JPH0620524B2 - Oscillating device for container containing liquid and / or solid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention mainly relates to a rocking device for vertically concentrating a cell culture bag to gently agitate a medium and cells to carry out large-scale concentrated culture of cells. Is.

Further, the present invention can be utilized for solvent extraction, mixing of liquid and solid, and of loosening minute components such as platelets separated in a liquid bag.

Hereinafter, specific examples using the cell culture bag will be described in detail.

(Prior Art) Various types of cell culture devices have been conventionally known. However, since these have problems that they cannot be mass-cultured and the apparatus is complicated and expensive, a new type of culture bag 40 was recently proposed by the present applicant (Japanese Patent Application No. 61-198898). ).

As shown in FIG. 19, this culture bag 40 is composed of an outer bag 41 made of vinyl chloride or other soft plastic and an inner bag 42 made of a semipermeable membrane of regenerated cellulose or the like.
The inner bag 42 is covered with a protective net 45, and the outer bag 4
1 is provided with an external liquid operation port 43, and an inner bag is provided with an internal liquid operation port 46. Then, by enclosing the cell suspension (internal solution) in the inner bag 42, enclosing the medium (external solution) in the outer bag 41, and enclosing sterile air in each bag 41, 42,
It allows nutrients to permeate through cells through a semipermeable membrane to carry out large-scale concentrated culture of cells in the inner solution.

(Problems to be Solved by the Invention) However, when the above-described cell culture bag 40 is simply allowed to stand and cultured, not only the inner solution and the outer solution do not contact each other uniformly, but also the cells in the inner solution adhere to the inner bag. However, there is a problem in that the cell recovery rate and the contact efficiency of the cells and nutrients are deteriorated due to the accumulation or aggregation. Therefore, the cell culture bag 40 described above needs to be cultivated while appropriately stirring the inner solution and the outer solution.

In order to solve such a problem, the applicant of the present invention has proposed a device for stirring the culture medium and cells by rotating the culture bag 40 with a certain inclination (actual application 62-4).
6745). As a result, the cells were prevented from adhering to the inner wall of the inner bag or from aggregating, and succeeded in increasing the cell recovery rate.

Although the above device is effective for culturing using only the cell culture bag 40 alone, the cell culture device recently proposed by the present applicant (trade name: CR-TISSUE system Japanese Patent Application No. 62
-80991), that is, a system for carrying out cell culture by connecting a cell culture bag 40 as an accessory to a medium bag, a cell suspension injection device, a cell collection bag after culture, a connecting tube, and a sterile air injection device. In the rotary culture method using the above apparatus, when connecting each accessory or measurement probe to the cell culture bag 40 during operation,
Due to the effect of rotation, it was difficult to operate by connecting the connecting tube twisted.

Therefore, as a result of studying a rocking device that enhances the operability of a continuous cell culture system without impairing the cell culture efficiency and recovery rate, the present invention has confirmed that it can be achieved by the rocking device described below.

(Means for Solving the Problems) (1) One end of the column 3a is pivotally supported at the stop point O of the rocking plate 3 via the link ball 4a, and the X axis of the rocking plate 3 and the Y axis are supported. One end of the push rods 5, 6 is pivotally supported at least at one position on each axis of the shaft via link balls 4b, 4C, and the other end of the support column 3a is fixed to the top surface of the housing 12, and the push rod 5 , 6 are connected to the other ends of links 7 and 8, the links 7 and 8 are connected to gears 9a and 9c, the gears 9a and 9c are meshed with the gear 9b, and the gear 9b is connected via a joint 10. To provide a rocking device for a container containing a liquid and / or a solid, which is connected to the motor 11.

