Advantage and Core Benefit
- Inexpensive and stable supply: Caco-2 cells, a standard model cell, are used, and stable supply is expected.
- Easy induction of concavity structure: concavity structure can be induced simply by combining air-liquid culture and supplements
- Improved expression of metabolic enzymes: The expression levels and patterns of metabolic enzymes such as MUC2 and CYP3A4, which were poorly expressed in conventional Caco-2 cells, were improved
|
Background and Technology
Intestinal models are widely used as important tools in pharmacokinetic studies. Among them, the evaluation system of intestinal cells with a concavo-convex three-dimensional structure is expected to be able to evaluate the human intestinal system as close as possible to the human biological system. Inventors have succeeded in culturing human intestinal cells (hIC) with a labial villi-like concavo-convex structure from human iPS cell-derived intestinal organoids, but there are issues such as high cost and difficulty in supplying a large quantity of cells with uniform quality.
Caco-2 cells derived from human colon cancer have accumulated a vast amount of data as a standard model cell that is inexpensive and can be stably supplied. Although there are reports of the formation of concavo-convex structures by culturing cells under mechanical loading, etc., there are no known cases of the formation of concavo-convex structures by normal culture.
The inventors applied the knowledge of Medium5 (M5) medium and gas-liquid culture obtained in previous studies on the induction and culture of intestinal organoids to the culture system of Caco-2 cells and succeeded in inducing Caco-2 cells to have a concavo-convex structure like that of the living intestinal tract. We identified the culture factors necessary for the formation of the concavo-convex structure, and by culturing the cells at the gas-liquid interface in M5 medium containing these factors, we formed the concavo-convex structure and confirmed that the cells exhibited TEER values closer to those of the living intestinal tract than those of monolayer Caco-2 layers. Furthermore, the expression levels of MUC2 and the metabolic enzyme CYP3A4, which had been an issue in Caco-2 cells, and the expression pattern of the carboxylesterase CES1/CES2 ratio were altered and were confirmed to approach that of the living intestinal tract. This invention is expected to lead to a stable and inexpensive supply as a model of an uneven three-dimensional intestinal evaluation system that closely resembles the human biological system.
Data
- Caco-2 cells seeded in cell culture inserts and cultured for 15 days in gas-liquid interface culture (ALI) or normal liquid phase culture (LL) using Caco-2 medium or M5 medium with supplement as culture medium (right panel) TEER values (A) CYP3A4 gene expression when cultured in ALI culture vs. small intestinal cells (B)
 |
 |
Patent
Pending
Researcher
Prof. Tamihide Matsunaga (Nagoya City University)
Expectations
We are looking for a cell product developer to license this invention to commercialize a three-dimensional culture test system. Detailed culture methods, including the composition of the supplements, can be disclosed under a nondisclosure agreement. We are also available to meet directly with researchers to discuss the invention/project.
Project No.WL-04826