Optimized culture systems for the preimplantation ICR mouse embryos with wide range of EDTA concentrations

Abstract

In this study, in vitro preimplantation embryo culture media especially for outbred stock mice (Institute of Cancer Research (ICR)) were optimized with different concentrations of ethylenediaminetetraacetic acid (EDTA). A plot with embryo development rates against EDTA concentrations ranging from 0 to 500 µM showed a unique pattern with two characteristic peaks. Two hundred micromolar was adopted as an optimal concentration of EDTA. The optimized media were also evaluated with two culture systems: conventional large volume culture system (1 ml) and micro-droplet culture system. In the conventional large volume culture system, the blastocyst development rates were compared among three different media (F-10, KSOM and KSOM with the optimized 200 µM EDTA). The rates were 0.4%, 16.7% and 57.6%, respectively. The development rates for the micro-droplet (10 µl) culture system were 73.9%. In conclusion, 200 µM EDTA concentration in 10 µl droplets in the KSOM medium was found as the most suitable culture conditions for ICR mouse embryos, as the blastocyst development rate was higher in the micro-droplet culture system than in the traditional conventional large volume culture system.

Keywords: EDTA; ICR mouse; KSOM; culture system; micro-droplet.

Figure 1.

Schematic diagram of overall assay procedure. (a) Preparation of mice by superovulating with 5 IU of PMSG and hCG, 16–18 h post hCG to collect zygote from ampulla, (b) two types of culture systems; large volume culture system and micro-droplet culture system to culture and observe the development of the collected zygotes and (c) stages of embryo development observed from Day 0–Day 5.

Figure 2.

Charts of embryo survival rates (morula and blastocyst) (a) Morula and blastocyst development rate of embryos cultured in KSOM (0) and different concentrations of EDTA (0–500 µM) in large volume culture system (1 ml), (b) development rate of morula and blastocyst in different droplet media volume in KSOM alone and EDTA optimized KSOM media, (c) development rate of embryos in KSOM and EDTA optimized KSOM media in 10 μl micro-droplet culture system and (d) comparison of morula block and blastocyst development rate in two culture systems with KSOM media alone and KSOM media with 200 μM EDTA. **Significantly different to medium KSOM (0); p < 0.01; *Significantly different in between two culture systems; p < 0.05.