Improvement Of The Method For In Vitro Culture Of Mouse Embryos

Despite advances in vitro manipulation of preimplantation embryos, there is still a reduction in the quality of embryosproduced leading to lower pregnancy rates compared with embryos produced in vivo. We hypothesized that a dynamic microenvironment embryoculture system would enhance outcomes by better mimicking the fluid-mechanical and biochemical stimulation embryos experience in vivo from ciliary currents and oviductal contractions.Objective To improve the embryo culture system for the quality of embryos and developmental potential.In this study a rotating plastic capillary with an inside diameter of 0.21 mm and an outside diameter of 0.28 mm were used to mimic oviduct properties and culture embryo in vitro. The capillary was inserted into a pipette tip, and then one to ten mouse 2-cell embryos in KSOM medium were drawn into a capillary by pipette. The capillary was immersed in a beaker containing a certain amount of autoclaved distilled water on a magnetic stirrer and then the device was placed in an incubator. After 48 hours culture the embryos were withdrew and their developmental potential were detected. Validated through a lot of experimental data,which indicate that the specification of the capillary is 34 G,The speed of the magnetic stirrer is 180r/min.1)Eighty-five and eight-two two-cells embryo were cultured in capillaries and microdrops, respectively. The blastocyst rate and average cell number are significantly higher in capillaries(85.4% and 57.0) than in microdrops(36.5%and 20.6). Capillary-cultured embryos formed bigger outgrowths than that of microdrop-cultured ones when they were seeded on matrigel-treated dishes.2)Capillary-cultured embryos,1-cell embryo, 2-cell embryo were cultured in capillaries to the blastocyst stage(E3.5 d) were use the non-surgical methods,transplanted in 3.5 d pseudopregnancy mice.The embryos(E3.5 d) producedin vivowere use the non-surgical methods, transplanted in 3.5d pseudopregnancy mice. Results showed that compared the Capillary-cultured embryos and in vivo embryos,development of synchronous.3)For naive i PSC injectioncells were trypsinized and microinjectedinto 8-cell stage embryos or E3.5 blastocysts(The embryo werecultured in capillaries and microdrops)of ICR diploid mouse embryo(6–10 cells per embryo. Approximately 15 injected embryos were transferredto each uterine horn of pseudopregnant females 2.5 days postcoitum.Embryos were dissected at E13–E14developmental stages for whole-mountstaining with anti-human nuclei antibodyunderthe whole-mount staining procedure from Abcam.The Chimeric embryos rate are significantly higher in capillaries(37/83)than in microdrops(28/79);For naive i PSCs injectioncells were trypsinized and microinjectedinto 2-cell stage embryos, then culture into capillaries, training to 8-cell/blastocyst stage(E3.5 d),The result showed that the capillary cultivate support chimeric embryos development.Capillary culture, a method for in vitro culture the mouse embryo, raises success rates following human assistedreproduction.It is good for the traditional microdrop-cultured.The Capillary-cultured was complied with the principle of experimental animal ethics.Importantly, the research model was provided for the embryo culture of biomedical research and assisted reproductive technologies in humans.