I try to grow Caco-2 cells monolayer and have tested a range of cell seeding densities, from 4 × 10⁶ to 4 × 10⁴ cells/mL.
I am using 12-well Transwell inserts with PET membranes (0.4 µm pore size), EMEM medium supplemented with 1% Penicillin-Streptomycin and 20% FBS, and cells at passage numbers ranging from 35 to 45.
However, even after 14 days, the TEER values remain below 150 Ω·cm². After 21 days, the TEER further decreases to around 60 Ω·cm². I change the media every two days and have tested monolayer integrity using Lucifer Yellow, which showed less than 1% permeability—suggesting the cell layer is intact.
Despite this, I have never observed TEER values exceeding 150 Ω·cm² within 21 days of culture. I’ve seen reports of TEER values reaching 200 Ω·cm² or even over 1000, so I’m unsure why my results are consistently lower.
Attached are microscope images of my cells at day 21.
I’d really appreciate any insights or suggestions.
Low TEER (Transepithelial Electrical Resistance) in Caco-2 cells usually indicates that the cell monolayer is not fully formed or is damaged. One common reason is that the cells haven't been cultured long enough—Caco-2 cells typically need about 21 days after seeding to form tight junctions properly. Using the wrong seeding density, either too low or too high, can also lead to uneven growth and poor barrier formation. Additionally, improper culture conditions such as low serum concentration, incorrect temperature or CO₂ levels, and infrequent media changes can affect cell health and junction formation. Over-passaging of cells can also reduce their ability to form tight junctions, so it’s best to use cells within the recommended passage range. If any drugs or chemicals are added to the cells, they may also disrupt the junctions and reduce TEER. Lastly, contamination with bacteria or mycoplasma can silently damage the cells and lower TEER without obvious signs. Regular checks, proper technique, and optimized conditions are essential to maintain high TEER values.
Hope this answer will be helpful to uhh
I am facing the exact same issue. I also have tested cells for mycoplasma, and they are negative.
Waiting to see if someone has an answer!