I'm using a colon cancer cell line, after loading in to the transwell, within a week it looks confluent under the microscope but it shows TER only around 200 ohms.
If the inserts are left for growing for 10-15 days the cells start coming out of the inserts in to the medium which is destroying the monolayer and there is even a decrease in TEER.
Could anyone suggest a reason for this?
Have you checked the pore of the inserts? There are many types and sizes. Cells can come out during the first days of culture, but after reaching confluency, there should not be leaks.
There are as Maya mentions many types of inserts (from plastic composition, through to pore size. You are probably considering what I am about to outline but just in case... be sure that your cell line is quality tested (in terms of contaminants such as mycoplasm - it may be that only on prolonged culture these issues are expressing themselves), be as careful as you possibly can be to fill / load the chambers with media (as they become cofluent they become more susceptible to hydrostatic forces acting across the whole 'sheet' causing lifting and tearing), and most obviously take care when measuring the TEER that you aren't inducing any damage (stick type probes can obviously stratch the cells, but chamber types can also be tricky). Also perhaps be super familiar with the cell line's literature it may be that cells are pushed out of the monolayer over time and that more complex cells organisations are required to form prior to TEER increase (bilayers / multilayers, channel expression patterns, and so forth). Stick with it and good luck.
Thanks Andrew.
still now i didnt find any contamination , the main problem is the decreasing of TEER , according to many articles the cell line im using shows 450 ohms at confluence but im unable to get proper teer readings.
Im using millipore TEER electrode,even after 1 month of seeding of cells the TEER value is not reaching 450 ohms.
Usually in flasks the same cell line takes just a week to become confluent dnt knw why not in transwell
please help me to solve this out
Have you coated the inserts? If not, you may want to consider collagen, fibronectin, or gelatin. Also, are you seeding enough cells so that insert is confluent asap? Take the greatest of care when changing medium within the insert. Also, check bottom of culture dish to make sure cells are not migrating out of insert and onto well of plate.
I absolutely agree with Connie, it is well worth trying out some of these suggestions to see if they make a difference. It is most likely that it is something in the preparation (choice of plastic insert (i.e. polycarbonate, polyester); niether particularly well mirror standard culture flask substrate), pore size, coating, seeding density, choice of media (some measure the TEER in HBSS, others Ringers w/glucose, when replacing the culture media for a short period for example), and so forth. Or that it is something about simple technique, either media changes or probing technique. You will likely have to try a few things I'm afraid [- you could perhaps post another question 're: growing your specific cell line in your specific inserts' and see if anyone has tried the combination].
PS. Remember temperature is important also, make sure you are warming and keeping warm your media and plates. If you serially test the resistance over 20mins or so the readings will alter fairly dramatically. If a lab is not well temperature controlled you may find this also effects readouts - I found this through bitter experience in my first year.
Hope all this is useful.
Concerning Andrews remarks, I stongly agree regarding medium temp, pore size, etc. Currently, I am at home or I would check construction of the inserts which I prepare for another lab. I must coat these inserts with fibronectin. We also utilize ECIS to measure TER. With ECIS, we have noted a rise in resistance immediately after medium is changed. The rise is brief and is quickly followed by a drop in resistance. We have been able to minimize such changes by warming medium in the incubator so that it is exposed to CO2 prior to any changes of medium or additions of reagents. Furthermore, the greatest care must be taken when adding reagents so that cell layer is not disturbed. I might also add that I depise the inserts in comparison to the ECIS arrays. Success with the inserts usually depends upon whether or not the cell layer can be observed under a microscope. In my case, the pore size is so small, the cells cannot be observed.
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