Mouse ES Cell Derivation Kit
Embryonic stem (ES) cells are derived from totipotent cells of the early mammalian embryos. ES cells have extensive proliferative potential and can differentiate into all tissue types in chimeras, including the germline (Evans, 1981; Martin, 1981; Robertson, 1986). The derivation of mouse ES cells from embryos is still not a fully defined process and there is substantial variation in success rates among different laboratories. Most protocols for ES cell derivation involved the use of metal or glass needle for ICM dissection that requires expertise and skills in handling the embryos. The isolation of ICM from blastocyst is a process that is often very inefficient and a success rate of 30% is regarded as high (Bryja, 2006).
Mouse ES Cell Derivation Kit is an optimized and simplified system for the derivation of embryonic stem cell lines from mouse blastocysts. The system is designed to selectively proliferate the totipotent cells from mouse embryos. This system has achieved 60-90% of the efficiency in obtaining ES cell lines for various mouse strains, including C57BL and ICR which are considered to be stringent and refractory for ES cell isolation. Using this system, a person with minimum knowledge of cell culture and embryo handling is sufficient to derive ES cells from blastocysts.
Product Description and Storage:
The Mouse ES Cell Derivation Kit includes the medium for the derivation of ES cells from 10 embryos.
When stored at 4- 8oC after thaw, the kit components are stable up to 2 weeks.
EDF1 medium 50 ml
Tyrode¡¯s solution 1 ml
Trypsin solution 5 ml
Materials Required But Not Supplied:
1. Feeder cells: Mouse embryonic fibroblasts (MEF) from E12.5 or E13.5 mouse embryos and inactivated with mitomycin C or irradiated.
2. DPBS (Dulbecco¡¯s Phosphate Buffered Saline, Cat # BSS-1005-B, Specialty media)
3. ES medium
4. Cell culture freezing medium (Specialty media, Cat# S-012-D)
ES Cell Derivation Protocol:
1. Place feeder cells of mouse embryonic fibroblasts (MEF) with serum-supplemented media in the 96-well plates and culture overnight. Wash with 1x DPBS to reduce serum just prior to plating embryos.
2. Remove zonae pellucidae of the blastocysts with brief exposure to Tyrode¡¯s saline solution.
3. Wash the denuded embryos 3 x in 30µl of EDF1 medium and plate individually into a well covered with feeder cells in the 96-well plates, and culture with EDF1 medium at 37oC in 5% CO2 in humidified air for 4-5 days.
4. Trypsinize cell clumps originated from the blastocysts in 20l of 0.025% Trypsin for 5-10 min, add 200l of EDF1 medium to stop the reaction.
5. Disperse the cell clumps by pipetting up and down at least 20 times with a 200l pipeteman, and transfer the whole medium with cell suspension to another well with freshly seeded feeder cells in the 96-well plate. Culture at 37oC in 5% CO2 in humidified air for 2-3 days.
6. ES cell colonies will form 2-3 days after the first trypsinization. Colony expansion of the putative ES cells proceed from 48-well plates to 12-well plates with feeder cells in EDF1 medium, and then switch to gelatinized 6-well plates and 25 cm2 flasks for routine culture in ES medium, with 15% fetal calf serum (FCS) and 1,000 IU/ml LIF.
7. Freeze the ES cells in cell culture freezing medium and store in LN2.
1. Evans M, Kaufman M. (1981). Nature 292, 154.
2. Martin G (1981). Proc Natl Acad Sci USA 78,7634.
3. Robertson E, Bradley A, Kuehn M, Evans M. (1986). Nature 323:445.
4. Bryja V, Bonilla S, Cajanek L, Parish CL, Schwartz CM, Luo Y, Rao MS, Arenas E. (2006). Stem cells 24:844.