JP6522346B2 - Clinostat - Google Patents

Description

The present invention relates to a clinostat used to culture cells.

In regenerative medicine, cells are cultured and proliferated in an undifferentiated state, and differentiation induction is carried out for transplantation into a living body.
Pluripotent stem cells are known to be able to proliferate in a state of reduced differentiation by culturing in a state of weightlessness (nondifferentiated maintenance culture). In addition, it is also known that differentiation is promoted by culturing with an appropriate medium in supergravity greater than the gravitational acceleration of the earth when differentiating into bones and cartilage, etc. (differentiation promoting culture). In order to perform such culture on the ground, a gravity dispersion-type culture device for culturing pluripotent stem cells in a weightless state or a heavy centrifugal culture device for culturing pluripotent stem cells in a hypergravity state are used.

For example, in Patent Document 1, the outer frame is rotated by a motor attached to the main body, and the inner frame is further rotated by a motor attached to the outer frame to rotate a culture vessel connected to the same body to the inner frame. Technology is disclosed. In the case of suppressing differentiation and culturing pluripotent stem cells, biaxial rotation is performed. On the other hand, in the case of inducing differentiation, uniaxial rotation is performed in which the outer frame is rotated while rotation of the inner frame is stopped. Thereby, undifferentiated maintenance culture and differentiation promotion culture are realized by one device.
In addition, in Patent Document 2, in order to avoid motor failure due to attaching a motor to a rotating frame, a motor attached to a fixed frame of the motor achieves two-axis rotation and a clinostat that performs undifferentiated maintenance culture Is disclosed.

JP 2003-9852 A Patent No. 4974283 gazette

In the technique of Patent Document 1, the motor is disposed on each of the outer frame and the inner frame, and the motor for rotating the inner frame is attached to the outer frame. growing. Usually, cell culture is performed in an incubator maintained at appropriate conditions of temperature, humidity and carbon dioxide concentration. The size of the incubator used in each research institute is generally 160-170 L class. When the apparatus of Patent Document 1 was placed in an incubator of class 160 to 170 L, it almost occupied a space in the incubator, and it was difficult to culture cells using a dish or the like in the vacant space.

The technique of Patent Document 2 is an apparatus for two-axis rotation, and therefore, is dedicated to gravity dispersion-type culture, and can not be uniaxially rotated. Therefore, heavy centrifugal culture can not be performed. In addition, since the rotational force is transmitted to the inner rotating body through the elastic body of the disk in the exposed state, it wears and causes rotation failure when used for a long time.
In addition, the present applicant proposed the krinostat which achieved size reduction by Japanese Patent Application No. 2014-121939. The clinostat disclosed in the above has not been provided with a clutch for transmitting or disconnecting the rotational force of the motor to the x-rotation axis by changing the rotational direction of the motor for rotating the y-rotation axis or increasing the rotational speed. The differentiation maintenance culture and the differentiation promotion culture can be performed with one crinostat.

The object of the present invention is to further miniaturize the crinostat so that the inside of the incubator in which the crinostat is to be installed can be effectively utilized, and to perform undifferentiated maintenance culture and differentiation promotion culture with one crinostat. Do.

According to the present invention, there are provided a rotary drive disposed in a housing, an x rotary shaft to which a rotational force of the rotary drive is transmitted, an x rotary body attached to one end of the x rotary shaft, and the x rotary body Concentrically in the hollow inside the x axis of rotation, the y axis of rotation having the axis of y direction orthogonal to the x direction, and the y axis of rotation attached to the y axis of rotation and housing the culture vessel And a rotational force transmission mechanism for transmitting the rotation of the central axis to the y rotational axis, and the x rotational axis and the central axis on the other end side of the x rotational axis. And a clutch disposed concentrically to switch between a state transmitting torque between the x rotation axis and the central axis and a state fixing the central axis to the housing, Operation mode of the undifferentiated maintenance culture by controlling the It is characterized by switching the operation mode of differentiation promoting culture.

According to the present invention, depending on the state of the clutch, undifferentiated maintenance culture (rotating the x rotating body and the y rotating body around the x rotation axis and y rotation axis respectively) and differentiation promoting culture with one rotation drive device (The x rotor and the y rotor can be rotated around the x rotation axis), and the number of rotary drive units is one, so the crinostat can be miniaturized and the crinostat occupies the incubator Volume decreases. Therefore, there is an effect that another culture can be performed in the vacant space.

