Remove Human ES cells from liquid nitrogen storage tank. Fill out a freeze/thaw form.
Thaw cryovial by gently swirling in waterbath until only a small ice pellet remains, being careful not to completely submerge the cryovial under water.
Completely submerge cryovial in 95% ethanol.
Very gently, pipet cells from the vial into a 15 ml conical centrifuge tube.
Slowly, add 9.5ml media dropwise to reduce osmotic shock. While adding media, gently mix the cells in the tube (by gently tapping the tube with a finger.)
Centrifuge 1000rpm for 5 minutes.
Wash cells by resuspending with 3ml media.
Centrifuge 1000rpm for 5 mintutes.
Resuspend in 2ml and add 0.5ml per well of a 4 well plate that has MEFs already plated on it.
Change media daily, however it may take 2 weeks before cells are ready to be expanded.
Note: Wear safety glasses when removing cells from liquid nitrogen until the cells are completely thawed.
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