Culture container for culturing epithelial cells and use thereof
An epithelial cell and culture container technology, applied in cell culture active agent, culture process, tissue culture and other directions, can solve the problems of artificial culture of intestinal bacteria and inability to clone.
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experiment example 1
[0166] (culture of epithelial cells)
[0167] use figure 2 (a)~ figure 2 The culture container for epithelial cell culture shown in the photograph in (c) is used for culturing epithelial cells. As the upper container, a commercially available insert cell culture dish (trade name "ThinCert", Greiner Bio-One) or a cell culture tank (trade name "Transwell", Corning Corp.) was used. In addition, the cover member is self-made. As the material of the cover member, butyl rubber was used. The oxygen permeability coefficient of butyl rubber is 0.99×10 -8 cm 3 cm / (cm 2 · seconds · air pressure). In addition, as the lower container, a commercially available 12-well plate or 48-well plate was used.
[0168]
[0169] As a cell culture medium, a medium was prepared as follows: Human recombinant R-sponpin was added to a commercially available Advanced DMEM / F-12 medium (manufactured by Thermo Fisher SCIENTIFIC) at a final concentration of 1 μg / mL 1 (manufactured by R&D systems), ...
experiment example 2
[0182] (measurement of oxygen concentration)
[0183] The same operation as in Experimental Example 1, with figure 2 (a)~ figure 2 Intestinal epithelial cells are cultured in the culture vessel for culturing epithelial cells shown in the photograph in (c). The interior of the upper vessel is maintained at anaerobic conditions and the interior of the lower vessel is maintained at aerobic conditions. In addition, for comparison, the following samples were also prepared, that is, the sample that was subjected to the same operation as Experimental Example 1 except that the epithelial cells were not seeded, and the sample that did not fit the lid member to the opening of the upper container. samples of cultured epithelial cells.
[0184] Next, 3 days after the epithelial cells were inoculated into the upper container, the dissolved oxygen concentration in the cell culture medium in the upper container and the dissolved oxygen concentration in the cell culture medium in the low...
experiment example 3
[0189] (2D organoid research1)
[0190] use figure 2 (a)~ figure 2 In the culture vessel for culturing epithelial cells shown in the photograph in (c), intestinal epithelial cells were cultured in a state where the upper vessel was kept under anaerobic conditions, and whether epithelial stem cells could be maintained or differentiated cells could be maintained. Research.
[0191] First, in the same manner as in Experimental Example 1, a layer of fused 2D organoids was formed on the surface of the upper container. Next, the culture medium in the upper vessel was replaced in the anaerobic chamber. Two sets of the same culture containers for epithelial cell culture were prepared, and in one culture container for epithelial cell culture, the lid member was fitted to the opening of the upper container, while in the other culture container for epithelial cell culture, the lid member was not used. Fits into the opening of the upper container. These cells were incubated at 37°C...
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