I am trying to compare and calculate the transport of my drug loaded nanoparticles and free drug using a caco2 cell transwell assay. I have 2 issues: 1. The drug concentration at 0 hours in the receiving or basolateral chamber is the highest despite having a high TEER values and a tightly packed monolayer of cells as confirmed by confocal imaging of controls. But, thereafter i see very negligible transport of drug from apical to basalateral chamber. Is this normal and if so what is the reason for initial increase in drug concentration in the basolateral chamber. 2. The formula to calculate apparent permeability or P aap= (dQ/dt)x 1/AC, where dQ/dt is change in concentration / change in time at steady state and A is the growth area and C is the initial conentration in apical chamber. is this formula correct. And if so what is steady state concentration and time?
One point I want to know:
1) Is your drug molecule hydrophobic or hydrophilic? What is log P value of your active?
To the first point:
The drug concentration at 0 hours in the receiving or basolateral chamber is the highest despite having a high TEER values. Have you compare the concentration of your dosing solution and the concentration oin the receiving chamber?
The second one:
The formula to calculate apparent permeability or P aap= (dQ/dt)x 1/AC is correct. dQ/dt is the slope of the cumulative concentration of the compound
in the recipient chamber over time. Assuming that the sampling point is in the linear range, you can even calculate using single point as compared with zero point.
There is a reference published in Nature Protocol titiled "Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers" (NATURE PROTOCOLS | VOL.2 NO.9 | 2007 | 2111). It may help a lot. Another good reference is Expert Opin. Drug Metab. Toxicol. (2005) 1(2):175-185.
Dear Jun-Ling,
To the first point: Yes, the concentration in dosing /apical chamber is 10 uM, and that in the receiving/basal chamber at the end of the experiment (6hours later) is 4 uM. I found that a lot is being taken up by the cells by imaging (but I have not quantified it).
But at time t=0, just as I add the drug to the apical chamber, the concentration in the receiving chamber is 0.3 uM. there after it drops to 0.01 uM and finally increasing slightly at 6 h to 0.03 uM (raw data attached).
1. Why is there a marked increase in the concentration of drug in the receiving chamber soon after the addition of the drug on the apical side at 0h, despite an average TEER value of >550 ohm cm2?
2. I would expect the amount of drug that crossed the membrane to remain in the basal chamber. But instead there is a decrease in the amount with time. Does it mean that there could be a basolateral to apical transport?
3. Since the transport of drug is not linear, how do I calculate slope in this case? which concentration and time-point represent steady state flux to calculate dQ/dt?
I do not understand your "Extrapolated concentration". I think propably the sampling step is incorrect. I remember that the compound you tested should be good in permeability. I show one of my tested compound as an example in the attachment.
Usually I don't sample at 0 time point. For highly permeable compounds, I sampled at 5, 10, 30 minuts. For compounds that shoed poor permeability, I sampled at 30 minute interval.
Dear Sasidharan,
Maybe you should try bidirectional permeability measurements. Some drugs are excreted by transporters from the cells.
You can look at this article:
Comparison of MDCK-MDR1 and Caco-2 cell based permeability assays
for anti-malarial drug screening and drug investigations. Journal of Pharmacological and Toxicological Methods 70 (2014) 188–194.
Best
What drug are you trying to measure here? The drug loaded nano-particles may not transport well depending on their composition. Do you have a positive control drug which has a high permeability (Papp) value?
Our company develops 3D cell barrier culture systems for in vitro study of virtually any cell barrier, and the system comes equipped with both a Fluid Perfusion Unit and a Trans-Endothelial/Epithelial Electrical Resistance (TEER) Measurement Unit! The use of a vessel-shaped 3D environment has been proven more effective than 2D systems like Transwell (
Article Santaguida S, Janigro D, Hossain M, Oby E, Rapp E, Cucullo L...
), and our advanced TEER Measurement Unit allows for frequency sweeps between 0.1-1,000 Hz, automated time point sampling, logging of data to Excel, and additional measurement of phase angle for cell capacitance calculations! For those that are committed to Transwell use, we also have a TEER Measurement Unit that is compatible with nearly all Transwell products (Endohm cell cup chamber, STX2 "chopstick" electrodes, etc) and allows the user to greatly expand their testing capabilities with Transwell equipment. Additionally, we have smaller modular systems that connect to microscope-friendly cell culture modules: i.e., a miniaturized 3D cell culture system that you can use right under your microscope! If you or anybody on this thread is interested in learning more, I encourage you to visit www.flocel.com or email me at [email protected], thank you!
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line