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Freezing-thawing behaviour and cell membrane ultrastructure of mouse embryos pre-cultured in B2-Menezo medium before
W Bernart1, T Rabe, E Schleiermacher
1Department of Obstetrics and Gynaecology, University of Heidelberg, FRG.
Abstract:
Mouse embryos were pre-cultured from the 2-cell to the 8-cell stage inserum-free B2-Menezo medium and in B2-Menezo medium with 15% fetal cord serum before cryopreservation with a slow freezing-thawing method (-0.3 degree C/min in 1.5 M DMSO). Viability after freezing-thawing was tested by continuing in-vitro culture and evaluating the percentage of embryos developing to the blastocyst stage. An extreme decrease in viability after freezing and thawing was found for the embryos pre-cultured in serum-free B2-Menezo medium compared with the embryos pre-cultured in B2-Menezo medium with 15% fetal cord serum (12 versus 81% blastocysts). Ultra-thin sections of the pre-cultured embryos and of freshly collected controls were prepared for transmission electron microscopy before and after the freezing-thawing procedure. A drastic decrease in the number of microvilli on the cell surface and a high degree of cell damage after freezing and thawing was observed in the embryos pre-cultured in serum-free B2-Menezo medium. In contrast a high number of microvilli and an intact cell structure after cryopreservation was observed in the embryos pre-cultured in B2-Menezo medium with 15% fetal cord serum and in the non-pre-cultured controls. It is suggested that the smaller cell surface and the resulting lower permeability of the cell membrane in the embryos with few microvilli are due to sub-optimal culture conditions and are the main reasons for the extremely decreased viability after freezing and thawing.
Insights
Pre-culturing mouse embryos in serum-free medium significantly reduces their viability after cryopreservation. Adding fetal cord serum to the culture medium improves embryo survival rates post-freezing and thawing.
Area of Science:
- Reproductive Biology
- Developmental Biology
- Cryobiology
Background:
- Cryopreservation is crucial for preserving embryos.
- Pre-culture conditions can impact embryo survival after cryopreservation.
- Serum-free culture media are increasingly used but may affect cryosurvival.
Purpose of the Study:
- To investigate the effect of pre-culture medium composition on mouse embryo viability after cryopreservation.
- To determine if serum supplementation in pre-culture medium enhances cryosurvival.
- To correlate cellular changes with cryopreservation outcomes.
Main Methods:
- Mouse embryos (2-cell to 8-cell stage) were pre-cultured in serum-free B2-Menezo or B2-Menezo with 15% fetal cord serum.
- Embryos underwent slow freezing-thawing (-0.3°C/min in 1.5 M DMSO).
- Post-thaw viability was assessed by in-vitro culture to blastocyst stage and transmission electron microscopy (TEM).
Main Results:
- Embryos pre-cultured in serum-free medium showed drastically reduced blastocyst development (12%) compared to those with serum (81%).
- TEM revealed fewer microvilli and significant cell damage in serum-free pre-cultured embryos post-cryopreservation.
- Embryos cultured with serum or non-pre-cultured controls maintained microvilli and intact cell structures.
Conclusions:
- Serum supplementation during pre-culture is critical for maintaining mouse embryo viability after cryopreservation.
- Reduced microvilli count in serum-free conditions likely decreases cell surface area, impairing membrane permeability and cryosurvival.
- Sub-optimal culture conditions negatively impact embryo resilience to cryopreservation stress.