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Hi,
We would like to thaw, expand and maintain MONO-ALLELIC mEGFP-TAGGED SOX2 cells in E8+ flex medium (Invitrogen) on Vitronectin-N coated wells, which is different method comparing to the protocol provided by the Coriell. Coriell suggests mTeSR medium and Matrigel coating. Could you give me some advice on how to adapt to the E8+flex culture successfully? Any suggestion and concerns? We follow the protocol @ Generation and characterization of vestibular inner ear organoids from human pluripotent stem cells | Nature Protocols . Thanks!
We do not normally go outside our recommended culturing conditions and have limited experience using other culturing methods, but we do have a few suggestions about how to make the transition to other cell culture media and substrates. We would recommend that you make the transition in a couple of stages. To start, we suggest that you follow our culturing protocol and thaw into mTESR and Matrigel and culture the cells for a recovery passage using our standard conditions and then freeze a bank of cells in the recommended mTESR+30%KSR+10%DMSO freeze media. At the next passage, we would suggest you make the change to vitronectin and then let those cells acclimate to the new substrate while still in mTESR media. This change in substrate might require another few passages until cells appear stabilized which might be assessed by both growth and morphology. The next stage would be to make the media change, but we are uncertain if you should do a total media switch or if you should gradually swap to your desired culture media. This step might take a bit of trial and error to see what works. You can always go back to your banked cells and retry if the cells do not recover well which is why it is very important to have that initial cell bank in the standard culturing conditions. We hope that these suggestions will be helpful and that you are successful in transitioning the cells!
