I would like to know a suitable method to check the intestinal absorption of some lipidic components such as fatty acids. I have read about the Caco-2 cell assay. Could you tell me your experience with this procedure, particularly the advantages and disadvantages that can be expected? Is there any other alternative?
more information about the Caco-2 cell model could be find in:
Caco-2 cells as a model for intestinal absorption.
Angelis ID, Turco L., Curr Protoc Toxicol. 2011 , doi: 10.1002/0471140856.tx2006s47
additional information about the intestinal absorption models could be find in the following book:
Drug Absorption Studies: In Situ, in Vitro and in Silico Models
edited by Carsten Ehrhardt, Kwang Jin Kim
Chapter8- page 188. Springer, 2008
I have tried this assay, and all I can say is I did not like it, my advisor was not impressed, and my committee did not care, and my department did not approve. There are more things that can go wrong then can go right. A lot of pre planning (2-3 months ahead) is needed.
First off, it takes too long. From the time you seed the cells to the time they are fully confluent and differentiated takes 21 to 28 days. It that time frame I had contamination problems, incubator failures, and power outages, all resulting in me having to start over. There are "fast culture" techniques that add growth factors to the serum, but peer reviewers and commentators will easily argue that your system is artificial and not representative of a true intestinal absorption.
Second, once the cells are ready, you must assay your molecules all at once (including replicates). All compounds must be assayed with 5 passages of the CaC0-2 cell line to discount phenotypic changes.
Third, you must have adequate controls (just like any other experiment). The tricky part here is you have to account for passive diffusion (apical to basal transport), active transport (apical to basal), PgP pumps (basal to apical), as well as receptor mediated transport, transport inhibition, and pH dependent absorption (pH varies in the intestinal lumen); There is no way to cover all the controls in one experiment.
Fourth, what would your data mean? Does it have value? Your experiment would show the absorption in a cell based assay, but would this translate in an animal model? Probably not. The can be profound differences between fed and fasted states in an animal that are not seen in a cell based assays. Also, the CaCo-2 cells themselves can have various morphologies (with or without cillia) and phenotypes (with or without mucosal lining).
All in all, if you need to do this assay, good luck, but if you do not, try using a PAMPA assay and be very clear that this is a simple in vitro assay. If you truly need oral absorbance, try a bioavailability study (blood/plasma concentration) on animals with various routes of administration.
What about the HT-29 cell line, with a clone secreting mucus ? You can have polarised cells using inserts. I hope this could help.
Best regards
Thank you all for your answers. Dr Sergent, could you suggest me any paper which they use HT-29 cells in for that purpose? I am just getting started with this topic and any reference will be welcome. Thank you.
You may find intersting information here :
http://www.pnas.org/content/85/1/136.full.pdf
http://jcs.biologists.org/content/112/16/2657.full.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1374586/pdf/gut00537-0064.pdf
http://onlinelibrary.wiley.com/doi/10.1111/j.1768-322X.1988.tb00740.x/abstract
http://link.springer.com/article/10.1290%2F04100061.1
Best regards
HT-29 cells are the same as CaCo-2. One is listed as HTB-37 and the other is HTB-38 at ATCC. Essentially, same cell type but from different hosts.
caco-2 cell lines are most exrensively used to study bioavailibilty invitro
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