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ES-D3 [D3]

CRL-11632

Product category
Animal cells
Organism
Mus musculus, mouse
Classification
Eukaryota, Animalia, Metazoa, Chordata, Vertebrata, Tetrapod
Cell type
pluripotent embryonic stem cell
Morphology
spherical colonies
Tissue
Embryo
Applications
3D cell culture
Stem cell research
Product format
Frozen
Storage conditions
Vapor phase of liquid nitrogen
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Documentation

Biosafety Icon Biosafety Level 1

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.

Required Products

These products are vital for the proper use of this item and have been confirmed as effective in supporting functionality. If you use alternative products, the quality and effectiveness of the item may be affected.

Detailed product information

Characteristics

Growth properties
Adherent
Strain
129/Sv+c/+p
Comments

The cells spontaneously differentiate into embryonic structures in the absence of a feeder layer or conditioned medium. They can be injected back into blastocysts and contribute to the germline.

Undifferentiated cells can be genetically modified by gene targeting techniques.

Technical information
ATCC Technical Services does not have technical information on patent deposits that are not produced or characterized by ATCC. Additional information can be found in the corresponding patent available from the patent holder or with the U.S. and/or international patent office.

Handling information

Unpacking and storage instructions
  1. Check all containers for leakage or breakage.
  2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.
Complete medium
Dulbecco's modified Eagle's medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose supplemented with 0.1 mM 2-mercaptoethanol, 85%; heat-inactivated fetal bovine serum, 15%
Temperature
37°C
Handling procedure

To insure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at –70°C.  Storage at –70°C will result in loss of viability.

NOTE:  Cells must be maintained on feeder layers of irradiated (12,000 rads) or mitomycin C treated (0.01 mg/ml for 90 minutes) primary mouse embryonic fibroblasts or STO cells. (see ATCC CRL-1503, STO or ATCC 56-X, irradiated STO cells). Plate feeder layers at 2 x 106/T25 or 6 x 106/T75 one or two days before plating ES-D3 cells.

  1. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water.  Thawing should be rapid (approximately 2 minutes).
  2. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
  3. Transfer the vial contents to a centrifuge tube containing  9.0 mL complete culture medium. and spin at approximately 125 x g for 5 to 7 minutes.
  4. Resuspend cell pellet with the recommended complete medium (see the specific batch information for the culture recommended dilution ratio). and dispense into a 25 cm2 or a 75 cm2 culture flask with feeder layer.
  5. Incubate the culture at 37°C in a suitable incubator.  A 5% CO2 in air atmosphere is recommended if using the medium described on this product sheet.
Subculturing procedure

Volumes are given for a 75 cm2 flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes.

Note: The cells can be maintained in the undifferentiated state by frequent subculture on feeder layers of mitomycin C (MITC) treated (0.01 mg/mL for 90 minutes) primary mouse embryonic fibroblasts or STO cells (see ATCC CRL-1503, STO or ATCC 56-X.2, MITC-STO cells).

  1. Remove and discard culture medium.
  2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
  3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
    Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
  4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
  5. Add fresh medium, aspirate and dispense onto fresh feeder layer cultures.
  6. Incubate cultures at 37°C.
    Subcultivation Ratio: A subcultivation ratio of 1:4 is recommended
    Medium Renewal: Every 2 to 3 days
    Reagents for cryopreservation
    Complete growth medium supplemented with 5% (v/v) DMSO (ATCC 4-X)

    Quality control specifications

    Mycoplasma contamination
    Not detected

    History

    Deposited as
    Mus musculus
    Depositors
    P Chambon
    Patent depository
    This material was deposited with the ATCC Patent Depository to fulfill U.S. or international patent requirements. This material may not have been produced or characterized by ATCC.  As an International Depository Authority (IDA) for patent deposits, ATCC is required to complete viability testing only at time of initial deposit of patent material. Patent deposits are made available on behalf of the Depositor when the pertinent U.S. or international patent is issued, but material may not be used to infringe the patent claims.
    Patent number
    6,486,381

    Legal disclaimers

    Intended use
    This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use.
    Warranty

    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.

    Disclaimers

    This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

    While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

    This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

    Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.

    Disclosures
    This material is cited in a US and/or international patent and may not be used to infringe the claims. Depending on the wishes of the Depositor, ATCC may be required to inform the Depositor of the party to which the material was furnished.

    Permits & Restrictions

    Import Permit for the State of Hawaii

    If shipping to the U.S. state of Hawaii, you must provide either an import permit or documentation stating that an import permit is not required. We cannot ship this item until we receive this documentation. Contact the Hawaii Department of Agriculture (HDOA), Plant Industry Division, Plant Quarantine Branch to determine if an import permit is required.

    MORE INFORMATION ABOUT PERMITS AND RESTRICTIONS

    References

    Curated Citations

    Doetschman TC, et al. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J. Embryol. Exp. Morphol. 87: 27-45, 1985. PubMed: 3897439

    Williams RL, et al. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336: 684-687, 1988. PubMed: 3143916

    Gossler A, et al. Transgenesis by means of blastocyst-derived embryonic stem cell lines. Proc. Natl. Acad. Sci. USA 83: 9065-9069, 1986. PubMed: 3024164

    Doetschman T, et al. Targeted mutation of the Hprt gene in mouse embryonic stem cells. Proc. Natl. Acad. Sci. USA 85: 8583-8587, 1988. PubMed: 3186749

    Chambon P, et al. Genetically engineered mice containing alterations in the genes encoding retinoic acid receptor proteins. US Patent 6,486,381 dated Nov 26 2002

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