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The following message was posted to: PharmPK
Dear All,
I am facing a peculiar problem in caco2 assay.
I am trying 7-day accelerated caco2 assay with high seeding density on
polyester filters.
On the day of experiment, the cells seemed to be fine under microscope
with a TEER of 250 ohms. But to my surprise after 30min incubation
with HBSS, the monolayer detached completely.
Can anyone help me in solving this problem
Thanks in advance
Warm Regards,
Pankajini
Drug Metabolism and Pharmacokinetics
New Chemical Entity Research
Lupin Research Park
46A/47A, Village: Nande, Taluka: Mulshi
Pune-411 042, India
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The following message was posted to: PharmPK
Pankajini,
You may know this already, but cell detachment may occur if the Hanks
buffer is calcium/magnesium free or there is a chelator in the medium.
Mark
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Check your HBSS and see whether it has Ca2+ and Mg2+. It has happened
to us when someone accidentally used a different kind of HBSS.
Good luck,
Sean
Sean Xiang Wu, Ph.D.
Associate Director, New Lead Discovery
Exelixis, Inc.
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It is good to know you are trying with high seeding density, but this
7 days model is not sufficient to study, cell grow in no.up to 10-11
days and then differentiation starts up to 20 days. So I feel it will
not work. If you are looking for short term model try MDCK. One tip
wash cell 2-3 times with media when you are seeding it in flask from
cryopreserved vials. Traces of DMSO cause detachment.
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The following message was posted to: PharmPK
Dear All,
Thanks a lot for your suggestions.
The HBSS we use is devoid of calcium/magnesium therefore i will try
the assay by supplementing these ions in HBSS.
In our lab we carry out MDCK for screening purposes but as caco2 have
an edge over MDCK, we are trying to accelerate the screening with 7-10
assay. Therefore can anyone suggest any differenciation media so that
we can acheive growth and differenciation of caco2 within 10days
without or minimal compromise with its properties.
Regards,
Pankajini Mallick
New Chemical Entity Research
Lupin Research Park
pankajini.mallick.at.gmail.com
46A/47A, Village: Nande, Taluka: Mulshi
Pune-411 042, India
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Dear All,
Thanks a lot for your suggestions.
The HBSS we use is devoid of calcium/magnesium therefore i will try
the assay by supplementing these ions in HBSS.
In our lab we carry out MDCK for screening purposes but as caco2 have
an edge over MDCK, we are trying to accelerate the screening with 7-10
assay. Therefore can anyone suggest any differenciation media so that
we can acheive growth and differenciation of caco2 within 10days
without or minimal compromise with its properties.
Regards,
Pankajini Mallick
New Chemical Entity Research
Lupin Research Park
pankajini.mallick.at.gmail.com
46A/47A, Village: Nande, Taluka: Mulshi
Pune-411 042, India
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Dear Mallick
check this paper
New and better protocols for a short-term Caco-2 cell culture system
Journal of pharmaceutical sciences 2002, vol. 91, no3, pp. 669-679 and
also this webste.
http://www.bdbiosciences.com/external_files/dl/doc/tech_bulletin/live/web_enabled/tb418.pdf
In advance I would like to let you know that I tried this approach but
I did not get comparable results with the traditional 21 days. But It
does not hurt try;
I hope it helps
Whocely Victor de Castro, PhD
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Dear Sir/Madam
Evaluation of an accelerated Caco-2 cell permeability model, J Pharm
Sci, 89(3), 2000, 336-345.
New and better protocols for a short-term Caco-2 cell culture system,
J Pharm Sci. 91(3), 2002, 669-679.
Development of a 7-Day, 96-Well Caco-2 Permeability Assay with High-
Throughput Direct UV Compound Analysis, Pharm Res, 20(12), 2003,
1961-1969.
This may help.
Regards
Sachin Ramrao Patil
Deptt of Pharm.Tech Formulations
NIPER, SAS Nagar
Mohali, Punjab-160062
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