Further, according to the present invention, (2) one end of the column 18a is pivotally supported on the center point O of the rocking plate 16 via the link ball 17a, and the push rod 18 of the push bar 18 is mounted on the X axis of the rocking plate 16 via the link ball 17b. One end is pivotally supported, one end of the push rod 19 is pivotally supported on the Y axis of the rocking plate 16 via a link ball 17c, and the other end of the pillar 18a is fixed to the top surface of the pedestal 24a.
The other ends of the push rods 18 and 19 are rotatably attached to racks 22 and 25, the racks 22 and 25 are attached to the pinion gears 21 and 23 so that they can move forward and backward, and the pinion gears 21 and 23 are attached to motors 20 and 23. The motors 20 and 23 provide a rocking device for a liquid and / or solid container which is controlled by a controller 26.

(Operation) In the rocking device of the present invention, after the container containing the liquid and / or solid is fixed to the rocking plate, the rocking plate moves up and down n times about the X axis at ti and then at ti + N. It moves up and down n times around the Y-axis, and the height hA of points A, B, C, D on the X-axis and Y-axis of the rocking plate with reference to the height ho of the center point O of the rocking plate. , HB, h
It is possible to perform the shaking of the container containing the liquid and / or solid in which C and hD are rocked while arbitrarily repeating the vertical movement.

Specifically, as a first aspect, the relationship between ho and hA, hB, hC, hD is expressed by the following formula (see the description of the first embodiment)
As shown by, the oscillating plate moves up and down once around the X axis, and moves up and down once around the Y axis.

In the following, the vertical movement about the X axis and the Y axis can be oscillated while repeating the same cycle once at a time.

In the second mode, the rocking plate repeats vertical movement about the X axis and the Y axis irregularly n times each so that the relationship between ho and hA, hB, hC, hD can be arbitrarily selected. Can swing.

As a third aspect, swinging can be performed by a combination of the first and second aspects.

According to the above-described embodiment, the cell culture bag 40 moves vertically and vertically along the X-axis and the Y-axis in accordance with the movement of the oscillating plate 1, and regularly and / or irregularly in any pattern in any combination. It can be carried out, and the inner solution and the outer solution in the culture bag 40 are agitated inside the respective bags. It is possible to easily connect a connecting tube such as a medium bag, a cell collection bag, and a measurement probe to the cell culture bag without twisting during the culture.

(Example) The principle of the rocking culture method when using the rocking apparatus of this invention is demonstrated below.

FIG. 1 shows an oscillating plate 1 for fixing a cell culture bag, a point O is a center point of the oscillating plate, and points A, B, C and D are on the X axis and the Y axis of the oscillating plate, respectively. That is the point above.

FIG. 2 is a side view showing the swinging method and the size of the swinging plate 1, whereby the swinging plate swings about the X axis and the Y axis, and the swinging amount is Swing points A, B, C,
The maximum amount of vertical movement of D relative to the height ho of the center point O is + hA, -hA, + hB, -hB, + hC, -hC, + h.
It is represented as D, -hD (+ display is the maximum value in the direction higher than ho, -display is the maximum value in the direction lower than ho).

The relationship between ho and hA, hB, hC, hD in the first embodiment is shown by the expressions (1) to (3) in which the rocking plate is centered on the X axis (actually, the X axis also makes a delicate circular motion). (See Fig. 3) Y while moving up and down once
It moves up and down once about the axis (actually, the Y axis also makes a subtle circular motion. See FIG. 3).

Hereinafter, a case will be described in which the vertical movement about the X-axis and the Y-axis is simultaneously repeated periodically and rocks.

In the equation (1), h is height, t is time, ω is angular velocity, and the relationship between ω and f is represented by ω = 2πf (f is the oscillation number).

At this time, the positional relationship between ho and hA, hB, hC, hD is expressed by equation (2).
It changes periodically as shown in.

,, and in equation (2) are t1 and t in FIG. 3, respectively.
Corresponds to 2, t3, t4.

FIG. 3 shows the relationship between the periodic changes of hA, hB, hC, and hD at each time ti. From this, it is understood that the vertical movements of the points A, B, C, and D of the oscillating plate have a phase relationship with each other (here, one vertical movement of the oscillating plate 1 means As shown in FIG. 3, the description will be made with reference to the point B.
Means to move in one cycle like ho → + hB → ho → −hB → ho).

