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I was wondering if anyone could give me some pointers on setting up and
implementing Caco-2 cells as a marker for oral bioavailability. I was
wondering what materials (type of transwell plate, manufacturer and
96vs24 well) and instruments (Ohm meter for Lucifer yellow for
monolayer confluency). Also if TEER/Lucifer yellow readings were fairly
consistent and if all wells were tested or only a few from each plate.
Another question is whether anyone has used MDCK cells for the same
type of testing and how they compare?
Any sort of information and personnel experiences would be welcome.
BC Research Complex
3650 Wesbrook Mall
Vancouver, BC V6S 2L2
phone: (604) 221-9666, ext.286
fax: (604) 221-9688
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We normally do not depend on the TEER values as the main criteria for
judging the monolayers' integrity. Permeability values for paracellular
markers (such as mannitol) always give more reliable indications.
Ideally it is better to determine the mannitol Papp value for all your
wells but for practical reasons we measure the TEERs for all the wells
(they all should be in the same range) then we select three at random
from the plate to determine the mannitol permeability.If you are
interested in transcellular transport, you may also check a lipophilic
marker (propranolol for example).
For the plates, many formats are being used. We use transwell costar
filters (1 cm^2 ) at a seeding density of 65000 cells/cm^2 (you may
also try 80,000 if 65,000 does not work well)
For TEER measurement , we use Millicell- ERS meter (Millipore
Corp.,Bedford, MA) with chopstick electrodes.
I hope this will help.
Ahmed Abdel-Fattah Othman.
Pharmacokinetics and Biopharmaceutics lab
School of Pharmacy.
University of Maryland at Baltimore.
20 Penn Street.HSF-2
Baltimore, MD 21201
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The following message was posted to: PharmPK
For Caco-2 cells, the culturing protcols are available at ATCC website.
for the type of transwells, it really depends on your application. in
words, on your drug, if you are assessing the transport of a drug that
high permeability then you probably would prefer to use a smaller pore
to capture the flux in the early time points. the transwells are
from Corning (through Fisher). I have used 12 well plates and 6 well
with Caco-2 and the results are basically consistent for the same probe
course using the same pore size). With regard to TEER, you will get
results depending on the integrity of the monolayer. If you do well
and distribution after seeding then using the TEER machine provided by
(Millicell-ERS-Millipore) you will get results within +/- 10 degrees.
you test the teer for all wells in the plate and test N=3-4 filters by a
paracellular marker like mannitol or inulin to ensure the integrity of
monolayer. there are several publications showing that results
Caco-2 cells are comparable to those with MDCK cells. But if you are
efflux, note that MDCK have a lower expression than Caco-2 cells. You
want to look up an article called (Cell culture as tools in Pharmacy)
compares both cell types.Hope this helps.
Best of luck.
Noha Nabil Salama, Ph.D.
DNA Sequencing and Gene Analysis Center
Department of Pharmaceutics
University of Washington School of Pharmacy
University of Washington
Department of Pharmaceutics,
Seattle, WA 98195-7610
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