FIG. 2 shows a crinostat stored in an incubator. Figure 2 shows a side cross section (Figure 2A) and a x1-x1 cross section (Figure 2B) of clinostat. It is a perspective view which shows the power transmission system in a housing. It is sectional drawing which shows the operation mode (FIG. 4A) of differentiation promotion culture | cultivation, and the clinostat of an idle state (FIG. 4B).

In FIG. 1, the crinostat 100 according to the present embodiment includes a housing 23, an x-rotating body 1, and a y-rotating body 3 accommodating the culture vessel 30. The culture vessel 30 contains cells and a medium. The clinostat 100 is installed inside an incubator 200 capable of maintaining temperature, humidity and carbon dioxide concentration under appropriate conditions.

The x-rotating body 1 is rotatable by an x-rotational axis 2 having an axis in the x-direction in the drawing, and the y-rotating body 3 has an y-axis in a direction different from the x-direction It can be rotated by the rotating shaft 16. Since the y-rotating body 3 is rotated by the x rotation axis 2 and the y rotation axis 16, the crinostat 100 can realize two-axis rotation. Reference numeral 31 denotes a stopper for preventing the culture container 30 from falling off when the culture container 30 is accommodated in the y rotating body 3.

FIG. 2 shows the side cross section (FIG. 2A) and the x1-x1 cross section (FIG. 2B) of the clinostat 100. FIG. In FIG. 2A, the rotary drive 22 and the housing 6 are fixed in the housing 23. The rotation drive device 22 is a stepping motor, and the housing 6 is a hollow member that accommodates the x rotation shaft 2 and a power transmission system such as a clutch lever 15 and a clutch 7 described later. At the inner peripheral side of the housing 6, a donut-shaped disc 6a protrudes. The housing 6 is disposed in the housing 23 parallel to the direction of the output shaft 22 a of the rotary drive 22 and substantially overlapping the length range of the rotary drive 22. A pulley 19 is attached to the output shaft 22 a of the rotational drive device 22, and transmits the rotational force of the output shaft 22 a to the x rotation shaft 2 in the housing 6 via the timing belt 24.

As main components of the power transmission system in the housing 6, an x rotation shaft 2, a clutch 7, a clutch lever 15 and a central shaft 10 are provided. The x rotation shaft 2 is rotatably attached to the housing 6 via the x rotation shaft support bearing 13. A timing belt 24 is wound around a pulley portion 2 b located at the center of the x rotation shaft 2. An x-rotating body 1 is fixed to one end side (right side in the drawing) of the x-rotational shaft 2 and is rotated. On the other end side (left side in the drawing) of the x rotating shaft 2, a wheel portion 2a which is radially expanded in a flange shape is provided. In addition, the x rotation shaft 2 is hollow, and the central shaft 10 is concentrically supported by the bearing 21. The central shaft 10 can be rotated independently of the x rotation shaft 2.

The clutch 7 is provided on the other end side of the x rotation shaft 2 and is concentric with the x rotation shaft 2 and the central shaft 10, and has a wheel portion 7a radially expanded in a flange shape on the left side in the drawing It has a disk unit 7b. The right disc 7b has a hexagonal through hole 7c at the center. The clutch lever 15 is C-shaped in a side view, and has claws 15a and 15b provided on the inside in an opposing manner. The claws 15a and 15b abut on the wheel 7a. The clutch lever 15 is pushed by the rotation of the cam 5 and can move left and right in the drawing. Further, the clutch lever 15 is provided with claws 15 c and 15 d toward the outside. One end of a spring 6b is in contact with the claws 15c and 15d, and the clutch lever 15 is biased in the right direction in the drawing. The other end of the spring 6b is fixed to the housing 6 by a locking piece 6c.

In the central shaft 10, the pulley 4 is fixed on the right side of the drawing, and on the left side, a shaft portion 10a having a hexagonal cross section passes through the through hole 7c. In addition, on the opposite side which penetrated, a spring 8 is disposed between the stop ring 10b and the disc portion 7b. The spring 8 acts to bias the clutch 7 in the right direction in the drawing.
In FIG. 2B, the clutch lever 15 slides in a guide groove 6 d provided in the housing 6. The cam 5 is rotated through the shaft 9 by turning a knob 14 provided outside the housing 23.

FIG. 3 is a perspective view of the x rotation shaft 2, the clutch 7, the clutch lever 15, the cam 5 and the central shaft 10 which constitute the power transmission system in the housing 6 shown in FIG. The clutch 7 has a donut-shaped wheel portion 7a and a disc-shaped disc portion 7b, and donut-shaped friction members 7e and 7g are provided on the opposing surfaces on the outer peripheral side of the disc portion 7b. The body 7f between the wheel 7a and the disk 7b has a cylindrical shape. By inserting the hexagonal shaft portion 10a of the central shaft 10 into the hexagonal through hole 7c of the disk portion 7b, the rotation of the clutch 7 is transmitted to the central shaft 10, and the clutch 7 is the shaft portion 10a. Slide. The position of the central shaft 10 in the left-right direction in the drawing is constant with respect to the housing 6, so the clutch 7 moves to the left and right.

