JP6474970B2 - Clinostat - Google Patents

Description

The present invention relates to a clinostat used for cell culture.

In regenerative medicine, cells are cultured and grown in an undifferentiated state, and appropriate differentiation is induced for transplantation into a living body.
It is known that pluripotent stem cells can be proliferated in a state in which differentiation is suppressed by culturing in a weightless state (undifferentiated maintenance culture). It is also known that when differentiation into bone, cartilage, etc., differentiation is promoted by culturing with an appropriate medium in hypergravity greater than the Earth's gravitational acceleration (differentiation promotion culture). In order to perform such culturing on the ground, a gravity dispersion type culture apparatus that cultures pluripotent stem cells in a weightless state or an over-centrifugation culture apparatus that cultures pluripotent stem cells in a hypergravity state is used.

For example, in Patent Document 1, an outer frame is rotated by a motor attached to a main body, an inner frame is further rotated by a motor attached to the outer frame, and a culture vessel connected to the inner frame is rotated. Technology is disclosed. When culturing while suppressing differentiation of pluripotent stem cells, it is rotated biaxially. On the other hand, when differentiation is induced, uniaxial rotation is performed in which the outer frame is rotated while the rotation of the inner frame is stopped. Thereby, undifferentiated maintenance culture and differentiation promotion culture are realized by one apparatus.
Patent Document 2 discloses a clinostat that performs biaxial rotation and performs undifferentiated maintenance culture with a motor attached to a fixed frame of the motor in order to avoid motor failure caused by attaching the motor to the rotating frame. Is disclosed.

JP2003-9852 Japanese Patent No.4974283

In the technique of Patent Document 1, a motor is arranged on each of the outer frame and the inner frame, and the motor that rotates the inner frame is attached to the outer frame. growing. Usually, cell culture is performed in an incubator in which temperature, humidity, and carbon dioxide concentration are maintained at appropriate conditions. The size of the incubator used in each research institution is generally 160 to 170 L class. When the apparatus of Patent Document 1 was placed in a 160-170 L class incubator, the space in the incubator was almost occupied, and it was difficult to culture cells using a dish or the like in the vacant space.
Since the technique of Patent Document 2 is a biaxial rotating device, it is dedicated to gravity dispersion type culture and cannot be rotated in a single axis. Therefore, over-centrifugation culture cannot be performed. In addition, since the rotational force is transmitted to the inner rotating body through the elastic body of the exposed disc, it is worn out for a long period of time and causes rotation failure.

The purpose of the present invention is to reduce the size of the clinostat so that the inside of the incubator where the clinostat should be installed can be used effectively, and to perform undifferentiation maintenance culture and differentiation promotion culture with a single clinostat. .

The present invention includes a rotation drive device, an x rotation shaft having an axis in the x direction,
An x rotating body attached to the x rotating shaft, a y rotating shaft attached to the x rotating body and having an axis in the y direction perpendicular to the x direction, and attached to the y rotating shaft and containing a culture vessel Y-rotating body,
A rotation main shaft that is rotated by the rotation driving device; a rotation transmission mechanism that transmits a rotation force of the rotation main shaft to the y rotation shaft; and a clutch that transmits the rotation force between the rotation main shaft and the x rotation shaft. A ratchet mechanism or a one-way clutch that transmits rotation in one direction of the rotation main shaft to the x rotation shaft and cuts rotation in the other direction; and an operation mode of undifferentiated maintenance culture or an operation mode of differentiation promotion culture And a control device that switches by controlling the rotation of the rotary drive device.

Just by controlling rotation, undifferentiated maintenance culture (rotating the x-rotator and y-rotator) and differentiation-promoting culture (rotating the y-rotator while the x-rotator is stopped) are performed with a single rotation drive device. ) And a single rotary drive unit, the size of the clinostat can be reduced, and the volume occupied by the clinostat in the incubator is reduced. Therefore, other cultures can be performed in the empty space.

It is a figure which shows the clinostat stored in the incubator. It is a figure which shows the cross section of a clinostat. It is a figure which shows a clutch. It is a figure which shows the example of another clutch.

