JP2016129493A - Clinostat - Google Patents

Abstract

PROBLEM TO BE SOLVED: To perform undifferentiation-maintaining culture and differentiation-promoting culture by a single clinostat.SOLUTION: A clinostat 100 has: a rotary driving device arranged in an enclosure 23; an x-rotary shaft 2 rotated by the rotary driving device; an x-rotor 1 attached to one end of the x-rotary shaft 2; a y-rotary shaft 16 which is attached to the x-rotor 1, and has a shaft center in y-direction; a y-rotor 3 which is attached to the y-rotary shaft 16, and houses a culture vessel 30; a central shaft which is housed concentrically in a hollow inside the x-rotary shaft 2 and independently rotatably; a torque transmission mechanism which transmits rotation of the central shaft to the y-rotary shaft 16; and a clutch which is arranged for the other end side of the x-rotary shaft 2 concentrically with the x-rotary shaft 2 and the central shaft, and switches a state where torque is transmitted between the x-rotary shaft 2 and the central shaft and a state where the central shaft is fixed to the enclosure, in which the clinostat controls the state of the clutch, and switches an operation mode of undifferentiation-maintaining culture or an operation mode of differentiation-promoting culture.SELECTED DRAWING: Figure 1

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 present applicant has proposed a clinostat with a reduced size according to Japanese Patent Application No. 2014-121939. The clinostat disclosed therein is not provided with a clutch that transmits or disconnects the rotational force of the motor to the x-rotating shaft by changing the rotational direction of the motor that rotates the y-rotating shaft or increasing the rotational speed. Differentiation maintenance culture and differentiation promotion culture can be performed with one clinostat.

An object of the present invention is to further reduce the size of the clinostat so that the inside of the incubator in which the clinostat should be installed can be used effectively, and to perform undifferentiation maintenance culture and differentiation promotion culture with a single clinostat. To do.

The present invention relates to a rotary drive device arranged in a housing, an x rotary shaft to which a rotational force of the rotary drive device is transmitted, an x rotary body attached to one end of the x rotary shaft, and the x rotary body And a y-rotating shaft having a y-direction axial center different from the x-direction, a y-rotating body attached to the y-rotating shaft and containing a culture vessel, and a hollow inside the x-rotating shaft A central axis that is concentrically and independently rotatably accommodated, a rotational force transmission mechanism that transmits the rotation of the central axis to the y rotation axis, and the x rotation axis and the other end side of the x rotation axis A clutch that is arranged concentrically with a central axis and switches between a state in which a rotational force is transmitted between the x rotational axis and the central axis and a state in which the central axis is fixed to the housing; By controlling the state, the operation mode of undifferentiated maintenance culture It is characterized by switching the operation mode of differentiation promoting culture.

According to the present invention, the undifferentiated maintenance culture (the x-rotator and the y-rotator are rotated around the x-rotation axis and the y-rotation axis, respectively) and the differentiation-promoting culture with one rotary drive device depending on the state of the clutch. (The x-rotating body and the y-rotating body can be rotated around the x-rotating axis) and the rotational drive device becomes one, so that the size of the clinostat can be reduced and the clinostat occupies the inside of the incubator. The volume becomes smaller. Therefore, there is an effect that another culture is possible in the vacant space.

It is a figure which shows the clinostat stored in the incubator. It is a figure which shows the side surface cross section (FIG. 2A) and x1-x1 cross section (FIG. 2B) of a 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, a clinostat 100 according to this embodiment includes a housing 23, an x-rotating body 1, and a y-rotating body 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.

