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Iscove's Modified Dulbecco's Medium (IMDM; ATCC^® No. 30-2005): RPMI-1640 Medium (ATCC^® No. 30-2001) (4:1) + 10% FBS (ATCC^® No. 30-2020) + additional 1% L-glutamine (ATCC^® No. 30-2214)

Coating description: Dilute rat tail type I collagen (BD Biosciences, Catalog No. 354236) to 50 μg/ml. Add 7.5 ml coating buffer to flask and incubate at room temperature for one hour. Carefully aspirate remaining solution. Rinse flask 2 times to remove acid, using 1x DPBS. Coated flasks may be used immediately or stored at 2-8^oC up to one week under sterile conditions.

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

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 5.0ml Ca++/Mg++ free Dulbecco's 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 5.0 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 5.0 ml of complete growth medium and aspirate cells by gently pipetting.
5. Transfer cell suspension to a centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes. Discard supernatant.
6. Resuspend the cell pellet in 10 ml fresh growth medium.
7. Add appropriate aliquots of the cell suspension to new coated culture vessels.
8. Incubate cultures at 37°C.

Rinner B, et al. Establishment and detailed functional and molecular genetic characterisation of a novel sacral cordoma cell line, MUG-Chor1. Int J Oncol. 40(2): 443-451, 2012. PubMed: 22002331