Subculturing
Cell culture dishes are coated with CellMatrix Basement Membrane Gel (ATCC® No. ACS-3035) to provide a surface for the attachment of iPSCs.
Coating Procedure:
- 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.
Volumes used in this protocol are for a 75 cm
2 flask.
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 on CellMatrix Gel-coated dishes containing 5 mL Pluripotent Stem Cell XF/FF medium/6-cm dish.
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.
Reconstitution of Stem Cell Dissociation Reagent:
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.
- 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.
- Filter sterilize through a 0.22 μm filter membrane.
- Aliquot into working volumes according to routine usage.
- 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.
- 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 DMEM:F12.
- Add 2 mL of Stem Cell Dissociation Reagent working solution to the dish.
- Incubate at 37°C for 2 to 5 minutes.
- 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 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 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. 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 on CellMatrix Gel-coated dishes containing 5 mL Pluripotent Stem Cell XF/FF medium/6-cm dish.
- 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).
