFs) may circumvent this problem. The aim of our work was to compare dimethyl sulfoxide (ME₂SO) and ethylene glycol (EG) as cryoprotectants (CPAs) for slow-freezing of isolated human PFs in alginate.

Methods

Ovarian biopsies from four women were processed for follicle isolation. PFs were embedded in alginate (5-15 per group). Follicles were frozen-thawed using 1.4 M ME₂SO or 1.5 M EG as CPAs. Fresh and cryopreserved isolated follicles were in vitro cultured (IVC) for 7 days. At different time periods (after isolation, cryopreservation and IVC), follicles were evaluated with live/dead assay (using fluorescent probes) and diameter measurement. Follicle viability was calculated according to the percentage of dead follicular cells and the presence of a live/dead oocyte.

Results

A total of 841 PFs were isolated, embedded in alginate and cryopreserved with ME₂SO (n = 424) or EG (n = 259), or used as controls (n = 158). After 7 days of IVC, a significant increase in follicle size was observed in the fresh and ME₂SO groups, but not in the EG group. The percentage of totally viable PFs was not significantly different before or after seven days of culture in fresh (100 % and 82 % ) or ME₂SO (93.2 % and 85.1 % ) tissue. The EG group showed significantly lower viability before (63.9 % ) and after IVC (66.2 % ) than the fresh and ME₂SO groups.

Conclusions

Our results show that 1.4 M ME₂SO yields better preservation of isolated PF viability after thawing and 7 days of IVC than 1.5 M EG. Alginate constitutes an easy, safe hydrogel matrix to handle and cryopreserve isolated human follicles using ME₂SO as a CPA.