JPH11239474A - Biochemical reaction device and biochemical reaction method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a biochemical reaction device capable of culturing animal cells in a high density, and maintaining and growing a living body tissue by being equipped with a fixing plate for fixing a physiologically active substance, a housing and a specific rotary means. SOLUTION: This biochemical reaction device is equipped with a fixing plate 6 for fixing a physiologically active substance, a housing for housing a reaction liquid supplied to the physiologically active substance and the fixing plate 6, and a rotary means 5, 7, 9, 10 capable of generating a centrifugal force capable of excluding the reaction liquid in the vicinity of the physiologically active substance by rotating or oscillating the fixing plate 6. In this case, it is preferable to be equipped with a reaction liquid supplying means 12, 13, 14 in the vicinity of the rotary shaft of the fixing plate 6 and a variable mechanism capable of changing the speed and/or moving direction of the rotation or oscillation of the rotary means 5, 7, 9, 10, and that the fixing plate 6 is a porous material having continuous cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biochemical reaction apparatus and a biochemical reaction method, and more particularly, to a biochemical reaction in which a physiologically active substance is immobilized, and a method for culturing cells and tissues of microorganisms, animals, plants, and the like. The present invention relates to a biochemical reaction device and a biochemical reaction method used for culturing, maintaining, and the like.

[0002]

2. Description of the Related Art
As a fixed-bed type biochemical reaction device, a device in which a reaction solution is supplied to a container having a fixed bed filled with a carrier on which a physiologically active substance is immobilized, and after the reaction, the solution is taken out of the container is used.

[0003] However, it is difficult to flow the reaction solution uniformly in the fixed bed, that is, it is difficult to flow the reaction solution with no flow rate distribution or with a reduced flow rate distribution. It is considered that it is difficult to reduce the flow velocity distribution because the force for flowing the reaction solution inside the fixed bed to the downstream is not uniformly applied due to the clogged state of the carrier.

In a reaction system in which a dissolved gas such as dissolved oxygen or dissolved carbon dioxide is consumed while the reaction solution flows through the fixed bed, it is difficult to supply the dissolved gas into the fixed bed. Therefore, only the dissolved gas content in the reaction solution at the time of supply can be involved in the reaction, and there is also a disadvantage that the flow path of the fixed bed cannot be set long. Particularly, when trying to culture cells in a fixed bed, oxygen was often the limiting factor in aerobic culture.

On the other hand, even in a reaction system involving the generation of gas, it is difficult to use the fixed bed because the gas accumulates in the fixed bed. If the flow rate of the reaction solution is too high, the physiologically active substance moves downstream. Eventually, she detached from the fixed bed. As a typical example, there is a phenomenon that even if the animal cells are fixed to the carrier, the animal cells are detached and move.

As another conventional example, there is one in which microorganisms are fixed to a disk-shaped rotating body, and a part or all of the rotating body is immersed in a reaction solution.
In addition to the disadvantage that only the surface of the rotating body affects the reaction,
Even if the microorganism fixed to the rotating body has a thickness, there is also a disadvantage that the liquid is not supplied in the thickness direction and only the surface reacts.

Further, in the above-mentioned conventional example, in order to supply a reaction solution and oxygen to the surface of a rotating body, a disk on which a physiologically active substance is fixed is set upright to the liquid surface, and a part of the disk is placed in a culture medium. For example, a disk having a diameter of about 10 cm is rotated at about 60 rpm. However, there is no idea of using centrifugal force, and the centrifugal force is small because the peripheral speed is small. The liquid was only moved by diffusion in the thickness direction of the rotating body.

Further, there is also used a basket in which a carrier containing a physiologically active substance immobilized therein is disposed in a reaction solution and a means for supplying the reaction solution into the basket is used. Is required to be smaller than the carrier, which may cause clogging and may become a limiting factor of the flow rate of the reaction solution. Also, the appearance of the carrier inside the basket cannot be seen,
In addition, it has been pointed out that it is difficult to aseptically sample the carrier.

