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Comparison of quartz vials with polypropylene vials for rapid cryopreservation of human ovarian tissue

View Article: PubMed Central - PubMed

ABSTRACT

Background: Because higher survival of follicles during the freezing/thawing procedure improves the quality of cryopreserved tissue reimplanted after oncological therapies, defining an optimal method for human ovarian tissue cryopreservation remains a major issue in this field. One option to improve the cryopreservation procedure is to use better materials, i.e., vials with better conductivity. The aim of this study was to compare polypropylene (PP) with quartz vials. Between September 2012 and January 2013, eight patients were recruited. The ovarian cortex was cut into 3 slices, assigned randomly to a fresh and a cryopreserved group in PP (method B) or quartz vials (method C). Histological and immunohistochemical (IHC) analysis were used. For IHC three antibodies were analyzed: Ki67 (proliferation index), Bcl2 (anti apoptotic index) and Hsp70 (stress index).

Results: The majority of GCs showed positive staining for Bcl2 in both cryopreservation device, with higher expression in group C than in group B. Oocytes and their nuclei showed intense positive staining for ki67 in both methods B and C, and also a patch positive stromal cells staining for Ki67. Expression of hsp70 was not increased after cryopreservation.

Conclusions: Cryopreservation using quartz vials led to larger numbers of good follicles while maintaining consistent preservation for stromal cells and vessels.

No MeSH data available.


Shows the used vials: a PP vial on the right side and a quartz vial on the left side
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Fig1: Shows the used vials: a PP vial on the right side and a quartz vial on the left side

Mentions: The ovarian cortex, cleaned from the ovarian medulla, was cut into 3 thin slices, which were assigned randomly to a fresh and a cryopreserved group. One of the slices was fixed immediately and used for fresh histological analysis, and the remaining pieces were cryopreserved rapidly in three solutions with increasing cryoprotectant concentration using the following protocol. All cryoprotectants were from Sigma-Aldrich: EG, ethylene glycol; DMSO, dimethyl sulfoxide; and PROH, 1,2-propanediol. Quartz and PP vials were refilled with solution 1, solution 2, and solution 3. Then, slices were immersed in both tubes (Fig. 1) in solution 1 (1 M EG in Leibovitz L-15 supplemented with 20 % HSA) for 8 min at RT, then passed through solution 2 (1 M EG, 1 M PROH, 1 M DMSO, 0.3 M sucrose in Leibovitz L-15 supplemented with 20 % HSA) for 5 min at RT, and finally equilibrated in both kind of tubes in fresh solution 2 at 4 °C for 15 min. We decided to use a rather long equilibration at 4 °C in step 3 in order to minimize toxicity, and because DMSO and EG penetrate tissue faster at 4 °C than do PROH and glycerol (GLY), as also suggested by Newton et al. [10]. At each step, tubes were rolled gently to allow penetration of cryoprotectants. All cryoprotectant solutions were filter-sterilized and stored for a maximum of 3 days at 4 °C prior to use. After removing all additional medium using sterile absorbent gauze, tissues were placed in the inner wall of sterile pre-cooled PP vials (1.8 ml NUNC cryotube, Sigma-Aldrich; method B), or pre-cooled quartz vials sterilized (H. Baumbach & Co Ltd., UK; method C), both closed, immersed in liquid nitrogen for 30 min and then stored in vapor storage tanks (CBS V1500-AB series). After a month, samples were thawed rapidly within 10 s at RT, followed by immersion in a water bath at 38 °C for 1 min. The contents of each vial were transferred at RT into solution I (Leibovitz L-15 supplemented with 0.5 M sucrose and 20 % HSA) for 5 min, then into solution II (Leibovitz L-15 supplemented with 0.25 M sucrose and 20 % HSA) for 5 min, and finally into solution III (Leibovitz L-15 and 20 % HSA) for 5 min, washed (3–5 times), and then incubated (HERA cell 150, Thermo Scientific) for 12–15 h in G-IVF medium (Vitrolife) in 6 % CO2 in air at 37 °C. The vial was agitated gently throughout the thawing steps. To allow immunohistochemical (IHC) analysis, all frozen/thawed samples were incubated for 12–15 h to provide time for protein expression, but not longer than 15 h to reduce any influence of culture on our cryopreservation results.Fig. 1


