Add 5 ml concentrates to 45 ml MEM medium.

Add 5 ml of the concentrated supplement to 45 ml MEM medium.

The shelf-life is at least 1 month at 4C. After one month storage at 4C, there is no obvious change in quality.

If the basal media is stored at 4C, it can last at least a year. If the supplements are stored at 4C, it can last 1-2 months. If the supplements are stored at -20C, it can last approximately one year as well. Multiple freezing and thawed is not encouraged and we recommend making aliquots before freezing. Based on our experience, there is no obvious change in quality after 1-2 freeze-thaw cycles.

The medium contains low-level serum and multiple supplements including growth factors. Generally, MSCs grow better in fibronectin-precoated non-TC plates and in hypoxia.

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).

No