The friction material 7e provided on one surface of the disk portion 7b abuts against the wheel portion 2a, whereby the rotation of the x rotation shaft 2 is transmitted to the clutch 7. The friction material 7g provided on the other opposite surface of the disk portion 7b abuts on the donut-shaped disk 6a protruding to the inner peripheral side of the housing 6 so that the clutch 7 is fixed to the housing 6 Do.

Returning to FIG. 2, the y rotating body 3 is attached to the y rotating shaft 16, and the y rotating shaft 16 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 16 are orthogonal to each other.

Transmission of rotational force from the central axis 10 to the y rotation axis 16 is performed by transmitting rotational force disposed in the x rotating body 1 including the pulley 4, the timing belt 25, the pulley 17, the transmission shaft 18 a and bevel gears 11 a and 11 b. It is done by mechanism 40. To explain from the y rotation shaft 16 to the upstream side, the rotational force is transmitted to the y rotation shaft 16 via the bevel gears 11 a and 11 b. The bevel gear 11b is attached to a transmission shaft 18a supported by a bearing 18b. A pulley 17 is attached to the transmission shaft 18a. A timing belt 25 is wound around the pulley 17 and the pulley 4. The pulley 4 is attached to the central shaft 10.

Next, the operation of the clinostat 100 will be described.
When the clinostat 100 is operated in the undifferentiated maintenance culture operation mode, the knob 14 is rotated to move the clutch lever 15 to the leftmost position as shown in FIG. 2A. Then, the housing 23 is installed in the incubator 200 so that the x rotation axis 2 is horizontal. In this state, the clutch 7 is moved to the left by the claws 15a and 15b, and the friction material 7g provided on the disc 7b of the clutch 7 is pressed against the disc 6a. On the other hand, the friction material 7e provided on the disk 7b is separated from the wheel 2a, and the transmission of the rotational force from the x rotation shaft 2 to the clutch 7 is interrupted. In this state, when the rotary drive 22 is activated, the rotation of the rotary drive 22 is transmitted to the pulley 19, the timing belt 24, and the pulley portion 2b of the x rotation shaft 2, and the x rotation shaft 2 Rotate. On the other hand, the clutch 7 is fixed to the housing 6 and the housing 23. In this state, when the x rotating body 1 rotates, the pulley 17 rotates around the pulley 4, and the pulley 17 rotates relative to the x rotating body 1. The rotational force is transmitted to the y rotation shaft 16 and the y rotating body 3 by the transmission shaft 18a and the bevel gears 11a and 11b.

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 time has passed, 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 in the entire direction of the culture vessel 30. It becomes 0. Therefore, in the undifferentiated maintenance culture, it is possible to obtain a simulated microgravity environment close to the space environment. When cell culture is performed under such a simulated microgravity environment, cells can be cultured while maintaining undifferentiated state.

When the clinostat 100 is operated in the differentiation promoting culture operation mode, the knob 14 is rotated 90 degrees from the angular position of the undifferentiated maintenance culture operation mode, and the cam 5 moves the clutch lever 15 most upward in FIG. 4A. Let it be in a state of being Then, as shown in FIG. 4A, the housing 23 is installed in the incubator 200 so that the x rotation axis 2 is vertical. In this state, when the rotary drive unit 22 is activated, the rotation of the rotary drive unit 22 is transmitted to the pulley 19, the timing belt 24, and the x rotary shaft 2, and the x rotary shaft 2 rotates the x rotary body 1. On the other hand, the clutch 7 is pressed against the wheel portion 2 a by the spring 8 so as to rotate in the same manner as the x rotation shaft 2. On the other hand, the friction material 7g is separated from the disc 6a. Further, in FIG. 4A, the spring 6b pushes the claws 15c and 15d upward, so that the claws 15a and 15b do not come in contact with the wheel 7a of the clutch 7. In this state, when the x-rotating body 1 rotates, the pulley 17 rotates around the pulley 4, but since the pulley 4 itself is also rotating, the pulley 17 is stopped relative to the x-rotating body 1 Be in a state of Therefore, the y rotation axis 16 does not rotate, and since the x rotation axis 2 is vertical, it rotates around the x rotation axis 2, and the rotation radius and the centrifugal force proportional to the square of the angular velocity are in the rotation radius direction Act on. The combined force of the centrifugal force and the gravitational acceleration acts on the culture vessel. In order to generate a resultant force about three times the gravitational acceleration of the earth, the y-rotating body 3 is rotated at a high speed of about 100 to 200 revolutions / minute.

