Effective method for in-vitro culture of cryopreserved human ovarian tissue

Abstract

It is widely accepted that it is possible to successfully cryopreserve human ovarian tissue by direct plunging into liquid nitrogen using permeable cryoprotectants only, without disaccharides. This study aimed to search for and test a new method for in-vitro culture of vitrified tissue. Ovarian biopsies were obtained during operative laparoscopy. Pieces of ovarian tissue were vitrified and warmed. After warming, tissue pieces were randomly distributed into three groups for further culture: in 2 ml of culture medium which was regularly renewed (group 1), in 30 ml of culture medium without agitation (group 2) and in 30 ml of culture medium with agitation (group 3). During the 2-week and 6-week culture, the growth of follicles within the vitrified-warmed ovarian tissue pieces was investigated. After 2 weeks of culture, mean numbers of non-degenerated follicles per mm(2) of tissue were 1.5, 1.7 and 4.5 for groups 1, 2 and 3 respectively (groups 1 and 2 versus group 3, P < 0.05). Agitation during culture of ovarian tissue is beneficial, and can be used as a prognostic tool for future warming and autotransplantation of ovarian tissue.

In vitro development of human primordial follicles to preantral stage after vitrification

“…This evaluation method might have skewed the viable follicles to a smaller number as determined by the fluorescent staining method compared to NR staining. To the best of our knowledge only a few studies on cryopreservation of ovarian tissue have been carried out culturing tissue strips in vitro for more than 24 h after thawing to evaluate the survival and development of follicles [25,44,61,65,66]. Finally, results of the present study showed that stainless steel mesh can be used as a carrier device in the vitrification procedure.…”

“…Other investigators have studied the effect of human ovarian cryopreservation on viability and development of follicles and on xenotransplantation [39][40][41][42]. Different strategies have been used to culture immature follicles in vitro using fresh or cryopreserved cortical tissue in situ [43][44][45][46]. It has been shown in a recent study that, after 6 days in culture, the number of primordial follicles decreased and the number of preantral follicles increased in cortical strips [46].…”

Human ovarian tissue vitrification versus conventional freezing: morphological, endocrinological, and molecular biological evaluation

“…Below, we briefly summarize the results of five investigations with different conclusions. Isachenko et al (2006Isachenko et al ( , 2007Isachenko et al ( , 2008bIsachenko et al ( , 2008c. BarZ300 mm.…”

“…Ovarian pieces were also conventionally frozen using 1.5 M DMSO or 1.5 M propylene glycol with post-thawing stepwise exposure of fragments in the respective cryoprotectants (DMSO or propylene glycol) of decreasing concentration (Newton et al 1996). Immediately after warming/thawing, ovarian Figure 3 Histological micrographs of follicles from: (A, a) fresh OP; (b) and (B) after vitrification and culture, (c) and (C) after conventional freezing and culture by Isachenko et al (2006Isachenko et al ( , 2007Isachenko et al ( , 2008a. BarZ35 mm.…”

Efficacy of ovarian tissue cryopreservation in a major European center

“…Using a continuous dilution protocol modified from Isachenko, [43] ovarian cortex strips were transferred to a sterile container with 10 ml Dulbecco's Phosphate Buffered Saline (DPBS; Gibco), 0.75 M sucrose (MP Biomedicals, Eschwege, Germany), 10 % Fetal Bovine Serum (FBS; PAA Laboratories GmbH, Cölbe, Germany), and 0.1 mg/ml Pen/ Strep (Lonza, Basel, Schwitzerland). After incubation under continuous agitation (15 min), a solution of 50 ml DPBS/10 % FBS/0.1 mg/ml Pen/Strep was added with 100 ml/h using a perfusion device.…”