Abstract
Procedures for cryopreserving embryos vary considerably, each having its specific advantages and disadvantages in terms of technical feasibility, embryo survival yield, temperature permissibility and species- or strain-dependent applicability. Here we report a high osmolality vitrification (HOV) method that is advantageous in these respects. Cryopreservation by vitrification is generally very simple, but, unlike slow freezing, embryos should be kept at a supercooling temperature (below –130°C) to avoid cryodamage. We overcame this problem by using an HOV solution containing 42.5% (v/v) ethylene glycol, 17.3% (w/v) Ficoll and 1.0 M sucrose. This solution is more viscous than other cryopreservation solutions, but easy handling of embryos was assured by employing a less viscous equilibration solution before vitrification. Most (>80%) embryos cryopreserved in this solution survived at –80°C for at least 30 days. Normal mice were recovered even after intercontinental transportation in a conventional dry-ice package for 2–3 days, indicating that special containers such as dry shippers with liquid nitrogen vapor are unnecessary. The HOV solution could also be employed for long-term storage in liquid nitrogen, as with other conventional cryoprotectants. Finally, we confirmed that this new vitrification method could be applied successfully to embryos of all six strains of mice we have tested so far. Thus, our HOV method provides an efficient and reliable strategy for the routine cryopreservation of mouse embryos in animal facilities and biomedical laboratories, and for easy and cheap transportation.
Highlights
With the advent of molecular genetics and modern developmental biology, thousands of new mutant strains of mice have been generated by genetic modification or induced mutagenesis
Because the 0.3 M sucrose solution containing ethylene glycol and Ficoll (EFS; Ethylene glycol–Ficoll–Sucrose) is the vitrification solution we routinely used for mouse embryo cryopreservation (EFS40; for composition, see Table 1), this result reinforces the notion that the typical vitrification solutions used for storage in LN2 are inappropriate for storage at –80uC
We have developed a new vitrification method for mouse embryos that enabled us to store the embryos at –80uC for several months and to transport them safely in dry-ice packages even over intercontinental distances
Summary
With the advent of molecular genetics and modern developmental biology, thousands of new mutant strains of mice have been generated by genetic modification or induced mutagenesis. Since the first report of successful mouse embryo cryopreservation by slow freezing in 1972 [1], many technical improvements have been made to increase the survivability of embryos and to reduce the labor required during the freezing/thawing procedures [2]. There are several embryo cryopreservation techniques, but each has specific disadvantages in terms of technical demands, embryo survival yield, temperature permissibility and applicability. Bulky and expensive and the cost of a single transportation including an empty shipper return trip is high. This has been one of the biggest drawbacks of embryo cryopreservation by vitrification in laboratories and mouse strain depositories
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
