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The following message was posted to: PharmPK
Hello,
Recently we have had difficulties obtaining adequate resistance
("TEERs") in
our Caco2 cells for permeability studies. Our cells are grown in fairly
standard conditions (passage 30-40, grown on collagen-coated 6.5 mm
Transwells for 14-21 days in DMEM containing 10-20% FBS, 1% sodium
pyruvate,
1% nonessential amino acids, 100 U/mL penicillin-G/streptomycin, 1%
HEPES).
We don't see any obvious contamination when we examine the cells
under the
microscope. Does anyone have any thoughts on what could be
contributing to
this? Any help would be very much appreciated.
thanks,
Dave
David R. Foster, Pharm.D.
Assistant Professor of Pharmacy Practice
Purdue University, School of Pharmacy and Pharmaceutical Sciences
Wishard Health Services, W7555 Myers Building
1001 W. Tenth Street
Indianapolis, IN 46202-2879
(317) 613-2315x309, fax (317) 613-2316
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Hello David!
Are you sure, you are seeding the cells at the right density?
After how long do you test for resistance?
I use MDCK cells regularly and find that adequate resistance is
developed at 3-5 days post-seeding at a density of 250,000 cells/insert!
Secondly, I would check the electrodes...
Regards,
Kavita
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The following message was posted to: PharmPK
Dear Dave,
Is this a recently occurring problem or did you never get high enough
TEER?
Susanne
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Hi David
there are multiple factors that may lead to inconsistent TEER values:
1- check the electrodes are clean and the machine is calibrated
2- maintain consistent method of measuring (approx. the same way the
electrode is immersed in the well, same individual, etc..)
3- maintain the same temperature at which you are measuring
4- confirm that you are not comparing measurements in PBS versus
media since the presence of nutients will affect readings
5- Examine the passage no of these cells: Caco-2 cells are tighter
according to passage no.
6- above all, TEER machine provides a general idea about the
integrity of the monlayer so even if you are showing super tight
readings for any reason, try to compare that with mannitol transport
data. try doing that a couple of times then Consequently you will
know when you get very high (or very Low) reading how much
(approximately) these data correspond to mannitol transport.
Hope this helps
Noha
Noha Nabil Salama, Ph.D.
Clinical Research Fellow
The University Of Michigan
Ann Arbor, MI 48109
Tel: 734-647-6256
Fax: 734-763-4480
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The following message was posted to: PharmPK
With such a high passage number the first thing that comes to (my) mind
is mycoplasma; have you recently checked for this?
Kind regards,
Frederik Pruijn
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The following message was posted to: PharmPK
David,
Have you checked for integrity of the monolayer? If you still have
the plates with low TEER values, I would suggest that you check the
permeabilty of mannitol or inulin (radioactive tracers could you used
for a quick check). If you find the monolayers to be leaky then the
cells could be a problem, else you can trouble shoot using some of
the valuable suggestions provided in this forum.
regards
Nagdeep
Nagdeep Giri
Dept. of Pharmaceutics, University of Minnesota
308 Harvard Street SE,3-140 WDH, Minneapolis, MN 55455
(612) 625-2446/626-3809 ; E-mail: giri0003.at.umn.edu
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Dear Dave
I would like to suggest you to check the integrety of the monolayer
with any of the markers, C14 mannitol would be a better choice. The
conditions and the media are perfectly ok, it is eveident from your
mail that you do not have contamination. If the permeability values
match with the standards a) check the TEER values by properly
cleaning the electrode with 0.1M KCL--70%Ethanol--and DMEM the values
should increase from day five b) You can replace the electrode( this
has been a case with me in the past).
Hope I have been of some help to you
Regards and best wishes
Naveed Shaik
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The following message was posted to: PharmPK
Once you have excluded a technical cause for your low TEER (e.g. faulty
electrode or equipment) I don't see much point in checking the integrity
of your monolayers other than to confirm what your TEER measurements
have already told you. It does not explain anything nor does it offer
you any solutions to the problem. Truly knowing (understanding) the
problem is more than 50% of getting to a solution.
Mycoplasma contamination cannot be ruled out by microscopic examination
unless you're name is Steve Austin. It is not like fungal or bacterial
contamination.
Other possibilities for sudden drop in TEER could be a new batch of
inserts, serum or serum supplements, culture medium, etc. Has anything
changed recently? Faulty thermostats are also notorious for leading
people down the garden path....
Good luck with this.
Frederik Pruijn
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Hello Dave,
may I ask what is an 'adequate resistance' (TEER-values) in you
laboratory and do the TEER-values increase or decrease with respect
to your earlier data.
In general, the average TEER-value is NOT automatically changing with
the age of the cells and the passage, but the culture conditions can
affect the growing of the cells on the filter support and, thus, the
measured TEER-values. Flux and/or permeability measurements are fun,
but will probably not solve your problem!
I would suggest to verify that you do control
- the seeding density in the flasks (stock) as well as for the
Transwell inserts (i.e. counting the cells and not simply splitting
the stock! It's annoying, but helpful!)
- the culture medium (especially the (heat-inactivated) serum, i.e.
batch)
- the transport medium/buffer (ion control, pH, temperature, additives)
- the quality of the electrodes (including background measurements of
'empty' filters)
- the trypsinisation/subculturing (i.e. no selection of an unsuitable
clone)
- and that you do not have any mycoplasma contamination or a mix (co-
culture) with other cells/cell lines, which you cultivate
simultaneously in your lab.
Why do you use collagen-coating for Caco-2 cells? Caco's will grow on
Transwell filter support (6.5 mm, No. 3413) without major problems.
Maybe the quality of the collagen is the problem?
My suggestion at the moment: Seed cells on one plate using filters
without collagen-coating (you could immediately try different seeding
densities, too) and prepare your cells for a mycoplasma test (without
PEST!!!). Do a mycoplasma test for (ALL!) your cell lines and don't
forget to consider your frozen stock as possible source of your trouble!
I hope that it is only the collagen!
Good luck!
Sibylle
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