ES-D3 [D3]

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.

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.

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.

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.

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.

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.

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.

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