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ATCC determines the biosafety level of a material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.
ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.
Complete Medium for Feeder Cells
Feeder cells may be grown in medium containing fewer growth factors than those required by the ES cells. Feeder cells are available from ATCC. Consult the product sheet provided for the feeder cells you wish to use for medium requirements.
Feeder cells should be initiated 24-48 hours prior to inoculating with embryonic stem (ES) cells.
ATCC recommends culturing J1 on mouse embryonic fibroblasts (MEFs) that have been mitotically arrested by either irradiation or treatment with Mitomycin-C. J1 cells have been cultured on mitotically arrested MEF (CF-1) (ATCC® SCRC-1040™).
Feeder cells should be used within one week of plating. It is best to use feeder cells within 24-48 hours of initiation.
Embryonic Stem (ES) Cells
Perform a 100% medium change every day. Passage the cells every 1 to 2 days. If the colonies are close to or touching each other the culture is overgrown. Overgrowth will result in differentiation.
Make sure that you have prepared a sufficient number of flasks pre-plated with MEF feeder layers to support frequent passage of the ES cells.
Note: To insure the highest level of viability, pre-warm media and Trypsin/EDTA to 37ºC before adding to cells. Volumes used in this protocol are for T75 flasks. Proportionally adjust the volumes for culture vessels of other sizes. A split ratio of 1:4 to 1:7 is recommended.
Feeder Cell Preparation for Subcultures
Dissociation and Transfer of ES Cells
The product is provided 'AS IS' and the viability of ATCC® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid. Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.
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Please contact Whitehead Institute for Biomedical Research at:
Whitehead Institute for Biomedical Research
Attn. Shoji Takahashi
Phone: (617) 258-8930
Stevens LC. A new inbred subline of mice (129-terSv) with a high incidence of spontaneous congenital testicular teratomas. J. Natl. Cancer Inst. 50: 235-242, 1973. PubMed: 4692863
Li E, et al. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69: 915-926, 1992. PubMed: 1606615
Krzyzanowski PM, et al. Identification of novel stem cell markers using gap analysis of gene expression data. Genome Biology 8(9): R193, 2007. PubMed: 17875203
Luikenhuis S, et. al. Antisense transcription through the Xist locus mediates Tsix function in embryonic stem cells. Mol. Cell Biol. 21(24): 8512?8520, 2001. PubMed: 11713286
Goto T, et al. Regulation of X-Chromosome inactivation in development in mice and humans. Micro. Molec. Biol. Rev. (ASM) 62(2): 362-378, 1998. PubMed: 9618446