The knob 14 is provided with a notch between the angular position in the operation mode of undifferentiated maintenance culture and the angular position in the operation mode of differentiation promoting culture, and the rotation angle of the knob 14 is temporarily set at this position. It is supposed to be held. FIG. 4B shows that the knob 14 has been rotated and the cam 5 has been tilted 45 degrees. In this state, the contact between the friction member 7e of the disk portion 7b and the wheel portion 2a is released, and the friction member 7g of the disk portion 7b is also separated (idle state). In this idle state, the clutch 7 is not fixed to the housing 6 (or the housing 23), and when the operator manually applies a force to the y rotating body 3, the y rotating body 3 becomes the x rotating body 1 And can be rotated independently.
The angular position of the knob 14 in the state in which the y rotor 3 is manually rotated independently of the x rotor 1 is referred to as an idle position. At the idle position, since the angle of the y-rotating body 3 can be freely changed manually, the culture vessel 30 can be mounted at an angle that makes it easy to mount the y-rotating body 3.

According to the present embodiment, the clutch 7 has an effect that the crinostat 100 can be used as an apparatus for undifferentiated maintenance culture or as an apparatus for differentiation promotion culture.

According to the present embodiment, the rotary drive 22 is disposed in parallel in the housing 23 so that the rotary drive 22 is accommodated within the length range of the clutch lever 15, the clutch 7 and the x rotary shaft 2. 22 lengths do not increase the entire clinostat 100

In addition, since the rotational force can be applied to the rotation of the x rotating body 1 and the y rotating body 3 with one rotation drive device 22, miniaturization of the crinostat becomes possible, and the crinostat occupies the inside of the incubator. Volume can be reduced. Therefore, other cultures can be performed in the vacant space.

In the above embodiment, in order to transmit the rotation of the central shaft 10 to the y rotation shaft 16, the rotational force transmission mechanism 40 including the bevel gears 11a and 11b, the pulleys 4 and 17, the transmission shaft 18a and the timing belt 25 is used. However, it is also possible to use a rotational force transmission mechanism that combines other known transmission elements. For example, a gear may be used instead of the timing belt 25 to transmit to the y rotation shaft 16 of the central shaft 10, and a crown gear or a screw gear may be used instead of the bevel gears 11a and 11b. Alternatively, the central shaft 10 and the y rotation shaft 16 may be connected by a flexible shaft.

In the embodiment, the hexagonal shaft portion 10a is caused to slide in the hexagonal through hole 7c of the disk portion 7b, but the rotational force is transmitted to the central shaft 10 In order to make the clutch 7 slide, the shapes of the through hole 7c and the shaft portion 10a may be another polygonal shape or an elliptical shape regardless of the hexagonal shape.

DESCRIPTION OF SYMBOLS 1 x rotating body 2 x rotating shaft 3 y rotating body 4, 17, 19 pulley 5 cam 6 housing 7 clutch 8 spring 10 central shaft 14 knob 15 clutch lever 16 y rotating shaft 23 housing 30 culture container

Claims (3)

A rotary drive disposed within the housing;
An x rotation axis to which the rotational force of the rotational drive is transmitted;
An x-rotating body attached to one end of the x-rotation axis;
A y rotation axis attached to the x rotor and having an axis in the y direction orthogonal to the x direction;
A y-rotating body attached to the y-rotating shaft and containing a culture vessel;
A central axis coaxially and independently rotatably accommodated in the hollow inside the x-axis of rotation;
A rotational force transmission mechanism for transmitting the rotation of the central axis to the y rotation axis;
A state of being concentrically arranged with the x rotation axis and the central axis on the other end side of the x rotation axis, and transmitting a rotational force between the x rotation axis and the central axis, and the central axis as the housing And a clutch that switches between the fixed state and the fixed state,
A crinostat characterized by switching an operation mode of undifferentiated maintenance culture or an operation mode of differentiation promotion culture by controlling the state of the clutch.
The crinostat according to claim 1, wherein the clutch does not transmit the rotation of the x rotation axis and does not fix the central axis to the housing.
In the clinostat according to claim 1,
The x-axis of rotation and the clutch are disposed in the housing such that the length of the rotary drive overlaps the direction of the output shaft of the rotary drive.