In FIG. 1, a clinostat 100 according to this embodiment has a fixed frame 23, a rotation drive device 22, an x-rotator 1, and a y-rotator 3 that accommodates a culture vessel 30. Cells and a medium are enclosed in the culture container 30. The clinostat 100 is installed inside an incubator 200 that can maintain temperature, humidity, and carbon dioxide concentration under appropriate conditions, and a control device 300 that controls the clinostat 100 is disposed outside the incubator 200. The rotation drive device 22 is a step motor, and the number of rotations can be controlled by the control device 300. As will be described later, the control device 300 switches between the operation mode of undifferentiated maintenance culture and the operation mode of differentiation promotion culture by controlling the rotation of the rotation drive device 22.

The x-rotating body 1 can be rotated by an x-rotating shaft 2 having an axis in the x direction in the figure, and the y-rotating body 3 has a y-rotation having an axis in the y direction in a direction different from the x axis in the x-rotating body 1. The shaft 4 can be rotated. Therefore, since the y-rotating body 3 is rotated by the x-rotating shaft 2 and the y-rotating shaft 4, the clinostat 100 can realize biaxial rotation. Reference numeral 31 denotes a stopper that prevents the culture vessel 30 from dropping off when the culture vessel 30 is accommodated in the y-rotator 3.

FIG. 2 is a side view showing a cross section of the clinostat 100. The x rotary body 1 is attached to the x rotary shaft 2, and the x rotary shaft 2 is attached to the fixed frame 23 via the x rotary shaft support bearing 13. The y rotating body 3 is attached to the x rotating shaft 2 that is not parallel to the x rotating shaft 2, and the y rotating shaft 4 is attached to the x rotating body 1 via the y rotating shaft support bearing 20. In the present embodiment, the x rotation axis 2 and the y rotation axis 4 intersect each other.

On the other hand, the rotation main shaft 19 is rotated by a rotation driving device 22 fixed to the fixed frame 23. The x rotation shaft 2 is hollow and is coaxial with the rotation main shaft 19. On the other hand, the rotation main shaft 19 is supported by a bearing 21 in the x rotation shaft 2 and can rotate independently of the x rotation shaft 2.

Rotational transmission from the rotation main shaft 19 to the y rotation shaft 4 is performed on the x rotator 1 by a rotational force transmission mechanism 40 including bevel gears 14a and 14b, a transmission shaft 15a, pulleys 16 and 18, and a timing belt. Describing upstream from the y rotation shaft 4, the rotational force is transmitted to the y rotation shaft 4 via the bevel gears 14 a and 14 b. The bevel gear 14b is attached to the transmission shaft 15a supported by the bearing 15b. A pulley 16 is attached to the transmission shaft 15a. A timing belt 17 is wound around the pulley 16 and the pulley 18. The pulley 18 is attached to the rotary main shaft 19.

Further, the rotation of the rotation main shaft 19 is transmitted to the gear 6 via the gear 5. The gear 6 is supported by a bearing 12 around the x-rotating shaft drive auxiliary shaft 10 and rotates. The clutch 9 is provided between the gear 6 and the x rotary shaft drive auxiliary shaft 10, and transmits or inhibits the rotation of the gear 6 to the x rotary shaft drive auxiliary shaft 10. A gear 7 is attached to the x rotation shaft drive auxiliary shaft 10, and the rotation is transmitted to the gear 8 attached to the x rotation shaft 2. The x rotary shaft drive auxiliary shaft 10 is attached to the fixed frame 23 via the x rotary shaft drive auxiliary shaft support bearing 11. That is, the clutch 9 switches whether or not to transmit the rotation of the rotation main shaft 19 to the x rotation shaft 2.

FIG. 3 is a diagram illustrating the clutch 9. The clutch 9 is a ratchet mechanism that transmits rotation only in one direction or a one-way clutch. In this embodiment, the clutch 9 includes a rotating plate 24, a stopper 26, a fulcrum pin 28, and a spring 29.

