CB-iPSC

Human iPSCs can be cultured with the Human iPSC Culture Medium Kit (Cat.No.: TM020) together with feeder cells such as MITC Inactivated Rat Embryonic Fibroblast (REF) Feeder Cells (T2017) or MITC Treated Immortalized Mouse Embryonic Fibroblast Feeder Cells (T2009).

To improve survival, it is recommended to use ROCK inhibitor (10uM) and conditioned medium immediately upon thawing. Conditioned medium can be made by adding Human iPSC Culture Medium (TM020) to inactivated MEF (T2009) or REF (T2017) for one day and harvest the supernatant.

To improve survival, it is recommended to use ROCK inhibitor (10uM) and conditioned medium upon thawing. Conditioned medium can be prepared by adding the Human iPSC Culture Medium (TM020) to inactivated MEF (T2009) or REF (T2017) for one day and harvest the supernatant.

1) One day before thawing or passaging iPSCs, prepare feeders in 0.1% gelatin-precoated 6-well TC plates (37C for ~1 hour). Seed ~3×10^5 feeder cells per well. 2) Thaw CB-iPSCs by immersing the vial in 37C water bath for 1-2 minutes. Gently pipette 2-3 times and transfer all the cells to a 15 or 50-ml tube with 5-10 ml TM020 medium. 3) Spin down at 200g for 3-5 minutes. Carefully aspirate all the supernatent and resuspend cells in 2 ml TM020 medium. Add Rock inhibitor to increase cell survival. Aspirate MEF medium in the wells with feeder and transfer thawed cells to the feeder for culture. 4) Starting the next day, change 2 ml medium every day or every other day without Rock inhibitor. 1-2 days later, you should be able to see survived small colonies. 5) Every 5-7 days, split iPSCs. Aspirate out medium and add 1 ml 0.5 mM EDTA/PBS. Incubate 3-5 minutes at 37C. When colonies start to curl up, use 1 ml pipettor to gently break down colonies into clumps of 10-50 cells. transfer 10-25% colonies into a new well precoated with feeders. 6) After 1-2 passages on feeder cells, iPSCs can be cultured with Matrigel-precoated 6-wells using TeSR medium.

1) 4-5 days after cell split, when iPSCs reach a confluency of 30-60%, aspirate out spend medium and add 1ml 0.5mM EDTA/PBS. Incubate cells at 37C for ~5min. 2) When colonies start to detach, gently harvest all the cells into a 5-ml or 15-ml or 50-ml tube by pippeting 1-2 times. Avoid multiple pippetting. Breaking down clumps into single cells may substantially decrease cell survival. May add iPSC medium to harvest all the cells. 3) Spin down at 200g for 2-3 minutes. 4) Carefully aspirate out supernatent and gently resuspend cells in 0.5ml iPSC medium. 5) Add 0.5ml freezing medium (TM023) and ROCK inhibitor. Mix well by flipping the vial and transfer the medium to a cryovial. 6) Finally transfer the vial to -80C freezer for short-term storage (days to weeks). After cells are frozen, you may then transfer the vial to liquid nitrogen tank for long-term storage (years).

IPSCs require seeder cells before thawing and plating the IPSC. Seed ~3×10^5 feeder cells in each well of a 6-well plate. After thawing the iPSCs, put all the cells from the vial into 1 well. After several days, you may start splitting the iPSCs into multiple wells depending on how many cells survived. The density mentioned above is for the seeder cells which should be at 3×10^5. Afterwards, please plate the entire IPSC into one well at 10^6 concentration.