On the other hand, in an animal cell culture apparatus, a carrier for animal cells is accommodated in a portion sandwiched between double cylindrical meshes, and a reaction solution is supplied radially from an outer cylinder to an inner cylinder. Although the device has been put into practical use, in addition to the same drawbacks as in the case of the above-described device provided with a cage, there is also a drawback in that when seeding cells, it is difficult to seed cells uniformly over the entire portion. Was. This is considered to be due to the fact that the carrier is housed in the net or basket.

The present invention has been made in view of the above-mentioned various problems in the prior art, and it is an object of the present invention to appropriately supply a reaction solution to the entire immobilized physiologically active substance and further dissolve the reaction solution. An object of the present invention is to provide an apparatus and a method capable of supplying a gas. Particularly, in the case of culturing animal cells, it is difficult to supply a substrate, a reaction solution and dissolved oxygen to each cell when the cell concentration is increased or when cells are dense as in a living tissue. Overcomes this problem and enables the cultivation of high-density animal cells and the maintenance and growth of living tissues.

[0011]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventor has found that a physiologically active substance is fixed and rotated, and a reaction solution is moved to the outside of a fixing plate by centrifugal force. The reaction solution in the vicinity of the physiologically active substance is eliminated by supplying a fresh reaction solution while or after eliminating the reaction solution, and by changing the rotation speed and the rotation direction, Active substance, particularly adjusting the force applied to the reaction solution contained in the living tissue, and discharging the reaction solution to the outside, and finding that the above-mentioned problem can be solved by supplying a fresh reaction solution thereto, The present invention has been completed.

That is, the biochemical reaction device of the present invention comprises a fixing plate on which a physiologically active substance is fixed, a housing for accommodating a reaction solution supplied to the physiologically active substance and the fixing plate,
A rotating means for rotating or swinging the fixing plate to generate a centrifugal force capable of removing a reaction solution near the physiologically active substance.

Further, a preferred embodiment of the biochemical reaction apparatus according to the present invention is characterized in that a reaction liquid supply means for supplying the reaction liquid is provided near the rotation axis of the fixed plate. Further, a preferred embodiment of the biochemical reaction device of the present invention is characterized in that the biochemical reaction device is provided with a variable mechanism capable of periodically changing the speed and / or the direction of the turning or swinging movement of the turning means.

Further, another preferred embodiment of the biochemical reaction apparatus of the present invention is characterized in that the fixing plate is a porous body having open cells. Further, another preferred embodiment is characterized in that the porous fixing plate is fixed to an upper surface of another disk. Further, another preferred embodiment is characterized in that a liquid-permeable spacer that is in contact with the physiologically active substance or the porous fixing plate is further added. Still another preferred embodiment is characterized in that the porous fixing plate is immersed in the reaction solution with the upper surface and the outer wall exposed to the gas phase.

Still another preferred embodiment of the biochemical reaction apparatus of the present invention is that a physiologically active substance fixed on the fixing plate is immersed in the reaction solution with a part thereof exposed to the gas phase. It is characterized by having. Also, another preferred form is
A reaction liquid collecting gutter is provided on an inner bottom surface edge of the housing. In this case, the reaction liquid collecting gutter is connected to the reaction liquid supply means via a reaction liquid adjusting tank provided outside the housing. Preferably it is in communication with Further, another preferred embodiment is characterized in that an installation angle of the fixed plate with respect to a horizontal plane is variable. In another preferred embodiment, the physiologically active substance is a living tissue, and the reaction solution is a culture solution.

In the biochemical reaction method of the present invention, the fixing plate on which the physiologically active substance is fixed is rotated or swung to generate a centrifugal force, thereby eliminating the reaction liquid near the physiologically active substance. The method is characterized in that a reaction liquid is supplied to the physiologically active substance.

