Human Embryonic Stem Cell Line H9 (Wa09) , #H9 Feeder Free

Recovery of Frozen Cells: For Suspension Cell passaging, the following methods can be used as reference: 1) Shake the cryotube containing 1mL of cell suspension quickly within seconds in a 37°C water bath to thaw. 2) Add it to a centrifuge tube containing 4-6mL of complete medium and mix well. Centrifuge at 1000rpm for 3-5mins. 3) Discard the supernatant, and resuspend the cells in complete medium. 4) Then add the cell suspension to a culture flask (or dish) containing 6-8ml of complete medium and culture overnight at 37°C. 5) Observe the cell growth and cell density under a microscope the next day. 6) Cell subculture: If the cell density reaches 80%-90%, subculture can be performed. For Adherent Cell passaging, the following methods can be used as reference: 1) Shake the cryotube containing 1mL of cell suspension quickly within seconds in a 37°C water bath to thaw. 2) Add it to a centrifuge tube containing 4-6mL of complete medium and mix well. Centrifuge at 1000rpm for 3-5mins. 3) Discard the supernatant, and rinse the cells 1-2 times with PBS without calcium and magnesium ions. 4) Add 0.25% (w/v) trypsin-0.53 mM EDTA to the culture flask (1-2 mL for T25 flask, 2-3 mL for T75 flask), 5) Place in a 37°C incubator for digestion for 1-2 minutes (difficult-to-digest cells can be properly prolonged for digestion time), 6) Observe the cell digestion under a microscope. If most of the cells become round and fall off, quickly take it back to the operating table, tap the culture flask a few times, and add 3-4 ml of culture medium containing 10% FBS to terminate digestion. 7) Gently mix and aspirate, centrifuge at 1000rpm for 3-5min, discard the supernatant. Add 1-2mL culture medium and mix evenly. 8) Divide the cell suspension into new T25 bottles at a ratio of 1:2. 9) Add 6-8ml of new complete culture medium prepared according to the instructions to maintain cell growth vitality, and perform subsequent passages at a ratio of 1:2~1:5 as per actual conditions.