Offspring from Mouse Embryos Developed Using a Simple Incubator-Free Culture System with a Deoxidizing Agent

<div><p>To culture preimplantation embryos <em>in vitro</em>, water-jacketed CO₂ incubators are used widely for maintaining an optimal culture environment in terms of gas phase, temperature and humidity. We investigated the possibility of mouse embryo culture in a plastic bag kept at 37°C. Zygotes derived from <em>in vitro</em> fertilization or collected from naturally mated B6D2F1 female mice were put in a drop of medium on a plastic culture dish and then placed in a commercially available plastic bag. When these were placed in an oven under air at 37°C for 96 h, the rate of blastocyst development and the cell numbers of embryos decreased. However, when the concentration of O₂ was reduced to 5% using a deoxidizing agent and a small oxygen meter, most zygotes developed into blastocysts. These blastocysts were judged normal according to their cell number, Oct3/4 and Cdx2 gene expression levels, the apoptosis rate and the potential for full-term development after embryo transfer to pseudopregnant recipients. Furthermore, using this system, normal offspring were obtained simply by keeping the bag on a warming plate. This culture method was applied successfully to both hybrid and inbred strains. In addition, because the developing embryos could be observed through the transparent wall of the bag, it was possible to capture time-lapse images of live embryos until the blastocyst stage without needing an expensive microscope-based incubation chamber. These results suggest that mouse zygotes are more resilient to their environment than generally believed. This method might prove useful in economical culture systems or for the international shipment of embryos.</p> </div

Preimplantation development of inbred strain (C57BL/6) mouse embryos cultured in a plastic bag without using an incubator.

*<p>5% CO₂, 5% O₂ and 90% N_2.</p

Full-term development of embryos cultured in a plastic bag using a warming plate.

*<p>5% CO₂, 5% O₂ and 90% N_2.</p

DeO₂-WP culture system and development of full-term offspring.

<p>(A, B) The culture dish was placed in a sealed plastic bag with a controlled O₂ concentration, placed on a warm plate at 37°C and then covered with aluminum foil during the culture period. (C) Two-cell-stage embryos cultured for 24 h; (D) 8-cell-stage embryos cultured for 48 h and (E) morulae/blastocysts cultured for 72 h on the warm plate. Because the plastic bag was transparent, it was possible to observe embryos directly using an inverted microscope. (F) Mice derived from embryos cultured in the DeO₂-WP culture system. (G) After two or three months, these offspring had grown to adulthood and randomly selected mice were proven fertile by natural mating.</p

Experimental procedure and culture systems.

<p>(A) Schematic diagram of the experimental procedure and each culture system. Air-Oven experiment: the bag was filled with air and kept in an oven at 37°C. DeO₂-Oven experiment: the deoxidizing agent was put into the bag, which was then kept in the oven. MixG-Oven experiment: the bag was filled with a gas mixture (see methodology) and kept in the oven. DeO₂-WP experiment: the deoxidizing agent was put into the bag, which was then placed on a warming plate at 37°C. (B) The hermetically sealed plastic bag with a deoxidizing agent is indicated by an arrow. (C) The O₂ concentration was regulated by the deoxidizing agent and measured using the oxygen meter (arrowhead). (D) After removal of the deoxidizing agent from the plastic bag.</p

Preimplantation development rates of embryos derived from IVF and cultured under each culture system.

<p>Rates of development to the 2-cell (A) and morula/blastocyst (B) stages for each culture system. Vivo-K: Zygotes produced <i>in vivo</i> and cultured in KSOM. IVF-C: IVF-derived zygotes cultured in CZB medium. IVF-K: IVF-derived zygotes cultured in KSOM. Within columns, values with different letters are significantly different (<i>P</i><0.05, χ² tests).</p

Full-term development of embryos fertilized <i>in vitro</i> and cultured in a plastic bag without using an incubator.

*<p>For fertility testing, three pairs were selected from each group at random.</p>a<p>vs. ^b, ^c vs. ^d; values with different superscript letters are significantly different (<i>P</i><0.05 by χ² tests or Student's <i>t</i>-tests).</p

Full-term development of embryos cultured in the warm box.

<p>Key: Gas perm. film, gas-permeable film; M/B, Morula/Blastocyst; Frag, fragmentation; implant., implantation</p><p>Full-term development of embryos cultured in the warm box.</p

Abnormalities in placentae derived from cloned embryos.

<p>(A) Placental weights from ICSI-derived control embryos and CB- or LatA-treated cloned embryos. Error bars indicate SD. Asterisks indicate significant difference at p < 0.05. (B) Hematoxylin and eosin staining of placentae derived from ICSI-derived control and CB- or LatA-treated cloned embryos. Abnormal distortion of the boundary between the spongiotrophoblast and labyrinth layers was observed in placentas derived from both CB- and LatA-treated cloned embryos. Bar = 500 μm.</p

Full-term development of embryos cultured in microtubes with or without a gas-permeable film.

<p>Values with different superscript letters are significantly different (<i>P</i> < 0.05 by χ² tests).</p><p>Key: Gas perm. film, gas-permeable film; M/B, Morula/Blastocyst; Frag, fragmentation; implant., implantation</p><p>Full-term development of embryos cultured in microtubes with or without a gas-permeable film.</p