ES-D3 [D3] (ATCC® CRL-11632)

Organism: Mus musculus, mouse  /  Cell Type: pluripotent embryonic stem cell  / 

Organism Mus musculus, mouse
Cell Type pluripotent embryonic stem cell
Product Format frozen
Morphology spherical colonies
Culture Properties adherent
Biosafety Level 1

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

Age embryo, blastocyst
Strain 129/Sv+c/+p
Storage Conditions liquid nitrogen vapor phase
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.

Complete Growth 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%
Subculturing

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
    Cryopreservation
    Freeze medium: Complete growth medium supplemented with 5% (v/v) DMSO
    Storage temperature: liquid nitrogen vapor phase
    Culture Conditions
    Temperature: 37°C
    Name of Depositor P Chambon
    U.S. Patent Number
    Disclosure This material is cited in a US or other Patent and may not be used to infringe the claims. Depending on the wishes of the Depositor, ATCC may be required to inform the Patent Depositor of the party to which the material was furnished.
    References

    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

    Basic Documentation
    References

    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