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. 2012 Dec;39(4):153-60.
doi: 10.5653/cerm.2012.39.4.153. Epub 2012 Dec 31.

Vitrification of mouse embryos using the thin plastic strip method

Eun Kyung Ryu  1 , Yong Soo Hur, Ji Young Ann, Ja Young Maeng, Miji Park, Jeong Hyun Park, Jung Yoon, San Hyun Yoon, Chang Young Hur, Won Don Lee, Jin Ho Lim
Affiliations

Vitrification of mouse embryos using the thin plastic strip method

Eun Kyung Ryu et al. Clin Exp Reprod Med. 2012 Dec.

Abstract

Objective: The aim of this study was to compare vitrification optimization of mouse embryos using electron microscopy (EM) grid, cryotop, and thin plastic strip (TPS) containers by evaluating developmental competence and apoptosis rates.

Methods: Mouse embryos were obtained from superovulated mice. Mouse cleavage-stage, expanded, hatching-stage, and hatched-stage embryos were cryopreserved in EM grid, cryotop, and TPS containers by vitrification in 15% ethylene glycol, 15% dimethylsulfoxide, 10 µg/mL Ficoll, and 0.65 M sucrose, and 20% serum substitute supplement (SSS) with basal medium, respectively. For the three groups in which the embryos were thawed in the EM grid, cryotop, and TPS containers, the thawing solution consisted of 0.25 M sucrose, 0.125 M sucrose, and 20% SSS with basal medium, respectively. Rates of survival, re-expansion, reaching the hatched stage, and apoptosis after thawing were compared among the three groups.

Results: Developmental competence after thawing of vitrified expanded and hatching-stage blastocysts using cryotop and TPS methods were significantly higher than survival using the EM grid (p<0.05). Also, apoptosis positive nuclei rates after thawing of vitrified expanded blastocysts using cryotop and TPS were significantly lower than when using the EM grid (p<0.05).

Conclusion: The TPS vitrification method has the advantages of achieving a high developmental ability and effective preservation.

Keywords: Apoptosis; Container; Embryos; Mice; Vitrification.

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Conflict of interest statement

No potential conflict of interest relevant to this article was reported.

Figures

Figure 1
Figure 1
Images of the electron microscopy grid, cryotop, and thin plastic strip (TPS) container (A), mouse embryos loaded onto TPS (B), TPS positioned into a cryovial (C), and the thawing procedure of TPS (D).
Figure 2
Figure 2
Morphology patterns of mouse embryos. Cleavage-stage embryos after being vitrified-warmed (A, ×200), expanded embryos after being vitrified-warmed (B, ×200), hatching-stage embryos after being vitrified-warmed (C, ×200), and hatched-stage embryos after being vitrified-warmed (D, ×200).
Figure 3
Figure 3
The fluorescent image patterns of total and apoptotic cells in mouse embryos. (A-C; ×400) Total cells were determined by DAPI (blue), (D-F; ×400) apoptotic cells were confirmed by TUNEL (green), and (G-I; ×400) colocalization with DAPI is indicated as blue-green. DAPI, 4', 6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase (TDT) mediated dUTP nick end labeling; EM, electron microscopy; TPS, thin plastic strip.
See this image and copyright information in PMC

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