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.

Coating Procedure:

Dilute CellMatrix in DMEM:F12 to a working concentration of 150 g/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 L CellMatrix directly in 4 mL cold DMEM: F-12 Medium

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.

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.

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.

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.

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ATCC-HYR0103 Human Induced Pluripotent Stem (IPS) Cells ATCC ...