ASC52telo, hTERT immortalized adipose derived Mesenchymal stem cells

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.

Recovery of Frozen Cells

To ensure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If storage of the frozen culture is necessary upon arrival, store the vial in liquid nitrogen vapor phase and NOT at -70°C. Storage at -70°C will result in loss of viability.

SAFETY PRECAUTION: Always use protective gloves and clothing and wear a full face mask when handling frozen vials. Some vials leak when immersed 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 vessel exploding or blowing off its cap with dangerous force creating flying debris.

1.     Prepare a 25-cm² or a 75-cm² culture flask containing the recommended complete culture medium. Prior to the addition of the vial contents, the vessel containing the growth medium should be placed in the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6) and to avoid excessive alkalinity of the medium during recovery of the cells.

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 out of the water. 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 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 of complete culture medium and centrifuge the cell suspension at approximately 275 x g +/- 125 x g for 5 to 7 minutes. 

5.     Discard the supernatant and resuspend the cells in fresh growth medium. Count the cells and seed new culture flasks at a density of 5,000 viable cells per cm².

6.     Incubate the culture at 37°C in a suitable incubator. 

7.     A 5% CO₂/95% air atmosphere is recommended if using the medium described on this product sheet.

Protocol: 

1. Passage immortalized adipose-derived MSCs when the culture has reached approximately 80% confluence.

 2.  Warm both the Trypsin-EDTA for Primary Cells (ATCC PCS-999-003) and the Trypsin Neutralizing Solution (ATCC PCS-999-004) to room temperature prior to dissociation. Warm the complete growth medium to 37°C prior to use with the cells.

 3. For each flask, carefully aspirate the spent media without disturbing the monolayer.

 4. Rinse the cell layer one time with 3 to 5 mL D-PBS (ATCC 30-2200) to remove residual medium.

 5. Add prewarmed trypsin-EDTA solution (1 to 2 mL for every 25 cm²) to each flask.

 6. Gently rock each flask to ensure complete coverage of the trypsin-EDTA solution over the cells, and then aspirate the excess fluid off of the monolayer.

 7. Observe the cells under the microscope. When the cells pull away from each other and round up (typically within 3 to 5 minutes), remove the flask from the microscope and gently tap it from several sides to promote detachment of the cells from the flask surface.

 8. When the majority of cells appear to have detached, quickly add an equal volume of the Trypsin Neutralizing Solution (ATCC PCS-999-004) to each flask. Gently pipette or swirl the culture to ensure all of the trypsin-EDTA solution has been neutralized.

 9. Transfer the dissociated cells to a sterile centrifuge tube and set aside while processing any remaining cells in the culture flask.

 10. Add 3 to 5 mL D-PBS (ATCC 30-2200) to the tissue culture flask to collect any additional cells that might have been left behind.

 11. Transfer the cell/D-PBS suspension to the centrifuge tube containing the trypsin-EDTA- dissociated cells.

 12. Repeat steps 10 and 11 as needed until all cells have been collected from the flask.

 13. Centrifuge the cells at 275 x g +/- 125 x g for 5 minutes.

 14. Aspirate neutralized dissociation solution from the cell pellet and resuspend the cells in 2 to 8 mL fresh, prewarmed, complete growth medium.

 15. Count the cells and seed new culture flasks at a density of 5,000 viable cells per cm².

 16. Place newly seeded flasks in a 37°C, 5% CO2 incubator for at least 24 to 48 hours before processing the cells further. 

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.

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.