Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

At ATCC, the cells have been shown to form small clusters when cultured in collagen gel. The cell clusters stain positive with Newport Green which would suggest some level of insulin expression.

Volumes are given for a 75 cm² flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with Ca++/Mg++ free Dulbeccos phosphate-buffered saline (D-PBS) or 0.25% (w/v) Trypsin - 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
3. 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.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
5. Add appropriate aliquots of the cell suspension to new culture vessels. An inoculum of 1 X 10⁴ to 2 X 10⁴ viable cells/cm² is recommended.
6. Incubate cultures at 37°C.

Subcultivation ratio: A subcultivation ratio of 1:3 to 1:4 is recommended.

Medium renewal: every 2 to 3 days

Freeze medium: complete growth medium, 95%; DMSO, 5%

Storage temperature: liquid nitrogen vapor phase

Temperature: 37°C

Atmosphere: air, 95%; carbon dioxide (CO₂), 5%