The x-rotating body 1 can be rotated by an x-rotating shaft 2 having an x-direction axis in the figure, and the y-rotating body 3 has a y-direction axis center different from the x-direction in the x-rotating body 1. The rotary shaft 16 can rotate. Since the y rotating body 3 is rotated by the x rotating shaft 2 and the y rotating shaft 16, the clinostat 100 can realize the 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 shows a side cross section (FIG. 2A) and a x1-x1 cross section (FIG. 2B) of the clinostat 100. In FIG. 2A, the rotation drive device 22 and the housing 6 are fixed in the housing 23. The rotational drive device 22 is a stepping motor, and the housing 6 is a hollow member that houses the x-rotating shaft 2 and a power transmission system such as a clutch lever 15 and a clutch 7 described later. On the inner peripheral side of the housing 6, a donut-shaped disk 6 a protrudes. The housing 6 is disposed in the housing 23 so as to be parallel to the direction of the output shaft 22 a of the rotary drive device 22 and to substantially overlap the length range of the rotary drive device 22. A pulley 19 is attached to the output shaft 22 a of the rotation drive device 22, and the rotational force of the output shaft 22 a is transmitted 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-rotating 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 an x rotation shaft support bearing 13. A timing belt 24 is wound around the pulley portion 2b located at the center of the x-rotating shaft 2. An x-rotating body 1 is fixed to one end side (right side in the figure) of the x-rotating shaft 2 and is rotated. On the other end side (left side in the figure) of the x-rotating shaft 2, a wheel portion 2a that is widened in a radial direction in a flange shape is provided. The x-rotating shaft 2 is hollow, and the central shaft 10 is supported by a bearing 21 concentrically. 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-rotating shaft 2 and concentrically with the x-rotating shaft 2 and the central shaft 10, and has a wheel portion 7a that is radially expanded in a flange shape on the left side in the drawing, and on the right side. It has a disk portion 7b. The right disk 7b has a hexagonal through hole 7c in the center. The clutch lever 15 is C-shaped in a side view, and has claw portions 15a and 15b provided on the inner side in a facing manner. The claw portions 15a and 15b are in contact with the wheel portion 7a. The clutch lever 15 is pushed by the rotation of the cam 5 and can move left and right in the drawing. The clutch lever 15 is provided with claw portions 15c and 15d facing outward. One end of the spring 6b is in contact with the claw portions 15c and 15d, and the clutch lever 15 is biased rightward in the drawing. The other end of the spring 6b is fixed to the housing 6 by a locking piece 6c.

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

3 is a perspective view of the x-rotating shaft 2, the clutch 7, the clutch lever 15, the cam 5, and the central shaft 10 constituting the power transmission system in the housing 6 shown in FIG. The clutch 7 includes a donut-shaped wheel portion 7a and a disk-shaped disc portion 7b, and donut-shaped friction materials 7e and 7g are provided on the opposing surfaces on the outer peripheral side of the disc portion 7b, respectively. A body portion 7f between the wheel portion 7a and the disk portion 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 connected to the shaft portion 10a. Slide. Since the position of the central shaft 10 in the left-right direction in the drawing is constant with respect to the housing 6, the clutch 7 moves to the left and right.

The friction material 7e provided on one surface of the disk portion 7b is brought into contact with the wheel portion 2a, whereby the rotation of the x-rotating shaft 2 is transmitted to the clutch 7. The friction material 7g provided on the other facing surface of the disk portion 7b is in contact with 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. To 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 this embodiment, the x rotation axis 2 and the y rotation axis 16 are orthogonal to each other.

The transmission of the rotational force from the central shaft 10 to the y rotation shaft 16 is performed by transmitting the rotational force disposed in the x rotating body 1 including the pulley 4, the timing belt 25, the pulley 17, the transmission shaft 18a, and the bevel gears 11a and 11b. This is done by mechanism 40. Describing upstream from the y rotation shaft 16, a 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 the transmission shaft 18a supported by the 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 operation mode of the undifferentiated maintenance culture is performed on the clinostat 100, the knob 14 is rotated so that the cam 5 moves the clutch lever 15 to the leftmost side as shown in FIG. 2A. And the housing | casing 23 is installed in the incubator 200 so that the x rotating shaft 2 may become horizontal. In this state, the clutch 7 is moved to the left by the claw portions 15a and 15b, and the friction material 7g provided on the disk 7b of the clutch 7 is pressed against the disc 6a. On the other hand, the friction material 7e provided on the disk portion 7b is in a state of being separated from the wheel portion 2a, and transmission of the rotational force from the x-rotating shaft 2 to the clutch 7 is stopped. In this state, when the rotation driving device 22 is started, the rotation of the rotation driving device 22 is transmitted to the pulley 19, the timing belt 24, and the pulley portion 2 b of the x rotating shaft 2, and the x rotating shaft 2 moves the x rotating body 1. Rotate. On the other hand, the clutch 7 is fixed to the housing 6 and the housing 23. In this state, when the x rotator 1 rotates, the pulley 17 rotates around the pulley 4, and the pulley 17 rotates relative to the x rotator 1. This rotational force is transmitted to the y rotating 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 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 the operation mode of the differentiation promoting culture is performed for the clinostat 100, the knob 14 is rotated 90 degrees from the angular position of the operation mode of the undifferentiated maintenance culture, and the cam 5 moves the clutch lever 15 to the uppermost side in FIG. Let it be in the state you let it. 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 rotation driving device 22 is started, the rotation of the rotation driving device 22 is transmitted to the pulley 19, the timing belt 24, and the x rotation shaft 2, and the x rotation shaft 2 rotates the x rotation body 1. On the other hand, the clutch 7 is pressed against the wheel portion 2 a by the spring 8 and is rotated in the same body as the x rotation shaft 2. On the other hand, the friction material 7g is separated from the disk 6a. 4A, the spring 6b pushes the claw portions 15c and 15d upward so that the claw portions 15a and 15b do not come into contact with the wheel portion 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 also rotates, the pulley 17 stops relative to the x-rotating body 1. It will be in a state. Therefore, since the y rotation axis 16 does not rotate and the x rotation axis 2 is vertical, the y rotation axis 16 rotates around the x rotation axis 2, and the centrifugal force proportional to the rotation radius and the square of the angular velocity is the rotation radius direction. Act on. 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.

