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Dear friend,
I m using monolayer of caco-2 cell to investigate the permeability of drug formulation. I m culturing the cell using hanging inserts on 24 wells plate. I have come to know that TEER value can be used to determine the degree of integrity of the monolayer. I m facing a problem where I m not sure when exactly I should be starting my permeability assay as different researchers uses different approaches. Some articles stating that experiment can initiate when TEER >350ohms , where some suggest to starts only when TEER range from 450-650 ohms and some restrict the TEER value to be at least 1000ohms before the starts of permeability assay. Also, most of the research culture the cell line on the inserts up to 14-21 days after seeding (to allow the growth of complete morphology to mimic the intestinal cell) and the reported TEER value is <= 1000ohms before the start of the assay. However, in my case the TEER value already exceeded 1400 ohm (after subtracting the TEER value without the cell) after 4th day of seeding.
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Dear friends,
Hi.
I m using monolayer of caco-2 cell to investigate the permeability of drug formulation. I m culturing the cell using hanging inserts on 24 wells plate. I have come to know that TEER value can be used to determine the degree of integrity of the monolayer. The problem is that i m not sure when should I starts my permeability assay after seeding. Some articles stating that experiment can starts when TEER range from 450-650ohms, where some suggest to start when TEER >350ohms and some restrict the TEER value to be 1000ohms before the starts of permeability assay. Also, most of the research culture the cell line on the inserts up to 14-21 days after seeding (to allow the growth of complete morphology to mimic the intestinal cell) and the reported TEER value is <= 1000ohms before the start of the assay. However, in my case the TEER value already exceeded 1400 ohm (after subtracting the TEER value without the cell) after 4th day of seeding (using the same number of cell/cm2). Hence,I do not know when my inserts are
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Dear Tan,
You should be aware that some references states TEER units as ohms/cm2 and other refers only Omhs. Normally, I consider values higher than 350 ohms as the threshold to perform my experiments. In your case if you divide 350 by 0.33 cm2 (the growth area of 24 well plate) you will get 1,060 ohms/cm2. Additionaly, at the end of the experiment it is recommend to use a hidrophlic probe (Lucifer yellow or Sodium Fluorescein) to the test the integrity of the membrane. If more than 1% of the probe is transported from apical do basolateral side then your experiment should be repeated for that especificaly insert.
Regarding the 21 days is the recommended period to the cells express the P-gp and other transporters. To check the suitability of your system use the hydrophilic marker at the different periods of culture as well as some marker compounds with low and high permeability with and without affinity to P-gp and others membrane transporters.
Therefore, you will be able to check about the confluence and the expression of specifc membrane transporter. Compare your data with what was already published. The FDA has a guidance where several marker compounds are suggested in order to validate in vitro systems for permeability studies.
I hope I could help
Victor
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Hi Seewei Tan
If i understand correctly, you are still in the process of standardizing your assay.
w.r.t to your question, i would go ahead, considering 21-day culture and the values are between 1000-1300 ohm/cm2. FYI, TEER values does not represent the true value of confluency (you can find in literature), therefore you need to run a positive control (lucifer yellow or C-14 mannitol) to assess the integrity of the cells.
Hope this information will help you
Regards
Khalid
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The following message was posted to: PharmPK
Dear Seewei Tan,
You have already received relevant answers, let me add a few words. Although there is extensive litterature about Caco-2 assays, there is significant inter-lab variability: one has to develop his own control criteria, especially regarding TEER values. Beware of any confusion with TEER units (ohms or ohms.cm2 or ohms/cm2).
You definitely have to rely on the testing of low permeability compounds to validate your model (lucifer yellow, radiolabelled mannitol); for such control, the endpoint can be either apparent permeability or % of transported compound for a given time. Use of high permeability compounds such as propranolol may help first validate the data handling and calculations. It is advisable to collect data at several time points, as it is the slope which is the main measurement to consider; eventually you may try to get down to a single-time measurement. Also, in the first attempts, use a large number of replicate wells (up to 6?) to assess the system variability. Lastly, you may first go for the recommended 21 days of culture and later try to monitor the quality of the monolayers at earlier times. Best of luck
Frederic Massiere, Novexel
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