| 研究課題/領域番号 |
15K18699
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| 研究機関 | 香川大学 |
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研究代表者 |
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| 研究期間 (年度) |
2016-04-01 – 2018-03-31
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| キーワード | Caco-2 / HT29-MTX / permeabiity assay / mucus layer / carotenoids |
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| 研究実績の概要 |
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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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
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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| 今後の研究の推進方策 |
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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| 次年度使用額が生じた理由 |
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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| 次年度使用額の使用計画 |
We will continue the project and make efforts to keep expenditures in line with the budget plan.
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