Regular maintenance of human pluripotent stem cells
Narayana Yadavalli, Shawn M. Ferguson
Abstract
This protocol describes the regular maintenance and passaging human iPSCs.
Attachments
Steps
Day 1
Pre coat a 6 well dish with Matrigel matrix for 24h 0m 0s or 1 hour.
Thaw the frozen iPSCs by placing the vial in 37°C water bath for 0h 2m 0s.
After thawing, spray the tube with 70% ethanol and place in biosafety cabinet.
Aspirate the cells into 15 ml falcon tube and add 4mL cold E8 media containing rock inhibitor.
Spin down the tube at 0.3rcf.
Aspirate the supernatant and resuspend in fresh E8 media containing rock inhibitor.
Remove Matrigel matrix and dispense appropriate number of cells onto Matrigel coated dishes.
Day 2
Remove the E8 media containing rock inhibitor and feed cells with E8 media without rock inhibitor.
Feed every day with fresh media till plate gets 60-70% confluency.
Cells were passaged every 5 days with E8.
iPSC passaging with 0.5 mM EDTA solution: Day 1
Pre coat a 6 well dish with Matrigel matrix for 24h 0m 0s or 1 hour.
Remove the culture media.
Rince 1X with sterile PBS.
Add 1mL of EDTA solution.
Place in the incubator for 5-7 minutes.
Bring the plate into biosafety cabinet and add 2mL E8 media containing rock inhibitor.
Dispense all the cell suspension into 15 ml falcon tube.
Spin down the tube at 0.3rcf.
Aspirate the supernatant and resuspend in fresh E8 media containing rock inhibitor.
Remove Matrigel matrix and dispense appropriate number of cells onto Matrigel coated dishes.
iPSC passaging with 0.5 mM EDTA solution: Day 2
Remove the E8 media containing rock inhibitor and feed cells with E8 media without rock inhibitor.
Cells were passaged every 5 days with E8.