(1) As shown in FIG. 4, point A has a height of + hA and point C has a height of −hA.
At the height of hC, points B and D are at the same height as ho (at t1 in FIG. 3).

The oscillating plate 1 starts to oscillate about the Y axis, and gradually becomes a point A,
C moves to the same position as the height of ho, and at the same time, points B and D start to move vertically in the + hB direction and point D in the −hD direction with the X axis as the center (at t1 + Δt in FIG. 3).

(2) After that, as shown in FIG. 5, the point B has a height of + hB, the point D has a height of −hD, and the points A and C have the same height as ho (third point).
(At t2 in the figure).

The oscillating plate 1 starts oscillating about the X axis, and points B, D
At the same position as the height of ho, and at the same time, point A starts moving in the -hA direction and point C in the + hC direction (at t2 + Δt in Fig. 3).

(3) After that, as shown in FIG. 6, point C becomes + hC, point A becomes −hA, and points B and D become the same as ho (at time t3 in FIG. 3). ).

The oscillating plate 1 starts to oscillate about the Y axis, and gradually becomes a point A,
C starts moving to the same position as the height of ho, and at the same time, point D starts moving in the + hD direction and point B moving in the -hB direction about the X axis (3rd position).
(At t3 + Δt in the figure).

(4) After that, as shown in FIG. 7, the point D is the height point B of + hD.
Is at -hB and points A and C are at the same height as ho (3rd
(At t4 in the figure).

Gradually, points B and D start moving to the same position as the height of ho, at the same time point A starts moving in the + hA direction and point C starts moving in the -hC direction (at t4 + Δt in Fig. 3) and returns to the state of (1) again, and then sequentially. The states of (1) to (4) are repeated.

FIG. 8 shows the rocking device 2 of the present invention for actually rocking the rocking plate to which the cell culture bag is fixed based on the principle described above.

The pillar 3 is attached to the center point O of the rocking plate 3 via the link ball 4a.
One end of a is pivotally supported.

One end of each of push rods 5 and 6 is pivotally supported at at least one position on each of the X axis and the Y axis of the oscillating plate 3 via link balls 4b and 4C.

The other end of the column 3a is fixed to the top surface of the housing 12, and the other ends of the push rods 5 and 6 are connected to links 7 and 8.

The links 7 and 8 are connected to gears 9a and 9c, respectively.
The a and 9c are meshed with each other together with the gear 9b.

The gear 9b is connected to the motor 11 via a joint 10.

The links 7 and 8 are adjusted by gears 9a, 9b and 9c so as to rotate at the same speed in phase with each other in accordance with the phase of the oscillating plate 3. These drives are transmitted from the motor 11 via the joint 10.

Next, an operation example of the rocking device 2 will be described in comparison with the rocking example of the rocking plate 1 described above.

Changes in the operation of the rocking device 2 in FIGS. 9 to 12 correspond to changes in the rocking of the rocking plate 1 in FIGS. 4 to 7, and FIG. 8A is a rear view of FIG. And (B) is a right side view of (A).

(1) Since the connecting point 13 between the link 7 and the push rod 5 is at the position (a), the push rod 5 pushes up the point A of the swing plate 3 to the height of + hA. Point C is lowered to the height of -hc by its recoil. At this time, since the connecting point 14 between the link 8 and the push rod 6 is at the position (), the points B and D of the oscillating plate 3 are at the same positions as the point O of the oscillating plate (FIG. 9).

(2) Next, when the links 7 and 8 rotate counterclockwise by 45 °, the connecting point 13 between the link 7 and the push rod 5 moves to the position (b), so that the points A and C on the rocking plate are the points O. It will be at the same height as. At this time, the connection point 14 between the link 8 and the push rod 6 is
Since it is in the (i) position, the point B of the oscillating plate is pushed up to the height of + hB, and the point D is lowered to the height of -hD by its reaction (Fig. 10).