In a preferred embodiment of the biochemical reaction method of the present invention, the rotation speed of the fixed plate, the supply speed of the reaction solution, and / or
Or the withdrawal speed, by adjusting at least one of the amount of the reaction solution, the physiologically active substance fixed to the fixing plate,
It is characterized in that a part thereof is immersed in the reaction solution in a state of being exposed to the gas phase.

[0018]

In the present invention, a physiologically active substance is fixed to a fixing plate, and this is rotated or swung to move a reaction solution contained in or near the physiologically active substance by centrifugal force. And the fresh reaction solution was supplied to the fixed plate again. Therefore, such a fresh reaction liquid is sufficiently supplied to the physiologically active substance, and even when the physiologically active substance is relatively thick, the reaction liquid is sufficiently supplied also in the thickness direction.

Further, in the present invention, it is possible to change the speed or direction of the turning or swinging motion of the turning means for turning the fixed plate or the like. By changing the acceleration, the force applied to the reaction solution near or inside the physiologically active substance can also be changed. Thereby, the physiologically active substance, in particular, the reaction solution contained near or inside the living tissue can be exchanged for a fresh reaction solution outside the living tissue. That is, the reaction liquid near or inside the living tissue is discharged to the outside due to a change in acceleration,
An external fresh reaction solution is easily supplied to the vicinity of or inside the living tissue generated by this discharge. Note that the supply is performed after the reaction liquid is discharged to the outside or at the same time when the reaction liquid is discharged to the outside.

Further, by changing the movement speed and the movement direction, the size and direction of the flow of the reaction solution inside the physiologically active substance can be changed, so that the reaction solution can be uniformly supplied into the physiologically active substance. Is possible. The discharge / supply of the reaction solution and the change of the flow rate and the flow direction are performed not only between the low-speed motion and the high-speed motion, but also between the motion state and the stationary state, and between the right-hand motion and the left-hand motion. Even between movements with opposite movement directions, such as between each other,
It can be carried out.

Further, in the present invention, the relative velocity between the physiologically active substance and the reaction solution contained therein can be reduced or set to zero by appropriately reducing or setting the kinetic acceleration to zero. Therefore, in the conventional culture, plant cells and animal cells were continuously exposed to the culture solution stream and continued to receive a shearing force, and the cells were sometimes damaged. In the present invention, such damage was avoided. can do.

Further, if the shape of the fixing plate is a disk shape or a donut shape, rotation and the like can be performed smoothly. Furthermore,
The fixing plate may be formed of a porous body having open cells to form a porous carrier. In this case, even if a physiologically active substance is carried inside the porous carrier, the reaction solution is sufficiently supplied. Further, when the porous carrier is in the form of a donut, the reaction liquid supplied to the central portion of the fixed plate is also supplied to the inside of the carrier from the inner peripheral wall of the donut-shaped porous carrier. Supply can be made. Further, such a porous fixing plate can be fixed to the upper surface of another disk having appropriate rigidity, whereby the porous fixing plate can be structurally reinforced. Furthermore, if a liquid-permeable spacer that comes into contact with the physiologically active substance or the porous fixing plate is further added, the reaction liquid can be easily supplied to the physiologically active substance.

In the present invention, a part of the physiologically active substance fixed on the fixing plate can be immersed in a reaction solution in a state of being exposed to a gas phase to carry out a biochemical reaction. If some of the physiologically active substances are brought into contact with the gas phase,
The flow rate of the reaction solution in the physiologically active substance can be increased as compared with the case where the reaction liquid is completely immersed in the reaction liquid, and the reaction liquid can be more easily discharged, and a part of the exposed physiologically active substance can be obtained. Various gases can be absorbed or released from. When the fixing plate is porous and a physiologically active substance is supported inside the porous fixing plate, the porous fixing plate is immersed in the reaction solution with the upper surface and the outer wall exposed to the gas phase. Thus, a biochemical reaction can be performed, and with such a configuration, the flow rate of the reaction solution flowing in the porous fixed plate can be increased. In addition, the exposure of a part of the physiologically active substance and / or the upper surface and the outer wall of the porous fixing plate to the gas phase as described above depends on the rotation speed of the fixing plate, the supply and / or withdrawal speed of the reaction solution, and the reaction rate. The adjustment can be performed by adjusting at least one of the usage amounts of the liquid.