The rotating plate 24 has an escape wheel shape, and several catching portions 25 are provided on the outer periphery. The rotating plate 24 is fixed to the x-rotating shaft drive auxiliary shaft 10, and when the rotating plate 24 rotates, the x-rotating shaft drive auxiliary shaft 10 also rotates together.

The stopper 26 is connected to the gear 6 by a fulcrum pin 28, and the stopper 26 can rotate around the fulcrum pin 28. The spring 29 is attached between the gear 6 and the stopper 26, and the stopper 26 receives a force in a direction in which the spring 29 is always pressed against the rotating plate 24. When the gear 6 rotates, the stopper 26, the fulcrum pin 28, and the spring 29 also rotate together.

Next, the operation of the clutch 9 will be described.
When the gear 6 rotates counterclockwise on the drawing, the stopper 26, the fulcrum pin 28, and the spring 29 together with the gear 6 rotate counterclockwise. At that time, the catch portion 27 at the tip of the stopper 26 rotates while being pressed against the rotating plate 24 by the force of the spring 29. As the rotation continues, the catching portion 27 at the tip of the stopper 26 comes into contact with the catching portion 25 on the rotating plate 24 side. When the rotation is further continued, the rotational force of the gear 6 is transmitted to the rotary plate 24 and the x rotary shaft drive auxiliary shaft 10 via the stopper 26, and the rotary plate 24 and the x rotary shaft drive auxiliary shaft 10 rotate counterclockwise.

When the gear 6 rotates clockwise in the drawing, the stopper 26, the fulcrum pin 28, and the spring 29 rotate clockwise together with the gear 6. At that time, the catch portion 27 at the tip of the stopper 26 rotates while being pressed against the rotating plate 24 by the force of the spring 29. When the rotation continues, the stopper 26 receives a force by the rotating plate 24 to rotate the fulcrum pin 28 toward the gear 6 side. Since the stopper 26 rotates to the gear 6 side while pushing and shrinking the spring 29, the catching portion 25 on the rotating plate 24 side and the catching portion 27 on the stopper 26 side are not caught. Therefore, even if the gear 6 continues to rotate in the clockwise direction, the rotating plate 24 and the x rotating shaft drive auxiliary shaft 10 do not rotate.

When the operation mode of undifferentiated maintenance culture is performed, after the culture vessel 30 is mounted on the y-rotator 3, the control device 300 causes the rotation drive device 22 to rotate in the direction in which the gear 6 rotates counterclockwise in the drawing of FIG. drive. The rotation of the rotation driving device 22 is transmitted to the y rotating shaft 4 and the y rotating body 3 by the rotating main shaft 19, the pulley 18, the timing belt 17, the pulley 16, the transmission shaft 15b, and the bevel gears 14a and 14b. The y-rotator 3 rotates.

The rotation of the rotary drive device 22 is transmitted to the gear 6 via the gear 5 attached to the rotation main shaft 19. When the gear 6 rotates counterclockwise on the drawing, the stopper 26, the fulcrum pin 28, and the spring 29 together with the gear 6 rotate counterclockwise. At that time, the catch portion 27 at the tip of the stopper 26 rotates while being pressed against the rotating plate 24 by the force of the spring 29. As the rotation continues, the catching portion 27 at the tip of the stopper 26 comes into contact with the catching portion 25 on the rotating plate 24 side. When the rotation is further continued, the rotational force of the gear 6 is transmitted to the rotary plate 24 and the x rotary shaft drive auxiliary shaft 10 via the stopper 26, and the rotary plate 24 and the x rotary shaft drive auxiliary shaft 10 rotate counterclockwise. When the x rotation axis drive auxiliary shaft 10 rotates, the x rotation shaft 2 and the x rotating body 1 rotate via the gear 7 and the gear 8.

The culture container 30 is rotated in an arbitrary posture, and the gravitational acceleration acting on the culture container 30 acts in all directions of the culture container 30. After a certain period of time, the gravitational acceleration acting on the culture vessel 30 acts uniformly in all directions of the culture vessel 30, and the time average value of the gravitational acceleration is relative to all directions of the culture vessel 30. 0. Therefore, in the undifferentiated maintenance culture, a simulated microgravity environment close to the space environment can be obtained. When cell culture is performed in such a simulated microgravity environment, cells can be cultured while maintaining undifferentiation.

