Box 1. Freezing and shipping skps

• SKPs can be frozen for long-term storage and then recultured with little change in growth characterisics. To initiate freezing, pellet SKP spheres from a culture that is ready to be passaged by centrifuging in a tabletop centrifuge at 1,200 r.p.m. for 5 min. Remove and save the conditioned medium (as described in text), and resuspend the sphere pellet in freezing medium consisting of 90% FBS and 10% DMSO. Put the resuspended cell suspension in a 2 ml screwtop cryovial, and place it in a slow freeze container at -80 °C. Do not dissociate the spheres prior to freezing because this decreases cell viability.
• Frozen SKPs should be shipped on dry ice. Upon receipt, frozen cells should be resuspended by thawing and immediately resuspending in 20 ml of wash medium to dilute the freezing medium. Mix the contents by repeated inversion of the tube.
• Pellet the spheres by centrifugation at 1,200 r.p.m. for 5 min.
• Resuspend spheres in 50% conditioned medium and 50% proliferation medium containing 40 ng ml^-1 FGF2, 20 ng ml^-1 EGF, and 2% B27. Put SKP suspension into a vented tissue culture flask and place in a 37 °C incubator. Cells should be not be disturbed for 24–48 h, at which time they should be fed with fresh proliferation medium.

As an alternative method, SKPs can be shipped at 4 °C in conical tubes:

• Remove all medium and floating spheres from a 7–14 d old SKP culture, and collect it in a 10 or 50 ml conical tube.
• Add 20% fresh proliferation medium (i.e., 2 ml in 10 ml total). Make sure the conical tube is completely filled with medium to minimize oxidation and changes in pH during shipping. Filtered, conditioned medium should be added if additional volume is required.
• Upon receipt, SKPs should be resuspended by trituration with a 10 ml disposable, plastic pipette. This will also dissociate any spheres that may have aggregated during transport.
• Remove entire contents of the tube into a vented tissue culture flask and place in a 37 °C incubator. Cells should be not be disturbed for 24–48 h, at which time they should be fed with fresh proliferation medium.