Isolation And Culture Of Mouse Embryonic Stem Cells And Embryonic Germ Cells

The major object of this paper is to increase the efficiency of establishment KunMingalbonine strain mouse ES/EG cell lines. Some factors influencing the efficiency of theisotation and culture of mouse ES/EG cells have been discussed. Using the primary murineembryo fibroblast as feeder cells, preimplantation embryo and PGCs derived from8.5dpc~12.5dpc genital ridge were cultured using the medium containing low glucose DMEM,15% FCS ,2.2g/L NaHCO3, 0.1mM 2-mercapothanol , 2mM glutamine and 1000IU/mL LIFas ES cell culture medium. And ES/EG-like colonies were identified, tripsinized and passaged.The results obtained were as following:1. 302 primary ES colonies were obtained from712 KunMing strain mouse preimplatation embryo and a cell line of the ninth passage of ES-like was obtained; 476 primary EG colonies were obtained from 184 genital ridge and a cell line of the eighth passage of ES-like was obtained.2. The rate of embryonic attaching, ICM and ES colony forming in ES cell medium adding 2.2g/L NaHCO3 are 75%,68% and 61% respectively ,while in ES cell medium adding 3.7g/L NaHCO3 those are only 27%,27% and 8% respectively ,having distinct difference.3. The rate of embryonic attaching , ICM and ES colony forming of 3.0dpc molara and 3.5dpc blastula are 30%,30%,18% and 75%,68%,61% respectively ,having distinct difference.4. The rate of embryonic attaching ,ICM and ES colony forming of blastula from nature mating are distinctly higher than those of blastula from superovulation mating , indicating that blastula from nature mating are fit to isolate ES cells.摘 要5. The medium supplied LIF can distinctly increase the rate of ICM colony and ES/EG colony comparing those without LIF, but having no effection on embryonic attaching rate, there are no digital dependent effection when supplied LIF concentration is from 250IU to 1000IU/ml.6. Average 1.89 EG cell colony were obtained in one genital ridge using "several times digestion" , while only 0.68 EG cell colony were obtained in one genital ridge using "one times digestion", having distinct difference .7. Using method III(using 0.1%tripsine-0.01%EDTA incubate ES/EG colonies for 2-3min, then vigorously tap the dish against the palm of hand to dislodge the cells into suspension. When colonies fully detached, pipette up and down to dissociate cells into small cells mass.) can obtained high ES/EG colony forming rate and passage, also is easy and practable.