Comparison of quartz vials with polypropylene vials for rapid cryopreservation of human ovarian tissue
Shows the used vials: a PP vial on the right side and a quartz vial on the left side
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5037623&req=5

Fig1: Shows the used vials: a PP vial on the right side and a quartz vial on the left side
Mentions: The ovarian cortex, cleaned from the ovarian medulla, was cut into 3 thin slices, which were assigned randomly to a fresh and a cryopreserved group. One of the slices was fixed immediately and used for fresh histological analysis, and the remaining pieces were cryopreserved rapidly in three solutions with increasing cryoprotectant concentration using the following protocol. All cryoprotectants were from Sigma-Aldrich: EG, ethylene glycol; DMSO, dimethyl sulfoxide; and PROH, 1,2-propanediol. Quartz and PP vials were refilled with solution 1, solution 2, and solution 3. Then, slices were immersed in both tubes (Fig. 1) in solution 1 (1 M EG in Leibovitz L-15 supplemented with 20 % HSA) for 8 min at RT, then passed through solution 2 (1 M EG, 1 M PROH, 1 M DMSO, 0.3 M sucrose in Leibovitz L-15 supplemented with 20 % HSA) for 5 min at RT, and finally equilibrated in both kind of tubes in fresh solution 2 at 4 °C for 15 min. We decided to use a rather long equilibration at 4 °C in step 3 in order to minimize toxicity, and because DMSO and EG penetrate tissue faster at 4 °C than do PROH and glycerol (GLY), as also suggested by Newton et al. [10]. At each step, tubes were rolled gently to allow penetration of cryoprotectants. All cryoprotectant solutions were filter-sterilized and stored for a maximum of 3 days at 4 °C prior to use. After removing all additional medium using sterile absorbent gauze, tissues were placed in the inner wall of sterile pre-cooled PP vials (1.8 ml NUNC cryotube, Sigma-Aldrich; method B), or pre-cooled quartz vials sterilized (H. Baumbach & Co Ltd., UK; method C), both closed, immersed in liquid nitrogen for 30 min and then stored in vapor storage tanks (CBS V1500-AB series). After a month, samples were thawed rapidly within 10 s at RT, followed by immersion in a water bath at 38 °C for 1 min. The contents of each vial were transferred at RT into solution I (Leibovitz L-15 supplemented with 0.5 M sucrose and 20 % HSA) for 5 min, then into solution II (Leibovitz L-15 supplemented with 0.25 M sucrose and 20 % HSA) for 5 min, and finally into solution III (Leibovitz L-15 and 20 % HSA) for 5 min, washed (3–5 times), and then incubated (HERA cell 150, Thermo Scientific) for 12–15 h in G-IVF medium (Vitrolife) in 6 % CO2 in air at 37 °C. The vial was agitated gently throughout the thawing steps. To allow immunohistochemical (IHC) analysis, all frozen/thawed samples were incubated for 12–15 h to provide time for protein expression, but not longer than 15 h to reduce any influence of culture on our cryopreservation results.Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Background: Because higher survival of follicles during the freezing/thawing procedure improves the quality of cryopreserved tissue reimplanted after oncological therapies, defining an optimal method for human ovarian tissue cryopreservation remains a major issue in this field. One option to improve the cryopreservation procedure is to use better materials, i.e., vials with better conductivity. The aim of this study was to compare polypropylene (PP) with quartz vials. Between September 2012 and January 2013, eight patients were recruited. The ovarian cortex was cut into 3 slices, assigned randomly to a fresh and a cryopreserved group in PP (method B) or quartz vials (method C). Histological and immunohistochemical (IHC) analysis were used. For IHC three antibodies were analyzed: Ki67 (proliferation index), Bcl2 (anti apoptotic index) and Hsp70 (stress index).

Results: The majority of GCs showed positive staining for Bcl2 in both cryopreservation device, with higher expression in group C than in group B. Oocytes and their nuclei showed intense positive staining for ki67 in both methods B and C, and also a patch positive stromal cells staining for Ki67. Expression of hsp70 was not increased after cryopreservation.

Conclusions: Cryopreservation using quartz vials led to larger numbers of good follicles while maintaining consistent preservation for stromal cells and vessels.

No MeSH data available.