JP-2023510681-A5 -

Dates

Publication Date

20230524

Application Date

20191119

Description

S1PR1 (F: 5'-TTTCCTGGACAGTGCGTCTC-3' (SEQ ID NO: 3), R: 5'-ACTGACTGCGTAGTGCTCTC-3' (SEQ ID NO: 4)). CXCR4 (F: 5'-CGTCTCAGTGCCCTTTTGTTC-3' (SEQ ID NO: 5), R: 5'-TGAAGTAGTGGGCTAAGGGC-3' (SEQ ID NO: 6)). VEGF (F: 5'-TACCGGGAAACTGACTTGGC-3' (SEQ ID NO: 7), R: 5'-ACCACATGGCTCTGCTTCTC-3' (SEQ ID NO: 8)). Figure 7 shows that (A) the conditioned medium was recovered when the microfluidic microwells were replaced. For the conventional flask, the conditioned medium was recovered at the same time. The exosome concentration in the conditioned medium in the cell culture system of this disclosure increased significantly more than the exosome concentration in the conditioned medium in the conventional flask at each time point in time when the microfluidic microwells were exchanged, and the overall exosome concentration in the conditioned medium in the cell culture system was more than twice the concentration in the conventional flask after four microfluidic microwell exchanges (7(A)-1 and 7(A)-2). (B) The expression of paracrine and autocrine-related genes SDF-1, S1PR1, CXCR4 and VEGF was detected by QPCR. The present invention provides the following: 1. A cell culture system comprising (i) one or more removable microfluidic microwells, and (ii) a culture device for holding the microfluidic microwells, wherein each microfluidic microwell has one or more hollow portions, each containing a partitioned microfluidic channel that has no bottom over the entire microfluidic microwell, and the microfluidic channel contains one or more cell inlets. 2. The cell culture system according to 1 above, comprising (i) a plurality of removable microfluidic microwells, and (ii) a culture device for holding the microfluidic microwells, wherein each microfluidic microwell has one or more hollow portions containing a partitioned microfluidic channel that has no bottom throughout the microfluidic microwell, the surface area of the microfluidic channel in the microfluidic microwell gradually increases; the surface area of the microfluidic channel in the plurality of microfluidic microwells increases in size by 2n, 3n, or 4n compared to the microfluidic channel in the plurality of microfluidic microwells having the minimum surface area, and n is an integer less than 1 the number of the plurality of microfluidic microwells; and the microfluidic channel contains one or more cell inlets. 3. The cell culture system according to 1 or 2 above, wherein the culture device is a culture plate or a culture flask. 4. The cell culture system according to 1 or 2 above, wherein the hollow portion is circular or has a polygonal pattern with 3 to 8 corners. 5. The cell culture system according to 1 or 2 above, wherein the hollow portion is arranged in a triangular, quadrilateral, pentagonal, hexagonal, octagonal, or nonagonal pattern. 6. The cell culture system according to 1 or 2 above, wherein the hollow portion has a hexagonal pattern. 7. The cell culture system according to 1 or 2 above, comprising at least three removable microfluidic microwells. 8. The cell culture system according to 1 or 2 above, comprising at least five removable microfluidic microwells. 9. The cell culture system according to 1 or 2 above, comprising 3 to 15 removable microfluidic microwells. 10. The cell culture system according to 1 or 2, wherein the plurality of removable microfluidic microwells are connected to one another in the culture device. 11. The cell culture system according to 1 or 2 above, wherein the microfluidic channel includes a plurality of cell inlets. 12. The cell culture system according to 1 or 2 above, wherein the microfluidic channels in the hollow portion are in fluid communication with each other. 13. A method for culturing cells, comprising: (1) loading cells into the cell inlet on the microfluidic channel of a removable microfluidic microwell of the cell culture system described in 1 or 2 above; and (2) culturing the cells under conditions suitable for cell proliferation. 14. The method according to 13 above, wherein the cells are adhesion-dependent cells. 15. The method according to 13 above, wherein the cells are stem cells, nerve cells, or fibroblasts. 16. The method according to 13, wherein the cells are loaded into the cell inlets on the microfluidic channels of the plurality of microfluidic microwells having the minimum surface area. 17. The method according to 13 above, wherein the cells are loaded at a density of 30% confluence. 18. The method according to 13, wherein the cells are loaded into the cell inlet on the microfluidic channel while the culture device is tilted or centrifuged. 19. The method according to item 13, wherein the angle of inclination is 120 degrees, 240 degrees, or 360 degrees for triangles and hexagons, or 90 degrees, 180 degrees, 270 degrees, or 360 degrees for quadrilaterals and octagons. 20. The method according to 13, wherein when the cells reach a confluence of more than 50%, the preceding microfluidic microwell is removed