When performing the operation mode of differentiation promotion culture, after the culture vessel 30 is mounted on the y-rotator 3, the rotation device is driven by the control device 300 while the x-rotator 1 is stopped at an arbitrary position by a braking mechanism (not shown). 22 is operated in the direction in which the gear 6 rotates clockwise in the drawing of FIG. The direction of the axis of the y rotation shaft 4 may not be perpendicular to the horizontal, and may be perpendicular. The rotation of the rotation drive device 22 is transmitted to the y rotation shaft 4 and the y rotation body 3 by the rotation main shaft 19, the pulley 18, the timing belt 17, the pulley 16, the transmission shaft 15a, and the bevel gears 14a and 14b. The y rotator 3 rotates. The rotational speed of the rotary drive device 22 may be a constant rotational speed or may be changed during the culture.

The rotation of the rotation drive device 22 is transmitted to the gear 6 via the gear 5 attached to the rotation main shaft 19. When the gear 6 rotates clockwise in the drawing, the stopper 26, the fulcrum pin 28, and the spring 29 rotate clockwise together with the gear 6. At that time, the catch portion 27 at the tip of the stopper 26 rotates while being pressed against the rotating plate 24 by the force of the spring 29. When the rotation continues, the stopper 26 receives a force by the rotating plate 24 to rotate the fulcrum pin 28 toward the gear 6 side. Since the stopper 26 rotates to the gear 6 side while pushing and shrinking the spring 29, the catching portion 25 on the rotating plate 24 side and the catching portion 27 on the stopper 26 side are not caught. Therefore, even if the gear 6 continues to rotate in the clockwise direction, the rotating plate 24 and the x rotating shaft drive auxiliary shaft 10 do not rotate. Since the x rotary shaft drive auxiliary shaft 10 does not rotate, the x rotary shaft drive auxiliary shaft 10, the gear 7, the gear 8, the x rotary shaft 2, and the x rotary body 1 also do not rotate.

The culture vessel 30 rotates around the y rotation axis 4 and a centrifugal force proportional to the rotation radius and the square of the angular velocity acts in the rotation radius direction. The resultant force of centrifugal force and gravitational acceleration acts on the culture container. In order to generate a resultant force that is about three times the gravitational acceleration of the earth, the y-rotator 3 is rotated at a high speed of about 100 to 200 revolutions / minute.

According to the present embodiment, it is possible to make the clinostat 100 an undifferentiated maintenance culture device or a differentiation-promoting culture device simply by changing the rotation direction of the rotary drive device 22 by the control device 300. effective. In addition, the rotation of the x-rotating body 1 and the y-rotating body 3 can be applied with a single rotational driving device 22 at a fixed position, so that the clinostat can be reduced in size. The volume occupied in the incubator can be reduced. Therefore, other cultures can be performed in the empty space.

FIG. 4 shows another embodiment in which the clutch 9 is replaced by a clutch 90.
In order to perform differentiation-promoting culture, the speed at which the y-rotator 3 is rotated in order to obtain an appropriate resultant force due to gravity acceleration and centrifugal force is determined, but in order to reproduce a simulated microgravity environment by undifferentiated maintenance culture Applicants' experiments have shown that there is no need to rotate quickly. For example, the x rotator 1 and the y rotator 3 may be rotated at a low speed of 10 revolutions / minute or less, and the difference in rotational speed is different by at least one digit.

The clutch 90 utilizes such a difference in the rotational speed to transmit the rotational force to the x-rotation shaft drive auxiliary shaft 10 when the rotation of the gear 6 is slow and to the x-rotation shaft drive auxiliary shaft when the rotation of the gear 6 is fast. The transmission of the rotational force to 10 is cut off. The clutch 90 includes a cylinder 91 concentrically fixed to the x-rotating shaft drive auxiliary shaft 10, a braking piece 92 facing the side surface of the cylinder 91, a guide path 93 that guides the braking piece 92 from the gear 6 to the cylinder 91, and a braking piece 92. And a spring 94 that urges the cylinder 91 to press against the side surface of the cylinder 91.

