Optimised CO2-containing medium for in vitro culture and transportation of mouse preimplantation embryos without CO2 incubator.

Yasuyuki Kikuchi,Daiyu Ito,Masatoshi Ooga,Sayaka Wakayama,Teruhiko Wakayama,Peter J. Hansen

doi:10.1371/journal.pone.0260645

Abstract

Conventional in vitro culture and manipulation of mouse embryos require a CO2 incubator, which not only increases the cost of performing experiments but also hampers the transport of embryos to the other laboratories. In this study, we established and tested a new CO2 incubator-free embryo culture system and transported embryos using this system. Using an Anaero pouch, which is a CO2 gas-generating agent, to increase the CO2 partial pressure of CZB medium to 4%-5%, 2-cell embryos were cultured to the blastocyst stage in a sealed tube without a CO2 incubator at 37°C. Further, the developmental rate to blastocyst and full-term development after embryo transfer were comparable with those of usual culture method using a CO2 incubator (blastocyst rate: 97% versus 95%, respectively; offspring rate: 30% versus 35%, respectively). Furthermore, using a thermal bottle, embryos were reliably cultured using this system for up to 2 days at room temperature, and live offspring were obtained from embryos transported in this simple and very low-cost manner without reducing the offspring rate (thermal bottle: 26.2% versus CO2 incubator: 34.3%). This study demonstrates that CO2 incubators are not essential for embryo culture and transportation and that this system provides a useful, low-cost alternative for mouse embryo culture and manipulation.

Highlights

The current mainstream method of embryo culture in vitro requires a CO2 incubator, which provides an optimal culture condition by stably maintaining both temperature and gas phase
Plastic tubes containing CZB medium were placed in a 5% CO2 incubator (Fig 1A), and the CO2 partial pressure of the medium increased with longer treatment duration, achieving equilibrium of approximately 4% after 24 h (Fig 1B)
For 2-cell embryos derived from In vitro fertilisation (IVF) cultured with OptC medium in a thermal bottle, we found that both 1- and 2-day incubations in a thermal bottle were possible and after subsequently cultured in a CO2 incubator for 3 or 2 days, most of embryo developed to blastocyst (100% and 91%, respectively, Table 5, Fig 3C)

Summary

Introduction

The current mainstream method of embryo culture in vitro requires a CO2 incubator, which provides an optimal culture condition by stably maintaining both temperature and gas phase. Using a CO2 incubator for culture has several weak points. Embryonic development in a CO2 incubator stops unless CO2 is continuously supplied [1]. In addition to the operating costs associated with a CO2 incubator, there is the risk of unintentional disruption of the gas supply due to unexpected accidents or events such as power outage.

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