In cacao2 cells grown on transwell inserts for around 21-30 days.
When seen under the microscope the transwell looks completely confluent but the TEER reading shows it was much less, by around 190 ohms than at confluence.
Usually it should be around 450 ohms after 21 days, but in my transwells I am growing them for a longer time because of the low TEER measurement.
Can any one help me to sort this out?
Is your cultured cells in good health after all the treatments at the end of incubation.
Secondly can you tell which type of multiwell you use for culture and the electrode used here.
I don't know what you mean by good health conditions, but they grow well in flasks.
Is there any test or some thing to determine the condition of the cells?
I am using polyester 6 well inserts, and Millipore - ERS electrode.
I gone through one of your another post where you mentioned about buzzing of the meter. So probably you should first check your electrode for any sign of broken ends and connection secondly you should clean the electrode with a cotton swab dipped in alcohol.
Secondly with the cells good health I meant, Are there more number of cells floating after such long day of culture or the cell shape under microscope doesnt look much in good shape (blebbing ).Probably then cells are stressed or almost on death bed. If so use a new fresh vial of cells or try one from neighbor's lab.
Lower resistance could also because of the bigger surface you use ie 6well place so larger surface area lower resistance and you also had problem reaching the complete confluence so you can also try to use 24well plate and check it.
Hope this should help you.
Good Luck!!!
Thanks for the reply Lijo.
i swabed the electrode with ethanol and even with the sand paper as given in troubleshooting of the electrode but still the buzzing is there, Will there be aby battery problem?
Usually after seeding the cells with in 5-7 days it looks fully confluent under the microscope, but the TEER is so less ,if i culture for a longer time, the cells start coming out of transwells it comes in such a way that 75% of the monolayer formed will come out and then again i have to grow till it attains a proper confluence and TEER reading i.e 450 - 500 ohms.
Since to attain a proper TEER reading , im growing the cells for a longer time more than 45 days, so in this period the cells will cum out and i have to leave them till its confluent.
I know Caco2 cell line very well. I think that you should not keep the Caco2 cells at confluence for 45 days. Once they reached the confluence, these cells differentiate in vitro starting from 10-15 days after which they begin to die or no longer in good health. This would also explain the low measurement of TEER. My experiments were no longer than 15 days.
Hi Maria,
Need a small clarification, in many papers regarding the permeability studies, they have grown caco2 cells for 21 days to attain a TEER reading of around 450 ohms. They say that only after 21 days cells will differentiate properly.
if possible can you please send me the papers regarding the TEER and incubating just for 10-15 days.
Thank you
In my experiments Caco-2 cells were already plated at optimum density to achieve quickly the confluence (the density varies depending on the diameter of the plate) and then after 10-15 day post-confluence i measured as TEER as a marker of differentiation namely alkaline phosphatase. These parameters indicated me that Caco-2 monolayer was intact and that cells were differentiated. Prolonging time of culture in this conditions cause a cellular stress that induce apoptosis or necrosis with rupture of monolayer and a lower TEER. Another cause of lower TEER is a late passage of your cells i mean > 70. Good luck for your experiments!
P.S. i send you this reference: Pharm Res. 2000 Apr;17(4):460-5.
Formation of multilayers in the caco-2 cell culture model: a confocal laser scanning microscopy study.
Rothen-Rutishauser B, Braun A, Günthert M, Wunderli-Allenspach H.
I'm not sure if this question is still relevant considering the fact that it’s older than a year. However let me share my piece of understanding with this cell line. First I agree with Maria on cell density which ultimately affects TEER readings. In my experience seeding cells at about 2 x 105 cells/cm2 is optimal to reach TEER reading above 350 ohms between 15-21 days hence recommended by many users. I also reckon you first check optimum seeding density before performing any experiments. Also to emphasise on passage number as Maria mentioned, recommended passage number is between 25 and 45 for permeability studies. So if you are not certain about how old your cells are, re-order new cells with known passage number.
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