Hi
Currently I'm working on a permeability study using Caco-2 cells.
When i cultivate the cells and start monitoring the TEER values after 7 days it starts to go up from 170 to 230 ohms in a few days. Unfortunately, between the eleventh and the fourteenth day, the value of the TEER begins to fall sharply, and the cells begin to detach from the transwell and give clumps shape.
Have you any ideas or suggestions ?
Thanks in advance.
NOTE:
*Passage number:45
*Seeding density:150.000/0.5 ml in transwell
*DMEM supplemented with with 10% FBS (Recently i used 20%), antibiotic(Strep. &Pen), L-glutamine.
*Transwell with 0.4 micro m in pore size with 12 mm in diameter
I attached images for the cells in the Transwell and an image for cells under Confocal microscopy with DAPI stain
Dear Mohammad Hatem Rasras ,
I think in the end you have overgrown your cultures and the monolayer is deteaching. You should either start your experiment earlier, so that you do not run into this problem, but you are still maintaining the barrier function.
Another way might be to stop the proliferation with something like Mitomycin C so that you can keep the mono cell cell layer longer.
That might be applied already earlier in the preparing culture and then you will have to seed the exact needed cell number to form a monolayer.
Kind regards Soenke
Thank you @Sönke Weinert
This idea is good in the case of high confluency, but the main problem I have is that the percentage of confluency does not exceed 30% to 40%, and after about two weeks the cells begin to be detached (confluency=10%) even if i increase the percentage of (FPS) to 20%.
Do you have any idea about this issue?
Dear Mohammad Hatem Rasras
Recently I got the similar problems. I used 24 well and seeded 1*105 cell/insert, then monitored the TEER values after day 14, but in day 19-22 all cells could only reach about 230 ohms, then starting to drop.
I had used microscope to observe the insert, it seemed there were many cells on the membrane.
Did you already solve this problems? And may I asked what might cause this situation?
Ching-Wen Tsai
Dear Mohammad Hatem Rasras ,
I don't have an answer to your question but I would like to ask you, how were you able to observe cells on a Transwell insert? In an inverted microscope, it seems impossible, as I only see the membrane.
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