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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.
The CE3 cell line was derived from the D3 ES cell line [PubMed: 12591158].
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 EDJ 22 on mouse embryonic fibroblasts (MEFs) that have been mitotically arrested by either irradiation or treatment with Mitomycin-C. EDJ 22 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.
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
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Please contact Washington University in St. Louis at:
Washington University Office of Technology Management
Attn. Associate Director
Matise M, et alProduction of targeted embryonic stem cell clonesIn: Matise M, et alGene Targeting: A Practical ApproachOxfordOxford University Press101-132, 1999
Adams LD, et al. Double lox targeting for neural cell transgenesis. Brain Res. Mol. Brain Res. 110: 220-233, 2003. PubMed: 12591158