Hello Fernanda Muckler,

The viability of cells on thawing can be affected if you haven’t carried out gradual freezing by reducing the temperature at approximately 1°C per minute using a controlled rate cryo-freezer or a cryo-freezing container such as Mr. Frosty. This is to prevent intracellular ice formation.

The viability of cells on thawing can also be affected if you haven’t carried out quick thawing to minimize any damage to the cell membrane. Quickly thaw the cells (< 1 minute) by gently swirling the vial in the 37°C water bath until there is just a small bit of ice left in the vial. Immediately transfer the cells to a large volume of pre-warmed cell growth medium (~10 mL/1-mL aliquot of cells). For the next step, you may go with/without centrifugation.

Rapid thawing is crucial for minimizing the formation of ice crystals within the cells, which can cause mechanical damage to

cell membrane. The optimal thawing rate is generally considered to be 37°C per minute, a rate that quickly surpasses the temperature range in which harmful ice crystals can form.

So, to get a good viability and cell revival after thawing, gradual freezing of cells and quick thawing must be followed.

Best.

Hello Malcolm,

Thanks for your answer, I'm afraid the freezing process probably was the issue. We don't have a Mr. Frosty, and as I freezed many vials in the same time, I probably let the cells suffer a little bit.

I'll try your tips next time.

Thanks a lot!

Hey Fernanda Muckler

I am not an expert with Caco-2 (I actually came across your question while searching about Caco-2 questions). However, I am also dealing with them lately. What I observe is similar to you, they grow vey slow but after 1 week they form more of "island like" structures and they got confluent afterwards. And when I passage after that (I divide a 80% confluent T25 to 2 x T25 flask). They became confluent in 3 days and now they are faster. So I think they need time after thawing.