Research Of Culture Conditionson Single Blastmeres Derived From Human Discarded Embros

Broken up embryo to form a lot of single blastomeres and obtain pregnancy has been reported in animal experiments. Because early embryo possess totipotency, Compared with the embryo before the embryo segmentation,The embryo which is derived from the cultivated single blastomeres has the same genome information,If we get the new embryos in vitro,and then transplant them, the offsprings must be exactly the same in genotype.if the development goes well, you can rely on this method to obtain the same genome information, At the same time,this method achieved with limited resources and get A lot of amplification. Granulocyte and macrophage colony stimulating factor is synthetized in the female reproductive tract, and this factor is closely related to the growth and development of rodents, such as livestock pre-transplant embryo, animal experiments suggest granulocyte macrophage colony stimulating factor can be used as development of "survival factors". In mice pre-transplant embryos can expressGM-CSF receptor alpha chains, to join in the cultures of recombinant granulocyte macrophage colony stimulating factor,the cleavage stage embryo growth, the formation of the blastocyst and subsequent stick wall adhesion promoted in mice. So blastomeres cultured in vitro achieve the basis of the above resources amplification, at first this study regards donated embryo as basic material, we explored the configuration time of the culture medium、culture time of the blastomere、the volume of droplets on the development of the blastomeres, the best culture conditions achieved。and then on this condition, in donated frozen embryos as experimental materials, obtained the optimum rhGM-CSF concentrations, finally based on this optimal cytokine concentrations of nutrient solution,we cultivated single blastomere derived from the donated embryo which were cultivated three daysafter fertilization and get satisfactory conclusions. The best culture conditions provide an early experimental research for subsequent cultivate and build lines of the stem cells.To get optimum blastomeres condition in vitro culture, after 120 thawed embryos using a laser to get rid of zona of high-quality, get 863 blastomeres, cultivate singlely to the sixth day. We investigate droplet volume of 10ul,20ul,30ul,40ul impact on the development of late stage blastomeres (40 embryos,339 blastomeres). Counterpoise time is 2h,4h,20h,24h on the late development of blastomeres (40 embryos,334 blastomeres). Blastomere incubation time was D3, D4. D5, D6 influence (40 embryos,340 blastomeres) on blastomere late development. Single blastomere cleavage rate, the densification rate and blastocyst formation rate between each groups, When to investigate the effect of incubation time to blastomere development, the last day of the remaining number of blastocyst is need to be discussed. The results are by the number of blastomeres formed blastocysts in vitro, when the droplet volume is 20ul, incubation time is D3, liquid counterpoise time is 20h achieved the highest blastocyst formation rate, thereby optimal culture conditions for droplet volume 20ul, incubation time is D3, liquid counterpoise time is 20 hours.To research rhGM-CSF concentration needed for the single blastomere cultivation, divided into five groups,0.2 ng/ml group,0.4 ng/ml group,0.6 ng/ml group,0.8 ng/ml group,1.0 ng/ml group,46 were thawed embryos, we apply the proceeds of the optimum cultivation conditions of single blastomere in this experiment, results show that although 0.4 ng/ml group cleavage rate is not high, but the densification rate and blastocyst formation rate is the highest, blastocyst count results show that the average number of blastocyst cells up to 30.36±12.75 in 0.4 ng/ml group, significantly higher than 0.2 ng/ml and 0.6 ng/ml group.Compared with the blank control group,0.4 ng/ml rhGM-CSF significantly improved the cleavage rate and blastocyst formation rate of the single blastomere.To study cytokines for inferior embryos (grade are IV), this study selected embryos on the third day following oocyte pick-up, the experimental group add cytokines, a total of 20 embryos, does not add any material in blank control group 10 embryo culture, the results showed that the experimental group’s cleavage rate, densification and blastocyst formation rate were significantly increased.