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.
