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ES-D3 [D3]

CRL-1934

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

Biosafety Icon BSL 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

General

Specific 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.


Characteristics

Growth properties
Adherent
Derivation
The clonal embryonic stem cell line ES-D3 was derived from blastocysts of a 129S2/SvPas mouse.
Strain
129S2/SvPas
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. 

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

The base medium for this cell line is Mouse ES Cell Basal Medium (ATCC SCRR-2011). To make the complete medium add the following components to 500 mL base medium and mix by swirling gently: 

  • 1 mL (0.1 mM final concentration) 2-mercaptoethanol (Life Technologies Cat. No. 21985-023)
  • 56 to 84 mL (10% to 15% final concentration) ES-Cell Qualified FBS (ATCC SCRR-30-2020)  
  • 1,000 U/mL mouse leukemia inhibitory factor (LIF) (Millipore Cat. No. ESG1107). *NOTE: LIF can be omitted from the culture media as long as 56-X.2 (MITC-treated STO) is used as a feeder layer since these cells produce LIF.

Complete Growth Medium for Mouse ES Cells is stable for 14 days when stored at 2°C to 8°C.

Temperature
37°C
Atmosphere
95% Air, 5% CO2
Handling procedure
Note: 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).
  1. It is recommended that the 56-X.2 MITC-treated feeder cells be plated 24 hours before use at 6 x 106/T75 flask or 2 x 106/T25 flask in order to obtain a 100% confluent monolayer for stem cells growth. 
  2. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap above the water level. Thawing should be rapid (approximately 2 minutes). 
  3. 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.
  4. 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. 
  5. Resuspend cell pellet with the recommended complete medium (see the specific batch information for the culture recommended dilution ratio)and dispense into a 75 cm2 culture flask.
  6. 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.
Subculturing procedure
Feeder Layer Preparation

56-X.2 cells should be seeded one day prior to use.

The medium to use when initiating the feeder layer is DMEM with 10% FBS.  This medium is prepared by aseptically combining:

56 mL FBS (ATCC 30-2020)

500 mL DMEM (ATCC 30-2002)

  1. Thaw the frozen feeder cell vial(s) per the ATCC product sheet. Wipe, spray, and/or soak the ampoule(s) with 70% ethanol (or equivalent disinfectant) and allow the ampoule(s) to dry.  
  2. Aseptically open the ampoule(s). Withdraw cells and transfer to a sterile 15 mL centrifuge tube. If more than one ampoule was thawed, the contents may be pooled into a single centrifuge tube. 
  3. Slowly add pre warmed feeder layer medium to the centrifuge tube by running 10 ± 2 mL down the side. Centrifuge the tube at 275 ± 125 x g for 10 ± 2 minutes.  Aseptically remove and discard the supernatant from the centrifuge tube. 
  4. Resuspend the cell pellet with feeder layer medium so that a final volume of 10 mL is achieved. Count the resuspended cells. Calculate the volumes of cell suspension and feeder layer medium needed to plate the feeder. Aseptically transfer the calculated volumes of cell suspension and feeder layer medium to appropriate vessel(s). 
  5. Incubate the culture in a CO2 incubator set to 5% ± 1% CO2 and 35.0 to 37.0 °C until ready for use.
  6. Plate irradiated (12,000 Rads) STO feeder layer at approximately 8.0 X 10e4 viable cells/ cm2 at least one day before plating the ES cells. After one day of incubation the vessel(s) are ready for use in CRL-1934 cultures.

Initiation of CRL-1934 Cell Culture

  1. Thaw a vial of CRL-1934 cells per the ATCC product sheet - Handling Procedure for Frozen Cells. Wipe, spray, and/or soak the ampoule(s) with 70% ethanol (or equivalent disinfectant) and allow the ampoule(s) to dry. 
  2. Aseptically open the ampoule(s). Withdraw cells and transfer to a sterile 15 mL centrifuge tube. 
  3. Slowly add pre-warmed complete growth medium to the centrifuge tube by running 12 ± 2 mL down the side. Centrifuge the tube at 275 ± 125 x g for 10 ± 2 minutes. Aseptically remove and discard the supernatant from the centrifuge tube. 
  4. Resuspend the cell pellet with 10 mL of culture medium. Aseptically transfer the contents of the centrifuge tube to a T75 flask containing the prepared 56-X.2 feeder layer. Add sufficient culture medium to the flask to bring the final volume to 15mL. NOTE: Remove the feeder layer media from the flask before adding the CRL-1934 cell suspension.
  5. Incubate the culture in a CO2 incubator set to 5% ± 1% CO2 and 35.0 to 37.0 °C. Observe and examine the culture every 1-2 days. If a fluid renewal/addition is needed, perform the fluid renewal/addition. Aseptically remove the culture medium from the flask and discard.  Add an equivalent volume of fresh culture medium to the flask.  Alternatively, perform a fluid addition by adding fresh culture medium to the flask without removing the existing medium. Return the culture to the incubator after fluid renewal/addition.

Subculture before the CRL-1934 colonies are close to or touching each other. The CRL-1934 cells should never become 100% confluent (although the 56-X.2 feeder cells may be 100% confluent). Attached cells are subcultured using 0.25% Trypsin 0.53 mM EDTA (ATCC 30-2101). The action of the 0.25% Trypsin – 0.53mM EDTA (ATCC 30-2101) is halted by adding culture medium to the detached cells. A split ratio of 1:3 to 1:10 is used when subculturing.


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
T Doetschman
Cross references
GenBank U20290 Mus musculus V-1 protein mRNA, complete cds.
GenBank NM_007795 Mus musculus cardiotrophin 1 (Ctf1), mRNA.
GenBank NM_008098 Mus musculus granule cell differentiation protein (Gcdp), mRNA.

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.

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

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