When undifferentiated maintenance culture is performed, after the culture vessel 30 is mounted on the y-rotator 3, the rotation driving device 22 is rotated by the control device 300 at a low rotation speed. Since the braking piece 92 is pressed against the spring 94 and the cylinder 91 and the gear 6 are relatively fixed, the rotation of the gear 6 is transmitted to the x-rotating shaft drive auxiliary shaft 10 and the x-rotating body 1. And the y-rotator 3 rotate.

When performing differentiation-promoting culture, after the culture vessel is mounted on the y-rotator 3, the rotation drive device 22 is rotated at high speed by the control device 300. The braking piece 92 pushes and contracts the spring 94 by centrifugal force, and the braking piece 92 cannot maintain braking with the side surface of the cylinder 91, and the rotational force of the rotary drive device 22 is not transmitted to the x-rotating body 1. Since the centrifugal force is related to the square of the angular velocity, if the rotation speed of the differentiation promoting culture is set to be different from the control apparatus 300 by at least one digit with respect to the rotation speed during the undifferentiated maintenance culture, the clutch 90 can operate as desired. Due to the gyro effect caused by the rotation of the y-rotating body 3, the axial direction of the y-rotating shaft 4 of the y-rotating body 3 is maintained in a substantially constant direction.

In the above embodiment, the rotational force transmission mechanism 40 using the bevel gears 14a and 14b, the pulleys 16 and 18, and the timing belt 17 is used to transmit the rotation of the rotation main shaft 19 to the y rotation shaft 1. You may use the rotational force transmission mechanism which combined these known transmission elements. For example, the rotation of the rotary main shaft 19 may be transmitted to the transmission shaft 15a using a gear instead of the timing belt 17, and a crown gear or a screw gear may be used instead of the bevel gears 14a and 14b. Alternatively, the rotation main shaft 19 and the y rotation shaft 4 may be connected by a flexible shaft. Moreover, although the y rotary body 2 was rotated inside the x rotary body 1, the reverse may be sufficient.

1: x rotating body 2: x rotating shaft 3: y rotating body 4: y rotating shaft 5-8: gear 9, 90: clutch 10: x rotating shaft drive auxiliary shaft 11-13, 20, 21: bearing 14a, 14b: Bevel gear 15a: Transmission shaft 16, 18: Pulley 17: Timing belt 19: Rotating main shaft
22: Rotation drive device 23: Fixed frame 24: Rotating plate 25, 27: Hook 26: Stopper 28: Support pin 29: Spring 30: Culture vessel 40: Rotating force transmission mechanism 91: Cylinder 92: Braking piece 93: Guide path 94: Spring 100: Clinostat 200: Incubator 300: Control device

Claims (2)

A rotary drive device;
an x rotation axis having an axis in the x direction;
An x-rotating body attached to the x-rotating shaft;
A y rotation axis attached to the x rotation body and having an axis in the y direction perpendicular to the x direction;
A y-rotator which is attached to the y-rotating shaft and accommodates a culture vessel;
A rotation spindle rotated by the rotation drive device;
A rotation transmission mechanism for transmitting the rotational force of the rotation main shaft to the y rotation shaft;
A clutch for transmitting a rotational force between the rotation main shaft and the x rotation shaft, wherein the ratchet mechanism transmits rotation in one direction of the rotation main shaft to the x rotation shaft and cuts off rotation in the other direction. Or with a one-way clutch ,
A clinostat comprising: a control device that switches an operation mode of undifferentiated maintenance culture or an operation mode of differentiation promotion culture by controlling rotation of the rotation drive device.
The clinostat according to claim 1,
The x-rotating shaft has a hollow in the x direction at the axis;
The clinostat is characterized in that the rotation main shaft is disposed concentrically in the hollow of the x rotation shaft.