(3) When the links 7 and 8 are further rotated counterclockwise by 45 °, the connecting point 13 between the link 7 and the push rod 5 moves to the position of (c), so that the point A of the rocking plate is -hA. It is pulled down to a height, and in response to that, point C is raised to a height of + hC. At this time, since the connecting point 14 between the link 8 and the push rod 6 is at the (ro) position, the points B and D of the rocking plate 3 are at the same height as the point O (FIG. 11).

(4) Next, when the links 7 and 8 are rotated counterclockwise by 45 °, the connecting point 13 between the link 7 and the push rod 5 is at the position (d), so that the points A and C of the swing plate 3 are at the point O. Will be in the same position as.

At this time, since the connecting point 14 between the link 8 and the push rod 6 is at the position of (), the point B of the oscillating plate is lowered to the height of -hB, and the point D is raised to the height of + hD by its reaction ( (Fig. 12).

When the links 7 and 8 further rotate counterclockwise by 45 °, the state returns to the above-mentioned state (1) and then the states (1) to (4) are repeated.

The case where the rocking plate is vertically moved along the X axis and the Y axis using the rocking device 2 has been described above.
As will be described later, the vertical motion and / or the forward / reverse motion can be rocked regularly and / or irregularly in any pattern in any combination.

Second Embodiment Next, in order that the relationship between ho and hA, hB, hC, hD can be arbitrarily selected, the oscillating plate repeats vertical movement about the X-axis and the Y-axis randomly n times each. An embodiment in the case of swinging will be described.

FIG. 13 is a front view of a rocking device 15 of the present invention for carrying out this embodiment, and FIG. 14 is a side view of FIG.

One end of a column 18a is pivotally supported at the center point O of the rocking plate 16 via a link ball 17a.

One end of a push rod 18 is pivotally supported on the X axis of the rocking plate 16 via a link ball 17b, and one end of a push rod 19 is pivotally supported on the Y axis of the rocking plate 16 via a link ball 17c.

The other end of the column 18a is fixed to the top surface of the pedestal 24a, and the other ends of the push rods 18 and 19 are rotatably attached to the racks 22 and 25.

The racks 22 and 25 are attached to the pinion gears 21 and 24 so as to be able to move forward and backward, the pinion gears 21 and 23 are attached to the motors 20 and 23, and the rotation speed directions of the motors 20 and 23 are interlocked and controlled by a controller 26. There is.

The movement of the push rod 18 is performed by the rack 22 moving forward and backward by the pinion gear 21 attached to the motor 20.

The movement of the push rod 19 is performed by moving the rack 25 forward and backward by the pinion gear 24 attached to the motor 23.

Since the control device 26 is adjusted to control the motor 20 and the motor 23 independently or simultaneously, the push rod 18 and the push rod 19 can be driven independently or simultaneously.

Next, an operation example of this embodiment will be described with reference to FIGS. 13 to 14 and FIG. 15 (the motor 20 and the motor 23 are independently controlled, and the push rod 18 and the push rod 19 are independently driven. To rock).

(1) The heights hA, hB, hC, and hD of the points A, B, C, and D are the same as the height ho of the point O (at t1 in FIG. 15).

Since the push rod 18 pushes up the oscillating plate 16 by driving the motor 20, the oscillating plate 16 starts oscillating about the Y axis, and gradually the point A starts moving up and down to + hA and point C to -hC (15th position). Figure t1 + Δt).

(2) After that, the point A becomes + hA and the point C becomes −hA (at t2 in FIG. 15).

Since the push rod 18 operates in the direction of pulling down the rocking plate 16 by the reverse driving of the motor 20, the rocking plate 16 starts rocking around the Y axis, and the points A, C, are gradually positioned at the same height as ho. It starts to move up and down to (at t2 + Δt in Fig. 15).

Points B and D are always at the same height as ho from time t1 to t2 + Δt.

(3) Points A, B, C and D return to the same height as ho (at t3 in Fig. 15).

Since the push rod 19 pushes up the oscillating plate 16 by driving the motor 23, the oscillating plate 16 starts oscillating around the X axis, and gradually the point B starts moving up and down to + hB and point D to -hD (15th position). (Figure t3 + Δt).

(4) After that, the point B becomes + hB and the point D becomes −hD (at t4 in FIG. 15).

