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Unpacking and storage instructionsCheck all containers for leakage or breakage.

Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.

Complete mediumEMEM (30-2003) supplemented with 10% FBS (30-2020)

Temperature37°C

Handling procedureTo insure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at -70°C.  Storage at -70°C will result in loss of viability.

Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water.  Thawing should be rapid (approximately 2 minutes). 

Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.

Transfer the vial contents to a centrifuge tube containing  9.0 mL complete growth medium and spin at approximately 125 x g for 5 to 7 minutes.

Resuspend cell pellet with the recommended complete growth medium (see the specific batch information for the culture recommended dilution ratio). and dispense into a 25 cm2 or a 75 cm2 culture flask.  It is important to avoid excessive alkalinity of the medium during recovery of the cells.  It is suggested that, prior to the addition of the vial contents, the culture vessel containing the complete growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6).

Incubate the culture at 37°C in a suitable incubator.  A 5% CO2 in air atmosphere is recommended if using the medium described on this product sheet.

Subculturing procedureVolumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.

Remove and discard culture medium.

Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.

Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes). Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach.  Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.

Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting. 

Add appropriate aliquots of the cell suspension to new culture vessels. 

Incubate cultures at 37°C.

Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:10 is recommended

Medium Renewal: Twice per week

Reagents for cryopreservationComplete growth medium supplemented with 5% (v/v) DMSO (ATCC 4-X)