Cells were frozen at 10% DMSO, slowly using Ms Frosty. Apn thaw smears and aggregates of cells were observed and the cells could not be extracted from them. Any suggestions?
In the protocol we use, speed is the critical issue. HUVECs in particular are very sensitive to shock, therefore we typically reconstitute the cells at twice their intended cell density in growth medium and leave them on ice for ~15 minutes. After this step, we slowly add medium containing twice the concentration of DMSO we want in the vial (20% DMSO in growth medium). It is very important to add the DMSO-containing medium dropwise and to mix the DMSO with the cells by inverting the tube several times during the addition. After the DMSO is added (should take no less than 10 minutes with the cells still on ice), we transfer the cells to cryo-vials (1mL per tube to avoid issues with expansion after freezing), freeze them in -80C overnight, and transfer to liquid nitrogen the next day or up to a few weeks after putting them in -80C. Basically, slow down with your procedure and this should clear up your problems.
Not sure what you are using, but contrary to David we use a freezing mix that consists of heat-inactivated FCS with 10% sterile tissue culture-grade DMSO. (we keep the freezing mix at 4C). For more detail I would like to point you to the following post :
https://www.researchgate.net/post/What_is_the_best_approach_for_freezing_and_thawing_HUVECs
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