Am suspecting the cells which are grown in DMEM will be revived in same medium alone or else we have to gradually reduce the concentration of reviving medium (DMEM:F12)
Could be that you are not diluting the DMSO enough when you thaw the cells. Try adding some more warm media to the centrifuge tube on the first dilution. Make sure you don't add it too fast since osmotic shock could also disrupt membranes.
Generally the cells became multinucleated when they are stressed and there are many possible factors for this:
1- the reviving medium should be well adjusted 1:1 v/v (DMEM:F12).
2- when removing the cells from liquid nitrogen container, it should be placed immediately into a 37°C water bath with agitation and don't leave the vials in water bath after most of the content is thawed.
3- DMSO could be not enough diluted as mentioned above by Dr. Reiserer.
I think if you see some amount of multinucleated cells, it could be due to a combination of the cell-cycle stage at the time of freezing coupled to the stress of DMSO/freezing. If after splitting, they look OK, then I wont bother too much.
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