Stem Cell Dissociation Reagent

Detailed protocols relating to this product are available online at ATCC Stem Cell Culture Guide.

Preparation of 0.5 U/mL Working Solution 

Lyophilized proteins tend to be hygroscopic. Bring the vial of Stem Cell Dissociation Reagent to room temperature before opening. The vial should not be cool to the touch. Once opened, the lyophilized material should be stored desiccated. The specific activity of the Stem Cell Dissociation Reagent is found on the product label. Prepare a 0.5 U/mL working solution by dissolving the Stem Cell Dissociation Reagent powder in the appropriate volume of DMEM: F-12 Medium (ATCC 30-2006). Filter sterilize the solution through a 0.22 µm membrane and aliquot into working volumes according to usage. Dissolve the appropriate amount of Stem Cell Dissociation Reagent in DMEM: F-12 medium to prepare a 0.5 U/mL working solution. 

Example:  To prepare 40 mL of a 0.5 U/mL working solution:

Specific activity of Stem Cell Dissociation Reagent (on certificate of analysis): 1.46 U/mg

[(40 mL)(0.5 U/mL)]/(1.46 U/mL)= 13.7 mg

Dissolve 13.7 mg Stem Cell Dissociation Reagent in 40 mL DMEM: F-12.

Store aliquots at -20°C for up to three months. Avoid repeated freezing and thawing. Once thawed, the aliquots may be kept at 2°C to 8°C for up to two weeks.

Dissociation Protocol

This protocol is designed for the dissociation of cells in a 6-cm dish. Volumes should be adjusted according to the size and number of the tissue culture vessels to be processed.

Stem cell culture medium: Pluripotent Stem Cell SFM XF/FF (ATCC ACS-3002) is recommended for feeder-free culture and Pluripotent Stem Cell SFM XF (ATCC ACS-3001) is recommended for feeder-dependent culture (e.g., mouse embryonic fibroblasts (MEF) or human foreskin fibroblasts (HFF).

1. Culture hESCs/hiPSCs in stem cell culture medium until cells reach 80% confluency.
2. Warm an aliquot of Stem Cell Dissociation Reagent working solution to room temperature.
3. Aspirate and discard the stem cell culture medium. 
4. Rinse (add and aspirate) the cells twice with 4 mL of Dulbecco’s Phosphate Buffered Saline (PBS) (ATCC 30-2200).
5. Add 2 mL of Stem Cell Dissociation Reagent working solution to the dish. 
6. Incubate at 37°C for 10 to 15 minutes or until the individual colonies begin to loosen and fold back. View the dish under the microscope starting at 5 minutes as incubation time may vary depending on the cell line and colony size.
7. Aspirate the Stem Cell Dissociation Reagent and gently rinse (add and aspirate) the colonies with 4 mL of DMEM: F-12 Medium, taking care not to dislodge the cells during manipulation.   
8. Add 2 mL of stem cell culture medium to the dish, and detach the cells by pipetting up and down several times with a 1 mL tip. Take care not to over-pipette the culture into a single-cell suspension as single cells will not establish colonies after seeding.
9. Transfer the cell aggregates to a 15 mL conical tube.
10. Add an additional 4 mL of stem cell culture medium to the dish to collect any remaining cells. Transfer this rinse to the 15 mL conical tube containing the cell aggregates.
11. Centrifuge cells at 200 x g for 5 minutes.
12. Aspirate the supernatant and discard. Gently tap the bottom of the tube to loosen the cell pellet.
13. For each dish processed, add 2 mL of stem cell culture medium in the presence of 10 µM ROCK Inhibitor Y27632* (ATCC ACS-3030) to the 15 mL tube.
    
    Note: Addition of ROCK inhibitor has been shown to increase the survival rate during subcultivation and thawing of human iPSCs. The use of ROCK inhibitor may cause a transient spindle-like morphology effect on the cells. However, the colony morphology will recover after subsequent media change without ROCK inhibitor.¹



14. Gently resuspend the pellet by pipetting up and down 2 to 3 times with a 1 mL tip, maintaining the small cell aggregates.
15. Plate the cells as desired on feeder or feeder-free cultures. The presence of 10 µM ROCK Inhibitor Y27632 in the stem cell culture medium is recommended.

References

1. Li X. et al, ROCK inhibitor improves survival of cryopreserved serum/feeder-free single human embryonic stem cells. Human Reproduction, Vol.24 No.3 pp 580-589, 2009 PubMed 19056770

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.