Hi Nur,

If you freeze any cells kind of cells, and thaw, part of them will lose viability and attachment, it might be related to what stage of cell cycle cells were at that time, that is my guess, some stage might be able to survive cryopreservation better and other stage might be less,

Hi Subhash,

Thank you for your answer. Do you have any recommendation on how can I improve my cryopreservation method? Our MSCs are derived from Wharton's jelly, adipose tissue and dental pulp. Usually, what I practice here is I will harvest the cells when they have reached 80 - 90% confluency.

Should I add other growth factors in culture media (I already add bFGF in my culture media.) or coated my culture dish with ECM to help the cells attach to the culture surface?

if the cells are not attaching and possibly non-viable why would you want to increase attachment??

I used to culture MSCs (human bone marrow derived) we rarely used antibiotics or growth factors when expanding. we used 5ml DMSO +45mL FBS for cryopreservation of the cells. yes we would lose some cells but nothing too much.

As passage increases above 4-6 we would loose more cells.

There is a lot of reasons for that. You must be specific about cryoperservation teqnique you are using and how you are defreezing them. I got 99% viable cells each time through this:

Freezing: trypsin-edta 0.25% for deattach them in 1 minute ( 1 ml for T25 flask to minimize trypsin exposure time). Then resuspend in 10% dmso-90% fbs and immediately freeze them in -80 overnight. After that, carry them to liquid N2.

Defreezing: first add 13 ml complete medium ( fbs 10%) to 15 ml centrifuge tube then warm up to 37 to be ready. In the following, melt down your cryovial in 40 degree water untill you see small piece of ice. Meanwhile, tranfer the melted cells to centrifuge tube and mix througly by up and down arrangement. Centrifuge them at 400g for 8 min thrn resuspend the pellet in complete pre-warmed medium.

Best

Some of the cells are going to lose their viability. But before thawing the cells, first you prepare 5-7 ml complete media in which you are going to suspend the cells for centrifuge that should be at around 37 degree temp. then you thaw your cells in water bath at 37 degree but dont keep for long time. It should remain cold but melted and you add that cells in to the complete media already prepared and further you continue with centrifuge at 2000 rpm for 7 min. and re suspend pellet as required.

This will help you.