Post thaw day 1, perform a 100% medium change and remove all cells that did not attach. Perform a 100% medium change every day. Passage the cells every 4 to 5 days (80% confluent) at an appropriate split ratio (a 1:4 split ratio is recommended). If the colonies are close to, or touching each other, the culture is overgrown. Overgrowth will result in differentiation.
ROCK Inhibitor Y27632 is not necessary each time the culture medium is changed. It is recommended when the cells are recovering from thaw or being passaged.
This protocol is designed to passage stem cell colonies cultured in a 6 cm dish, using Stem Cell Dissociation Reagent to detach the cell colonies. Stem Cell Dissociation Reagent is stored as a 0.5 U/mL working solution at -20°C. 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. 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 product label): 1.46 U/mg
(40 mL)(0.5 U/mL) = 13.7 mg.
Dissolve 13.7 mg Stem Cell Dissociation Reagent in 40 mL DMEM: F-12.
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.
- Warm an aliquot of Stem Cell Dissociation Reagent working solution to room temperature.
- Aspirate and discard the stem cell culture medium.
- Rinse the cells twice by adding and discarding 4 mL of D-PBS.
- Add 2 mL of Stem Cell Dissociation Reagent working solution to the dish.
- Incubate at 37°C for 10 to 15 minutes or until the edges of the individual colonies begin to loosen and fold back. View the dish under the microscope starting at 5 minutes as incubation time will vary depending on the cell line and colony size.
- 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.
- Add 2 mL of stem cell culture medium with ROCK Inhibitor Y27632 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. If cells are not detaching, use a cell scraper to detach the cells.
- Transfer the cell aggregates to a 15 mL conical tube.
- Add an additional 3 mL of stem cell culture medium with ROCK Inhibitor Y27632 to the dish to collect any remaining cells. Transfer this rinse to the 15 mL conical tube containing the cell aggregates.
- Centrifuge the cell aggregates at 200 x g for 5 minutes.
- Aspirate the supernatant and discard.
- Add 1 mL of stem cell culture medium with ROCK inhibitor Y27632. 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.
- Plate the cells as desired on feeder or feeder-free cultures.
- 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.
- 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.
- Take the Stem Cell Freezing Media from storage and swirl to mix. Keep cold. Decontaminate by dipping in or spraying with 70% alcohol.
- 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.
- Immediately transfer 1 mL each of the cell suspension into two labeled cryovials.
- 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.
- 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.
CellMatrix Basement Membrane Gel, ATCC ACS-3035
Pluripotent Stem Cell SFM XF/FF, ATCC ACS-3002