2016 Fiscal Year Research-status Report
Development and application of a cell based assay to assess the intestinal absorption of phytochemicals in the presence of mucus layer
| Project/Area Number |
15K18699
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| Research Institution | Kagawa University |
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Principal Investigator |
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| Project Period (FY) |
2016-04-01 – 2018-03-31
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| Keywords | Caco-2 / HT29-MTX / permeabiity assay / mucus layer / carotenoids |
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| Outline of Annual Research Achievements |
Caco-2 cell-based absorption assays can fairly predict the bioavailability of several phytochemicals and trace metals in humans. The luminal surface of the human intestine is covered by a mucus layer, however Caco-2 cells produce only minute amounts of mucins. We have developed a co-culture system using Caco-2 combined with the HT29-MTX cell-line, which form a mixed population of mucus-secreting goblet cells and enterocytes upon confluency. Simultaneous seeding of Caco-2 to HT29-MTX at 8:2 and 9:1 ratios formed monolayers with acceptable electric resistance. On the other hand, the key feature of this co-culture system is the presence of an overlaying mucus layer on top of the monolayers. We applied several methods to visualize que mucus layer, based on classical staining techniques and optical microscopy on fresh and fixed cell monolayers, but none provided a satisfactory proof of the mucus layer presence. In due course, we have confirmed the presence of a mucus layer on top of the Caco-2/MTX co-culture monolayers by scanning electron microscopy. HPLC methods for carotenoids and polyphenols were developed and trial permeability assays were run using beta-carotene incorporated in artificial micelles.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Although initial plans were slightly delayed due to an unavoidable lot change of foetal bovine serum, proper growth has been verified with a new lot. Published protocols for verification of mucus layer formation, based on Alcian blue staining, could not discriminate between the Caco-2 and the Caco-2/MTX co-culture monolayers. A new research collaborator specialized in poultry intestinal histology (Assoc. Professor Matsumoto Yoshiki) joined the project and we have successfully confirmed the presence of a mucus layer on top of the Caco-2/MTX co-culture monolayers by scanning electron microscopy.
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| Strategy for Future Research Activity |
Further tests are being conducted to confirm mucus layer formation (staining and optical microscopy) and to develop a fast method for detection of mucus layer in Caco-2/MTX co-culture, using a pH-sensitive fluorescent probe. The fast mucus detection will be used as a routine quality control test for the monolayers produced by the co-culture method. Other aspects of the project will be conducted as planned.
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| Causes of Carryover |
We have decided not to purchase the CO2 incubator as initially planned, and start the project with the small incubators already in place in the lab. Instead, a probe sonicator had to be purchased to enable proper extraction of phytochemicals from the cells.
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| Expenditure Plan for Carryover Budget |
We will continue the project and make efforts to keep expenditures in line with the budget plan.
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