Co-culturing device, motor neuron culturing device, multi-well plate, fabrication method of in vitro evaluation model of neuromuscular disease, and screening method of therapeutic agent against neuromuscular disease

1 . A co-culturing device for a motor neuron and a skeletal muscle cell, the device comprising:

a first unit for skeletal muscle tissue formation;

a second unit for motor neuron culture;

at least one third unit for causing the first unit and the second unit to communicate with each other; and

at least one pillar serving as a scaffold for the skeletal muscle tissue formation,

wherein the first unit includes

a first base material, and

a first culture tank formed in the first base material,

wherein the second unit includes

a second base material, and

a second culture tank formed in the second base material,

wherein the third unit includes

a third base material, and

an axon channel which is formed in the third base material and includes an inlet opening and an outlet opening, and through which a bundle of axons passes,

wherein the inlet opening of the third unit is configured to be connected to the second unit and configured to cause the axon channel and the second culture tank to communicate with each other when the third unit is connected to the second unit,

wherein a first opening member is attached to a periphery of the outlet opening to close the outlet opening of the third unit, and includes one or more first axon passage holes that pass through the first opening member from a first surface of the first opening member facing the axon channel to a second surface of the first opening member opposite to the first surface,

wherein the outlet opening of the third unit faces the first unit,

wherein the first axon passage hole has a size through which an axon passes while neither a motor neuron nor a skeletal muscle cell passes, and

wherein at least a part of the pillar is arranged in the first culture tank.

2 . The device according to claim 1 ,

wherein a height of the axon channel is 30 μm to 100 μm.

3 . The device according to claim 1 ,

wherein a height of the third unit is lower than a height of the first unit and/or second unit.

4 . A co-culturing device for a motor neuron and a skeletal muscle cell, the device comprising:

a first unit for skeletal muscle tissue formation;

a second unit for motor neuron culture;

at least one third unit for causing the first unit and the second unit to communicate with each other; and

at least one pillar serving as a scaffold for the skeletal muscle tissue formation,

wherein the first unit includes

a first base material, and

a first culture tank formed in the first base material,

wherein the second unit includes

a second base material, and

a second culture tank formed in the second base material,

wherein the third unit includes

a third base material, and

an axon channel which is formed in the third base material and includes an inlet opening and an outlet opening, and through which a bundle of axons passes,

wherein the inlet opening of the third unit is configured to be connected to the second unit and configured to cause the axon channel and the second culture tank to communicate with each other when the third unit is connected to the second unit,

wherein a first opening member is attached to a periphery of the outlet opening to close the outlet opening of the third unit, and includes one or more first axon passage holes that pass through the first opening member from a first surface of the first opening member facing the axon channel to a second surface of the first opening member opposite to the first surface,

wherein the outlet opening of the third unit faces the first unit,

wherein the first axon passage hole has a size through which an axon passes while neither a motor neuron nor a skeletal muscle cell passes,

wherein at least a part of the pillar is arranged in the first culture tank,

wherein the axon channel includes

a first channel, and

a second channel having an inlet opening and an outlet opening and disposed inside the first channel,

wherein the first opening member is formed so as to be in contact with the first channel,

wherein the inlet opening of the second channel is configured to communicate with the second culture tank, and a second opening member is attached to a periphery of the outlet opening of the second channel to close the outlet opening of the second channel,

wherein one or more second axon passage holes are formed in the second opening member, which pass through the second opening member from a first surface of the second opening member facing the second channel to a second surface of the second opening member opposite to the first surface of the second opening member, and each of the second axon passage holes has a size through which an axon passes while neither a motor neuron nor a skeletal muscle cell passes,

wherein in a cross section cutting the first channel and the second channel, the first channel and the second channel are separated from each other by walls, and

wherein a part of the first channel exists between the first surface of the first opening member and the second surface of the second opening member.

5 . The device according to claim 1 , wherein two or more third units are stacked.

6 . The device according to claim 1 ,

wherein a positional relationship of the first unit, the second unit, the one or more third units, and the pillar is arranged such that

when two or more pillars are included, any of virtual planes connecting the two or more pillars intersects a virtual axon channel of the axon channel virtually extending to the first culture tank, or

when only one pillar is provided, a virtual plane formed by the one pillar or a virtual plane formed by the pillar and the outlet opening of the third unit intersects a virtual axon channel of the axon channel virtually extending to the first culture tank.

7 . The device according to claim 6 , wherein two pillars are included, one end of each of the pillars is connected to the first base material, and the other end is arranged in the first culture tank in a cantilevered manner.

8 . The device according to claim 1 ,

wherein the size of the first axon passage hole is formed such that

the minimum distance is greater than or equal to 0.5 μm and less than or equal to 2.5 μm, or

the minimum distance is greater than or equal to 0.5 μm, and the sectional area is less than or equal to 60 μm 2 .

9 . The device according to claim 1 ,

wherein motor neuron culturing devices are each fabricated by the second unit and the third unit being connected to each other, and

wherein two or more of the motor neuron culturing devices are arranged to the first unit.

10 . The device according to claim 1 ,

wherein a motor neuron culturing device is fabricated by connecting two or more third units to the second unit, and

wherein the first unit is arranged to the outlet opening of any of the third units.

11 . The device according to claim 1 ,

wherein a height of the axon channel is lower than a height of the first culture tank and/or the second culture tank.

12 . The device according to claim 2 ,

wherein a height of the axon channel is lower than a height of the first culture tank and/or the second culture tank.

13 . The device according to claim 2 ,

wherein a height of the third unit is lower than a height of the first unit and/or second unit.

14 . The device according to claim 11 ,

wherein a height of the third unit is lower than a height of the first unit and/or second unit.

15 . A co-culturing device for a motor neuron and a skeletal muscle cell, the device comprising:

a first unit for skeletal muscle tissue formation;

a second unit for motor neuron culture;

at least one third unit for causing the first unit and the second unit to communicate with each other; and

at least one pillar serving as a scaffold for the skeletal muscle tissue formation,

wherein the first unit includes

a first base material, and

a first culture tank formed in the first base material,

wherein the second unit includes

a second base material, and

a second culture tank formed in the second base material,

wherein the third unit includes

a third base material, and

an axon channel which is formed in the third base material and includes an inlet opening and an outlet opening, and through which a bundle of axons passes,

wherein the inlet opening of the third unit is configured to be connected to the second unit and configured to cause the axon channel and the second culture tank to communicate with each other when the third unit is connected to the second unit,

wherein a first opening member is attached to a periphery of the outlet opening to close the outlet opening of the third unit, and includes one or more first axon passage holes that pass through the first opening member from a first surface of the first opening member facing the axon channel to a second surface of the first opening member opposite to the first surface,

wherein the outlet opening of the third unit faces the first unit,

wherein the first axon passage hole has a size through which an axon passes while neither a motor neuron nor a skeletal muscle cell passes,

wherein at least a part of the pillar is arranged in the first culture tank, and

wherein when the third unit is connected to the second unit and the second unit is connected to the first unit, the third unit is not in direct contact with the first unit.