Since the push rod 19 operates in the direction of pulling down the rocking plate 16 by the reverse driving of the motor 23, the rocking plate 16 starts rocking around the X axis, and the points B and D gradually move up and down to the same position as ho. Start (at t4 + Δt in Fig. 15), points A and C
Is at a position of ho and height from t3 to t4 + Δt.

(5) A, B, C and D return to the same height as ho and at t5 and t5 +
At the time of Δt, at the time of t6 and t6 + Δt of the above (3),
During the operation t7 and t7 + Δt in the above (4), during the operation t8 and t8 + Δt in the above (1), the oscillation is performed while repeating the above operation (2). In this embodiment, as can be seen from FIG. 15, the swing plate 16 swings up and down by repeating 1/2 times around the X axis and 1/2 time around the Y axis from t1 to t8 + Δt. Is to do.

Furthermore, the rocking | fluctuation apparatus 15 can also collectively rock the rocking | fluctuation of 1st Example. The swing will be described below.

(6) Points A, B, C, D are at the same height as ho (t9
Time).

By driving the motor 23 in the reverse direction, the push rod 19 operates in the direction of pulling down the swing plate, so that the swing plate 16 moves in the X direction.
Oscillation starts about the axis, and points B and D gradually move up and down to the positions of -hB and + hD (t9 + Δ in Fig. 15).
at t).

(7) Points B and D are −hB and + hD, and points A and C are at the same height as ho (at t10 in FIG. 15).

The motor 20 operates in the direction of pushing up the oscillating plate 16 via the push rod 18, and the motor 23 in the direction of depressing the oscillating plate 16 via the push rod 19, and hereafter, from time t10 + Δt to time t14 in FIG. 15, t4 + Δt in FIG. The same swing from time to t8 can be performed.

Further, in this embodiment, the motors 20 and 23 are connected to the push rod 18,
It is also possible to swing up the swing plate 16 at the same time via 19 or simultaneously rotate it in the direction of pulling it down to perform swinging.

FIG. 15 is an assembly exploded view showing an example of a fixture 30 used when attaching the cell culture bag 40 to the oscillating plate 1.

A pin mounting hole 1a extending in the central direction of the oscillating plate 1 is formed in a corner portion of the oscillating plate 1. A plurality of semi-circular cutouts 1b are formed in the mounting hole 1a so that the mounting position of the pin 31 can be set in several steps.

The lower portion 31b of the pin 31 is inserted into the mounting hole 1a from the upper surface side of the rotary plate 8 and the pin 31 is fixed to the swing plate 2 by tightening with the tightening screw 32 from the lower surface side. That is, the flange 31a is provided near the lower end of the pin 31, and the pin 31b below the flange 31a has a diameter adapted to the notch 1b of the mounting hole 1a. A screw groove (not shown) is formed inside the lower pin 31b.

Further, the tightening screw 32 is also provided with a flange 32a, and the handle portion 32b formed at the tip thereof has a diameter adapted to the cutout portion 1b of the mounting hole 1a. Further, the screw portion 32c is adapted to be screwed with the screw groove of the lower pin 31b. By screwing the tightening screw 32 into the pin 31, the flanges 31a, 32
The side edge of the mounting hole 1a is tightened with a to fix the pin 31 to the swing plate 1.

When adjusting the mounting position of the pin 31, loosen the tightening screw 32, move the pin 31 in the length direction of the mounting hole 1a, move the lower pin 31 to the desired notch 1b, and then use the tightening screw 32 again. It may be fixed to the oscillating plate 1.

A cylindrical stopper ring 33 is slidably inserted into the pin 31. This stopper ring 33
A tightening screw 33a is attached to the side of
The stopper ring 33 can be fixed at an arbitrary position of the length of the pin 31 by tightening the screw 33a.

FIG. 16 shows a cross-sectional view of the cell culture bag 40 attached to the oscillating plate 1 using the fixture 30.

