Dear all,
For my last two experiments, my supposedly endothelial cells (differentiated from bone marrow-derived mesenchymal stem cells, at passage ~35) have detached from Transwell inserts 1-2 days following seeding, looking as if I trypsinized them, and creating some cell clumps.
I expand (for 2 days) and differentiate (for 3 days) them in 48-well plates. Then I expose them to endothelial medium for one day. On the second day of endothalial medium, I transfer them to Transwell inserts that have been coated with Fibronectin and Collagen Type I. When I check 3-4 hours after seeding, I observe that they nicely attach. However, either the next day or the other day, they detach from the Transwells (Corning 3740) and I can't find the reason why.
In both of the experiments, I changed the media of the Transwells the following day after seeding. I inspected the cells both before and after the medium change. In one of them, the cells detached right after medium change although I aspirated the old medium very slowly (on the minimum speed of the vacuum suction and without touching to the membrane). In the other experiment, the cells were (mostly) fine after the medium change. But the next day after medium change (two days after seeding onto Transwells) they had detached.
The possibilities I could rule out are:
- There should be no problem with the medium contents/temperature/CO2 concentration/coating because I'm seeding the same cells to coated 48-well plates as well and applying the same conditions on them; and they stay healthy & alive.
- There is no contamination in the plates.
- It's not because they are over-crowded, I'm trying to form a monolayer indeed but they are sparsely distributed and thus they shouldn't be dying from over-confluency.
- I believe it is not about the force my medium change exerts on the cells either, because in one of the experiments cells looked fine after the medium change.
What do you think the reason could be?
Thanks in advance!
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