ES-D3 [D3] (ATCC® CRL-1934)

Organism: Mus musculus, mouse  /  Cell Type: embryonic multipotent stem cell  /  Tissue: embryo  / 

Organism Mus musculus, mouse
Tissue embryo
Cell Type embryonic multipotent stem cell
Product Format frozen
Morphology spherical colony
Culture Properties adherent
Biosafety Level 1
Age embryo, blastocyst
Strain 129S2/SvPas
Applications

The cells spontaneously differentiate into embryonic structures in the absence of a feeder layer or conditioned medium. 

Undifferentiated cells can be genetically modified by gene targeting techniques.


Storage Conditions liquid nitrogen vapor phase
Derivation
The clonal embryonic stem cell line ES-D3 was derived from blastocysts of a 129S2/SvPas mouse.
Comments

The cells spontaneously differentiate into embryonic structures in the absence of a feeder layer or conditioned medium.  They can be maintained in the undifferentiated state by frequent subculture (every 2 to 3 days) on confluent feeder layers (STO cells) arrested with Mitomycin-C (see ATCC 56-X.2; MITC- STO cells).

Fibroblast-like feeder layer cells are present in the ampules sent by ATCC.

Note: These ES-D3 cells are not germline competent. 

Complete Growth Medium Grow ES cells in Mouse ES Cell Basal Medium (ATCC SCRR-2011) that has been supplemented with the following components:
1. 0.1 mM 2-mercaptoethanol (Life Technologies Cat. No. 21985-023)
2. 1,000 U/mL mouse leukemia inhibitory factor (LIF) (Millipore Cat. No. ESG1107)
3. 10% to 15% ES-Cell Qualified FBS (ATCC® SCRR-30-2020) or an ES cell qualified serum replacement
Complete Growth Medium for Mouse ES Cells is stable for 14 days when stored at 2°C to 8°C.
Subculturing
  1. Plate irradiated (12,000 Rads) STO feeder layer at approximately 5.0 to 6.0 X 106 cells/ 75 cm2 (confluent monolayer) at least one day before plating the ES cells.
    Note: If the colonies are close to or touching each other the culture is overgrown
  2. Remove and discard culture medium.
  3. Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum, which contains trypsin inhibitor.
  4. 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.
  5. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
  6. Add appropriate aliquots of the cell suspension to new culture vessels pre-plated with new feeder layer.
  7. Incubate cultures at 37°C.
Subcultivation Ratio: A subcultivation ratio of 1:4 to 1:10 is recommended
Medium Renewal: Every 2 to 3 days
Cryopreservation
Complete growth medium supplemented with 5% (v/v) DMSO
Cell culture tested DMSO is available as ATCC Catalog No. 4-X.
Culture Conditions
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37°C
Growth Conditions: To maintain the cells in the undifferentiated state they must be grown on confluent feeder layers of irradiated STO cells (see 56-X, irradiated STO cells).
Name of Depositor T Doetschman
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

Cross References

Nucleotide (GenBank) : U20290 Mus musculus V-1 protein mRNA, complete cds.

Nucleotide (GenBank) : NM_007795 Mus musculus cardiotrophin 1 (Ctf1), mRNA.

Nucleotide (GenBank) : NM_008098 Mus musculus granule cell differentiation protein (Gcdp), mRNA.

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