As a development of the present invention, a more efficient culture can be performed by storing the rocking culture device together with the temperature control device 36 in a closed container 37 and performing the culture under the conditions most suitable for the culture conditions of each cell. You can

(Effect of the Invention) As described above, in the present invention, since it is easy to connect the cell culture bag to the connecting tube or the like during the culture, it is possible to continuously exchange the medium during the culture. This can contribute to the development of an automatic medium exchange system in the future.

Since the measurement probe can be attached to the cell culture bag during culturing, it becomes easy to measure the culturing state of cells and the like.

Since the inner liquid and the outer liquid are uniformly stirred by using a constant period and / or an at random period together, the components in the outer liquid are uniformly dispersed, and the contact efficiency of both liquids through the semipermeable membrane is improved. Since the cells in the inner solution are improved and are less likely to adhere or aggregate on the inner wall of the inner bag, the cell recovery rate is increased. It is an invention having excellent effects such as.

[Brief description of drawings]

1 is a perspective view of a rocking plate used in the present invention, FIG. 2 is a side view of FIG. 1, and FIG. 3 is a center point O and a point A of the rocking plate 1,
Graphs showing changes in height of B, C, and D with time, FIGS. 4 to 7 are schematic diagrams showing periodic movement of the oscillating plate 1, and FIG. 8 is a schematic diagram of the oscillating device of the present invention. 9 to 12 are schematic views showing an example of periodic operation of the rocking culture apparatus, FIG. 13 is a schematic view of the rocking apparatus of the present invention, FIG. 14 is a right side view of FIG.
FIG. 5 is a graph showing the temporal change of the height of the center point O of the rocking plate 16 and the points A, B, C, and D, FIG. 16, an assembled exploded view of the fixture, and FIG. 17 is a cell culture bag. FIG. 18 is a perspective view of the device 40 attached to an oscillating plate, FIG. 18 is a schematic view of an apparatus for culturing in a closed container, and FIG. 19 is a schematic view of a cell culture bag. In the figure, 1, 3 and 16 are rocking plates, 3a and 18a are columns, 2,
15 is a rocking culture device, 4a, 4b, 4c, 17a, 17
b, 17c are link balls, 5, 6, 18, 19 are push rods, 7, 8 are links, 9a, 9b, 9c are gears, 10 is a joint, 11, 20, 23 are motors, 12 is a housing, 1
3, 14 are connecting points, 21 and 24 are pinion gears, 22,
25 is a rack, 24a is a pedestal, 26 is a control device, 31 is a pin, 32 is a screw, 33 is a stopper ring, 36 is a temperature control device, 37 is a closed container, and 40 is a cell culture bag.

Claims (2)

[Claims] 1. One end of a column 3a is pivotally supported by a center point O of the rocking plate 3 via a link ball 4a, and the link ball is provided at at least one position on each of the X axis and the Y axis of the rocking plate 3. One end of the push rods 5, 6 is pivotally supported via 4b, 4C, the other end of the pillar 3a is fixed to the top surface of the housing 12, and the other ends of the push rods 5, 6 are connected to the links 7, 8. The links 7 and 8 are connected to gears 9a and 9c, the gears 9a and 9c are intermeshed together with the gear 9b, and the gear 9b is connected to the motor 11 through a joint 10. A rocking device for a container containing a liquid and / or a solid. 2. A link ball 17 is provided at a center point O of the oscillating plate 16.
One end of the pillar 18a is pivotally supported via a, the one end of the push rod 18 is pivotally supported on the X axis of the rocking plate 16 via the link ball 17b, and the link ball 17c is pivoted on the Y axis of the rocking plate 16. One end of the push rod 19 is pivotally supported through the other end, and the other end of the pillar 18a is fixed to the top surface of the pedestal 24a.
The other ends of the push rods 18 and 19 are rotatably attached to the racks 22 and 25, the racks 22 and 25 are attached to the pinion gears 21 and 23 so as to be able to move forward and backward, and the pinion gears 21 and 23 are attached to the motors 20 and 23. The motors 20 and 23 are controlled by a control device 26 in conjunction with each other, and a rocking device for a liquid and / or solid container.