The knob 14 is provided with a notch between the angular position in the operation mode of the undifferentiated maintenance culture and the angular position in the operation mode of the 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 is rotated and the cam 5 is inclined at 45 degrees. In this state, the friction material 7e of the disk portion 7b and the wheel portion 2a are separated from each other, while the friction material 7g of the disk portion 7b is also separated (idle state). In this idle state, the clutch 7 is not in a state of being fixed to the housing 6 (or the casing 23), and when the operator manually applies a force to the y-rotating body 3, the y-rotating body 3 is changed to the x-rotating body 1. It is in a state where it can be rotated independently.
The angular position of the knob 14 in a state where the y rotating body 3 is manually rotated independently of the x rotating body 1 is referred to as an idle position. At the idle position, the angle of the y rotator 3 can be freely changed manually, so that the culture vessel 30 can be mounted at an angle at which it can be easily mounted on the y rotator 3.

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

According to the present embodiment, since the rotary drive device 22 is disposed in parallel so as to be within the length range of the clutch lever 15, the clutch 7 and the x rotary shaft 2, the rotary drive device The length of 22 does not enlarge the entire clinostat 100

Further, since the rotational force can be applied to the rotation of the x-rotating body 1 and the y-rotating body 3 by one rotation driving device 22, the clinostat can be downsized, and the clinostat occupies the inside of the incubator. The volume can be reduced. Therefore, other cultures can be performed in the empty space.

In the above embodiment, in order to transmit the rotation of the center 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, the timing belt 25, and the like is used. However, you may use the rotational force transmission mechanism which combined the known transmission element other than this. 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. Or you may connect between the center axis | shaft 10 and the y rotating shaft 16 with a flexible shaft.

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

1 x Rotating body 2 x Rotating shaft 3 y Rotating bodies 4, 17, 19 Pulley 5 Cam 6 Housing 7 Clutch 8 Spring 10 Center shaft 14 Knob 15 Clutch lever 16 y Rotating shaft 23 Housing 30 Culture vessel

Claims (3)

A rotational drive device disposed in the housing;
An x-rotating shaft to which the rotational force of the rotational drive device is transmitted;
An x-rotating body attached to one end of the x-rotating shaft;
A y rotation axis attached to the x rotation body and having an axis in the y direction in a direction different from the x direction;
A y-rotator which is attached to the y-rotating shaft and accommodates a culture vessel;
A central shaft accommodated concentrically and independently in a hollow space inside the x-rotating shaft;
A rotational force transmission mechanism for transmitting rotation of the central axis to the y rotation axis;
The other end side of the x rotation shaft is arranged concentrically with the x rotation shaft and the central axis, and a state in which a rotational force is transmitted between the x rotation shaft and the central axis, and the central shaft is connected to the casing And a clutch for switching between the fixed state and
A clinostat characterized by switching the operation mode of undifferentiated maintenance culture or the operation mode of differentiation promotion culture by controlling the state of the clutch.
The clinostat according to claim 1, wherein the clutch has a state in which the rotation of the x rotation shaft is not transmitted and the central shaft is not fixed to the casing.
The clinostat according to claim 1,
The clinostat, wherein the x rotation shaft and the clutch are arranged in the casing so that the length of the rotation drive device overlaps in the output shaft direction of the rotation drive device.