Good evening,
I have started my culture with HUVEC cells and HAoEC, I use Endothelial Cell Medium
(Cat1001) from sciencell, so the complete medium contains FBS, antibiotics and endothelial cell growth supplement (ECGS). I thawed HUVEC and seeded in T25 without fibronectine and I used fibronectine to cover the bottom of the T25 for HAoEC. Both cell types don't want to grow properlly, there are a lot of dead cells in the medium and very little, round shaped attached to the bottle. Any advices? I would be very grateful.
Hi Anna,
From your description I assume the problem is in cell thawing. If your ECs do not adhere to fibronectin-coated dish(es) it suggests that they are inured/dead :(
If the "classical" cell thawing/seeding procedure is not working for you, consider the following to minimize cell death/loss :
1) Thaw your frozen EC without "shaking". Don't keep the vial in the water bath (+37oC) for too long (just long enough to dissolve "ice crystals").
2) Once the" ice crystals" in the vial will be gone, gently suspend/transfer the cells in to the tube with 10-20 ml of complete medium (depending on the cell culture dish you are planning to use). At this point it is important to dilute DMSO (present in the cell freezing medium) as much as possible.
3) DO NOT centrifuge!
4) Transfer the whole content of the tube (10-20ml) in to T25/T75 dish. Allow cells to adhere for 2-3 hrs. This period of time is long enough for EC to adhere and spread on Fibronectin-coated surfaces (in most cases 1 hr is long enough for cells to adhere). You can check cell adhesion under phase contrast microscope.
5) Once the cells are attached, collect the medium with remaining not attached cells into a separate tube, and place new fresh EC growth medium to the attached cells. The attached cells should be happy :)
6) At this point you may centrifuge the collected medium (with remaining not attached cells) to remove DMSO and plate them in to a new T25. With this step you will maximize recovery of your original cell stock.
Best of luck!
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