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ATCC iPSCs have been adapted to feeder- and serum-free culture conditions.

The base medium for this cell line is Pluripotent Stem Cell SFM XF/FF (ATCC^® No. ACS-3002) which is a ready-to-use medium for serum-free and feeder-free iPSC culture. 

This protocol is designed to passage stem cell colonies cultured in a 6 cm dish, using Stem Cell Dissociation Reagent (ATCC ACS-3010) to detach the cell colonies. The recommended spilt ratio is 1:4. Volumes should be adjusted according to the size and number of the tissue culture vessels to be processed. 

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 reagent is found on the certificate of analysis. Dissolve the appropriate amount of Stem Cell Dissociation Reagent in DMEM: F-12 Medium to prepare a 0.5 U/mL working solution.

1. 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) x (0.5 U/mL)/(1.46 U/mg) = 13.7 mg Dissolve 13.7 mg Stem Cell Dissociation Reagent in 40 mL DMEM: F-12 Medium. 
2. Filter sterilize through a 0.22 μm filter membrane. 
3. Aliquot into working volumes according to routine usage. 
4. Store aliquots at -20°C for up to three months. Avoid repeated freezing and thawing. Thawed aliquots may be kept at 2°C to 8°C for up to two weeks. 

Note: Addition of ROCK inhibitor has been shown to increase the survival rate. 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. Warm an aliquot of Stem Cell Dissociation Reagent working solution to room temperature.
2. Aspirate and discard the stem cell culture medium.
3. Rinse the cells twice by adding and discarding 4 mL of DMEM:F12.
4. Add 2 mL of Stem Cell Dissociation Reagent working solution to the dish.
5. Incubate at 37°C for 2 to 5 minutes.
6. Aspirate the Stem Cell Dissociation Reagent and gently rinse the colonies with 4 mL of DMEM: F-12 Medium, taking care not to dislodge the cells during manipulation.  Aspirate the DMEM: F12 rinse and discard.
7. Add 2 mL of stem cell culture medium to the dish, and detach the cells by pipetting up and down 2 to 3 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. 
8. Transfer the cell aggregates to a 15 mL conical tube.
9. Add an additional 3 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.
10. Centrifuge the cell aggregates at 200 x g for 5 minutes.
11. Aspirate the supernatant and discard.
12. Add 1 mL of stem cell culture medium. Gently resuspend the pellet by pipetting up and down 2 to 3 times with a 1 mL tip, maintaining the small cell aggregates. Take care not to over-pipette the culture into a single-cell suspension as single cells will not establish colonies after seeding.
13. Plate the cells on CellMatrix Gel-coated dishes containing 5 mL Pluripotent Stem Cell XF/FF medium/6-cm dish.
14. Incubate the culture at 37°C in a humidified 5% CO2/95% air incubator. Perform a 100% medium change every day. Passage the cells every 4 to 5 days (80% confluent).

For optimal results, cryopreserve stem cell colonies when the cell cultures are 80% confluent. This protocol is designed to cryopreserve stem cell colonies cultured in a 6 cm dish.

1. Detach stem cell colonies from the dish as described in the recommended subculturing protocol (steps 1-11). Gently tap the bottom of the tube to loosen the cell pellet.
2. Take the Stem Cell Freezing Media from storage and swirl to mix. Keep cold. Decontaminate by dipping in or spraying with 70% alcohol.
3. Add 2 mL of cold Stem Cell Freezing Media to the tube. Gently resuspend the pellet by pipetting up and down 2 to 3 times with a 1 mL tip, maintaining the cell aggregates.
4. Immediately transfer 1 mL each of the cell suspension into two labeled cryovials.
5. Freeze the cells gradually at a rate of -1°C/min until the temperature reaches -70°C to -80°C. A cryopreservation container (e.g., CoolCell^® freezing container) may also be used.
6. The cells should not be left at -80°C for more than 24 to 48 hours. Once at -80°C, frozen cryovials should be transferred to the vapor phase of liquid nitrogen for long-term storage.