Cell culture method and cell culture device used with cell culture method

A cell culture device comprising:

• a plurality of cell culture chambers, each of the plurality comprising a pair consisting of an upper cell culture chamber and a lower cell culture chamber, the pair being defined by a tubular inner wall divided by a porous membrane (3), the porous membrane (3) having two surfaces to which a living organism material is to be fixed, the upper cell culture chambers being coupled and integrated with each other, and the lower cell culture chambers being coupled and integrated with each other;
• a membrane holder (27) including a second inner wall (27b) having one end closed by the porous membrane (3), the second inner wall (27b) being the inner wall, the membrane holder (27) being one of the plurality of pairs of upper cell culture chambers and lower cell culture chambers;
• a plate (22) including a planar first base (22a), a plurality of first through-holes (22b) in the planar first base (22a), and a plurality of first cylinders (22c) extending perpendicularly to one surface of the planar first base (22a), the plurality of first cylinders (22c) being continuous with the plurality of first through-holes (22b) and receiving the membrane holder (27) inside, the plurality of first cylinders (22c) each being the inner wall together with a corresponding first through-hole (22b) of the plurality of first through-holes (22b);
• a plate cover (23) including a planar second base (23a) and a plurality of first protrusions (23b) on one surface of the planar second base (23a) to simultaneously fit into the plurality of first cylinders (22c), the plate cover (23) being the cell culture chamber cover to cover the plurality of first through-holes (22b) each receiving the membrane holder (27) inside;
• a spacer cover (24) including a planar third base (24a) and a plurality of second protrusions (24b) on one surface of the planar third base (24a) to simultaneously fit into the plurality of first cylinders (22c), the spacer cover (24) being the cell culture chamber cover to cover the plurality of first through-holes (22b) each receiving the membrane holder (27) inside;
• a spacer (25) including a planar fourth base (25a) and a plurality of second through-holes (25b) in the planar fourth base (25a), the plurality of second through-holes (25b) being as many as the plurality of first through-holes (22b), the plurality of second through-holes (25b) each being the inner wall, the spacer (25) including a plurality of second cylinders (25c) extending continuously with the plurality of second through-holes (25b) and perpendicularly to one surface of the planar fourth base (25a), the plurality of second cylinders (25c) defining the upper cell culture chambers and the lower cell culture chambers together with the plurality of first cylinders (22c), being simultaneously receivable in the plurality of first cylinders (22c), and each being the inner wall together with a corresponding second through-hole (25b) of the plurality of second through-holes (25b);
• and
• a pusher (26) including a planar push plate (26a) and a plurality of push portions (26b) extending perpendicularly to one surface of the planar push plate (26a) to be simultaneously received in the plurality of first through-holes (22b).

A cell culture device comprising:

• a plurality of cell culture chambers, each of the plurality comprising a pair consisting of an upper cell culture chamber and a lower cell culture chamber, the pair being defined by a tubular inner wall divided by a porous membrane (3), the porous membrane (3) having two surfaces to which a living organism material is to be fixed, the upper cell culture chambers being coupled and integrated with each other, and the lower cell culture chambers being coupled and integrated with each other;
• a membrane holder (18) including a third inner wall (18b) having one end closed by the porous membrane (3), the third inner wall (18b) being the inner wall, the membrane holder (18) being one of the plurality of pairs of upper cell culture chambers and lower cell culture chambers;
• a plate (14) including a planar base (14a) and a plurality of through-holes (14d) in the planar base (14a), and the plurality of through-holes (14d) each receiving the membrane holder (18) inside and being the inner wall of each of the upper cell culture chambers and the lower cell culture chambers, the plate (14) being one of the plurality of pairs of upper cell culture chambers and lower cell culture chambers;
• two planar plate covers (15) placeable on the plate (14) to cover the plurality of through-holes (14d) each receiving the membrane holder (18) inside, the two planar plate covers (15) each being the cell culture chamber cover;
• and
• a pusher (17) including a planar push plate (17a) and a plurality of push portions (17c) extending perpendicularly to one surface of the planar push plate (17a) to be simultaneously received in the plurality of through-holes (14d).

A cell culture device comprising:

• a plurality of cell culture chambers, each of the plurality comprising a pair consisting of an upper cell culture chamber and a lower cell culture chamber, the pair being defined by a tubular inner wall divided by a porous membrane (3), the porous membrane (3) having two surfaces to which a living organism material is to be fixed, the upper cell culture chambers being coupled and integrated with each other, and the lower cell culture chambers being coupled and integrated with each other;
• a plurality of cell culture inserts (1) each including a fourth inner wall (1e) having one end closed by the porous membrane (3), the fourth inner wall (1e) being the inner wall, the plurality of cell culture inserts (1) being the upper cell culture chambers and the lower cell culture chambers;
• a cell culture case (11) including an open top and a bottom board (11a), the bottom board (11a) having a plurality of through-holes (11b) each being the inner wall and receiving the plurality of cell culture inserts (1), the cell culture case (11) including the upper cell culture chambers and the lower cell culture chambers;
• an insert holder (10, 12) configured to hold the plurality of cell culture inserts (1) while allowing the plurality of cell culture inserts (1) to be simultaneously received in the plurality of through-holes (11b) in cell culture case (11);
• and
• a case lid (13) configured to cover the open top of the cell culture case (11), the case lid (13) being the cell culture chamber cover.

A cell culture method for fabricating a three layer blood brain barrier in vitro model including a vascular endothelial cell, a pericyte, and an astrocyte performed with a cell culture device comprising:

• a plurality of cell culture chambers, each of the plurality comprising a pair consisting of an upper cell culture chamber and a lower cell culture chamber, the pair being defined by a tubular inner wall divided by a porous membrane (3), the porous membrane (3) having two surfaces to which a living organism material is to be fixed, the upper cell culture chambers being coupled and integrated with each other, and the lower cell culture chambers being coupled and integrated with each other;
• and a cell culture plate (33) having a plurality of first through-holes (33d), each through-hole having an inner circumferential surface that forms the inner wall, the porous membrane (3) covering the plurality of first through-holes (33d);
• the method comprising:
• seeding, with the astrocyte, a first surface of the two surfaces of the porous membrane (3) facing upward;
• culturing the astrocyte;
• simultaneously closing upper end openings of the plurality of pairs of upper cell culture chambers and lower cell culture chambers;
• simultaneously inverting the plurality of pairs of upper cell culture chambers and lower cell culture chambers to seed a second surface of the two surfaces of the porous membrane (3) with the pericyte;
• culturing the pericyte;
• laying a sheet of the vascular endothelial cell on the second surface of the porous membrane (3);
• and
• culturing the vascular endothelial cell.