Complete Growth Medium
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.
Cell culture dishes are coated with CellMatrix Basement Membrane Gel (ATCC® No. ACS-3035) to provide a surface for the attachment of iPSCs.
Volumes used in this protocol are for a 75 cm2 flask.
- Thaw CellMatrix Gel on ice and swirl gently to mix. Important: CellMatrix Gel will solidify in 15 to 30 minutes above 15°C. Keep CellMatrix Gel, vials and pipette tips on ice at all times to prevent CellMatrix Gel from solidifying. If air bubbles form, they may be eliminated by centrifuging CellMatrix Gel at 300 x g for 10 minutes at 2°C to 8°C.
- Determine the appropriate volume per aliquot based on concentration and usage.
Example: 2 mL of CellMatrix at 150 μg/mL is required to coat one 6-cm dish. To coat two 6-cm dishes, prepare as follows:
Dilute CellMatrix in DMEM:F12 to a working concentration of 150 μg/mL. For instance, if the protein concentration of CellMatrix (on certificate of analysis) is 14 mg/mL, then:
(4 mL) x (0.15 mg/mL)/(14 mg/mL) = 0.043 mL. Therefore, add 43 μL CellMatrix directly in 4 mL cold DMEM: F-12 Medium
- Cell culture dishes coated with CellMatrix Basement Membrane Gel should be incubated at 37°C for one hour. Aspirate coating solution and immediately plate the cells. It is critical that the coating does not dry out.
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 required when cells are recovering from thaw; it is recommended when the cells are being passaged.
This protocol is designed to passage stem cell colonies cultured in a 6 cm dish, using EDTA Dissociation Reagent 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.
EDTA Dissociation Reagent:
500ul 0.5M EDTA
in 500ml Calcium/Magnesium free PBS
Sterile filter and store at 4°C
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 EDTA 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 EDTA Dissociation Reagent working solution to the dish.
- Incubate at 37°C for 2 to 5 minutes.
- Aspirate the EDTA 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.
- 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