(AICS-0074-026) MONO-ALLELIC mEGFP-TAGGED SOX hiPSCs Thawing/freezing question

1

Hello everyone, we recently ordered MONO-ALLELIC mEGFP-TAGGED SOX catalog ID AICS-0074-026.
We have technical questions regarding handling the cells right after thawing and when freezing:

  1. Can the cells be directly thawed in Essential 8 Flex medium and plated on vitronectin-coated plates rather than mTSER medium and Matrigel coating? If this is not possible, should we gradually transition from mTSER to Essential 8 Flex?
  2. According to the protocol, the freezing medium is mTeSR1 with 30% KSR, and 10% DMSO, so I assume the vial we ordered is in the same condition, could you confirm it?
  3. Since I plan to culture the cells in E8 flex, shall I freeze them, according to the above, using E8 Flex +30% KSR +10% DMSO?

Thank you very much in advance.

2

Hello and sorry for the late response. To address your questions:

  1. I would not recommend to thaw directly into E8 and onto vitronectin but rather onto Matrigel coated plates in mTESR medium. I personally don’t have experience on how to transition the cells onto Vitronectin and E8 but I do know someone who has purchased this line and adapted them onto Vitronectin and I believe they culture in E8 medium. I am happy to start an email to put you two into contact. Email me at jacquelines@alleninstitute.org.
  2. Yes, the vial you ordered is frozen in mTESR1 with 30% KSR and 10% DMSO.
  3. I would recommend that you first thaw them into MG coated plates in mTESR1 medium, freeze several vials from the thaw (with the freeze media we use as I addressed in question 2), then freeze additional vials from a recovery passage using (mTESR1, 30% KSR and 10% DMSO). That way you have cells to go back to in case anything goes wrong during the transition to Vitronectin and E8.

Best, Jacqueline

3

Thank you very much for your response.

I will I was initally planning to transition the cells by first plating them in mTESR1/vitronectin, and then gradually switch to E8 flex as follows: day 2: 75% mTESR 25% E8 + Ri + antibiotic, day 3: 50-50 E8-mTESR + antibiotic, day 4: 25% mTESR 75% E8 + antibiotic and finally day 5: 100% E8 + antibiotic, all days in vitronectin-coated plates. Do you think that this is a good idea?
As I don’t see the point transitioning in such a way the coating as well.

In any case, I will definetly freeze some cells in mTESR to make sure.
In this context, I was wondering if it is ok to use mTESRPlus instead of mTESR1.

Looking forward to hearing from you.