May be you can try 4 h leakage test: 1. creating 1 cm liquid difference between transwell and 24-well (200 μL medium in upper well and 500 μL medium in lower well ). 2. waiting for 4 h, if the model has been maitaining liquid difference, monolayer integrity is achieved.

Another method is by doing immunofluorescence for a cell adhesion marker.

for example in my case (lung epithelium) I'm staining for Epcam. There are also dye based assays of movement across the membrane.

Note that if you can't measure TEER because a volt-ohmmeter is too expensive, there's a protocol for making your own (this is what I use, and it works well!)

https://app.jove.com/t/60087/a-simple-approach-to-perform-teer-measurements-using-a-self-made-volt-amperemeter-with-programmable-output-frequency

Good luck!

Update: After ten or so days of culturing cells in the transwell, I noticed that the media color in the top compartment would change faster to yellow (acidification) than in the bottom compartment. I think this is a good indication that the monolayer is tight and the cells are polarized. I was also able to visualize the cells using light microscopy at 40X. Thanks!

Sounds like you have the information you need!

Thanks for the update!

Here you find a method to evaluate paracellular permeability