its depend on many factors which you should check on your sample, firstly the permeability of cryoprotective inside your sample, secondly the minimum toxicity of your vitrification solution (and this depend on type and percentage of CPAs, as well to exposure time), then warming procedure after quenching in LN......

Robert,

Yes, you can. However if your plan is to work on differentiated adipocytes. The differentiation capacity decline progessivelly to be almost none at P4. 75% at P1, 60% at P2, 25% at P3, less than 5% at P4 . If you don't freeze and split to use it increase at 85% on P1, 65% on P2, 35% on P3 and less than 5% at P4

We grow them in DMEM:F12 that contains 10% FCS, 33 µM biotin, 17 µM D-pantothenate, 100 nM insulin (stock 1000X in 10mM HCL), 10 nM hydrocortisone (stock 5000X in PBS), 1 nM bFGF (Stock 1000X in PBS), 1 nM EFG (stock 1000X in PBS). Without EGF the differentiation yield drop by 50% to be almost none at P3.

We freeze them in the same media without the growth factors, add an extra 10% of serum and 10% DMSO. Store them for 24 hours at -80C in Mr Frosty and move them to liquid nitrogen the next day,

Hope it can help.

Jacques

i would suggest you to store your cells at P3, when pre-adipocytes are pure. Differentiation potential of these cells is maintained even till P20.

Good Luck

Fantastic answers, thank you. I will let you know how I get on, and the details of our protocol.

Hi Robert, I came upon your interesting question a little late. I have not worked with preadipocytes but I have worked with many other cell types. The suggestion to work with the media you culture in is often the way to get started in combination with 5-10% DMSO. There are also commercially available serum-free solutions available that you can use if you are working serum-free. If you are working with small cell numbers yield may be important as well as the relative viability of the cells post-cryopreservation. Another issue we see with many cell types is a significant drop in cell numbers on the first and second days after placing back in cell culture due to apoptosis. These issues can be minimized by using optimized cryopreservation solutions.

Hi,

Somebody have worked with Human primary pre-adipocytes of ATCC (ATCC® PCS-210-010™)??

We are going to work with them, and I don´t know if these answer are appropriate for them.

Thank you!

How do passage and cryopreserve the cells after isolation?? Whats the next step after isolation and purification of the cell? I already cultured the cells after tissue extraction but what is next??