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Cell Journal، جلد ۱۳، شماره Supplement، صفحات ۰-۰
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عنوان انگلیسی P-2: Optimization of Murine Blastocyst Isolation and Its Implantation on Mouse Embryonic Fibroblast (MEF) for Establishing an Embryonic Stem Cell Line
چکیده انگلیسی مقاله Objective: The process of ESCs derivation is often very inefficient and requires high specialization, training, and expertise. To circumvent these limitations, we aimed to optimize a protocol for isolating and culturing of mouse embryonic fibroblast (MEF) as feeder layer and mESC culture on MEF. Materials and Methods: Mature NMRI male mice were coupled for one night with two groups of females (either naïve or those pre-injected with different doses of Folligon (PSMG). At 12.5 days after gestation (E12.5), MEF cells were obtained from mice embryos using two different methods Chemical (using trypsin/EDTA) or mechanical (using 18G syringe). Different combination of supplements/media were used to optimize MEF culture. The viability of cells was measured by trypan blue and MTT-assay and the doubling time was calculated. To obtain blastocyst, time-mated females were sacrificed at E3.5, and then blastocysts were flushed-out of the uterous and collected using an inverted microscope. The blastocysts were implanted on mitomycin inactivated MEF feeders. After 4-6 days the inner cell mass of hatched blastocysts were isolated from trophoblast cells by pipetting the ICM. Results: We obtained blastocysts from both hormone injected and not injected mice with the same success rates. Obtained MEF cells grow better in cultivation medium with 15% FBS and 0.1% 2ME and they retain higher viability after freeze/thawing. Implanted blastocysts, obtained by both procedures, hatched and expanded for derivation of mESCs similarly. The blastocysts were positive for alkaline phosphatase. Conclusion: The protocol described above seems to be the most efficient and practical procedure for blastocysts isolation and MEF culture as its feeder layer for establishing a mESC line in our laboratory
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نشانی اینترنتی http://celljournal.org/journal/article/abstract/1534
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