Aggregating MSC's - (Jun/10/2009 )

Hello,

When passaging over my hMSC's from human umbilical cord donors a lot of the cells aggregate in a foggy aggregate not able to be spinned down by centrifugation.

I culture them in Alpha-MEM +10% FBS, 2% penstrep, 1% L-glutamine and add 10ng/ml bFGF as well as 205uM Ascorbic acid.
When cultures is confluent (in T175) i detach them with 15ml 2,5x Trypsine. After 5min cells are all detached and i rinse
with 15ml culture medium. I see sheets of cells aggregating then. After spinning for 5min at 300xg all cells but the aggregate
are pelleted. The aggregate is still floating in media.

i tried different things: pipetting up and down with 5ml pipet, 1ml pipet, 200ul pipet tip and needle with syringe. The first one didnt work, even
after more then 20 times, all other options the tips got clogged up with the aggretate I also tried vacuum the media off and then scratch it over the rails of the cabinet and shaking it. I also lengtened the icubation with Trypsin to 30min, seeing all cells loose from eachother (and pretty unhappy) but they still clogged up. I lost about 5/6 of my cells in this way, so if someone has some similar problems or has any ideas to solve it please gimme a post.

Thanks in forward,

Rutger

Hello,
Reading through your post I see you have tried all of the methods which I would try.
But perhaps after detaching cells or even before you could try with some of a trypsin-free enzyme mix
meanted for processing a really gentle tissue.

Best,
kk