Further, in the present invention, it is possible to provide a reaction liquid collecting gutter on the inner bottom edge of the housing for accommodating the reaction liquid and the fixing plate, and discharge the liquid outward by rotating the fixing plate. The reaction solution thus collected is collected in the reaction solution collection gutter. The reaction solution is appropriately extracted from the reaction solution collection tube, sent to a reaction solution adjustment tank added outside the housing, and after adjusting the components, returned into the housing. Can be efficiently and continuously supplied to the physiologically active substance.

Further, it is possible to change the installation angle of the fixed plate with respect to the horizontal plane.
This is convenient for immobilizing the physiologically active substance, and can easily adjust the flow rate of the reaction solution flowing through the collecting trough. Furthermore, when seeding a physiologically active substance, in particular, various cells, and at the beginning of the culture, the fixing plate is kept horizontal and not rotated, and thereafter the cells grow and the cell concentration increases, and the If the supply becomes insufficient, a culturing method of rotating the fixing plate to sufficiently supply the reaction solution to the cells may be employed.

[0026]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with some preferred embodiments with reference to the drawings. In the present invention, a physiologically active substance, that is, an active substance derived from an organism such as an enzyme or a coenzyme, or a substance having a physiological activity such as a living cell or a living tissue such as a microorganism, a plant or an animal cell, is added to the fixed plate. Use fixed. In addition, when such a physiologically active substance has a certain size like a living tissue,
The fixed plate may be fixed (spread) in contact with the surface, or when the fixed plate is porous having open cells, it may be dispersed and fixed to the entire inside or surface thereof.

(Embodiment 1) FIG. 1 shows an embodiment of the biochemical reaction apparatus of the present invention. In FIG. 1, a reaction solution tank 2 of this biochemical reaction device 1 is formed in a bottomed cylindrical shape, and a reaction solution is accommodated therein. A bearing 3 is provided at the center of the reaction solution tank 2 and supports a shaft 4 described later. In addition, as a reaction liquid, for example,
When the physiologically active substance is a living cell, a culture solution is used.

The disk 5 is a porous disk made of ceramics and has a shaft 4 at the center of the bottom surface. The shaft 4 is supported by the bearing 3. On the upper surface of the disk 5, a porous carrier 6, which is an example of a fixed plate and has a donut shape and has open cells, is fixed.

The material and form of the porous carrier 6 are not limited as long as it has a function of immobilizing a physiologically active substance and a function of passing a reaction solution.
Inorganic compounds such as glass, metal, cellulose, plastics, gelatin, zeolite and silica gel and the like, and combinations thereof. Further, those using these as a base material and binding collagen and components having various ion exchange functions, etc. Can be used.

As the porous carrier 6, the carrier material itself may be a porous body having open cells, or a plurality of carriers may be aggregated to form the porous carrier 6 having open cells. You may. For example, the material itself may be porous, such as urethane having open cells, or a plurality of carriers having no pores, such as sintered glass spheres, may be aggregated to form a porous material having open cells. The carrier 6 may be formed.

When an adhesive cell or tissue is used as the physiologically active substance, it may be desirable that the porous carrier 6 serves as a scaffold for adhesion. Cells and tissues can proliferate.

In FIG. 1, a disk-shaped magnet 7 is fixed to the lower surface of the disk 5. A disk-shaped drive magnet 9 fixed to a disk 8 is disposed at a position facing the magnet 7 with the bottom surface of the reaction solution tank 2 interposed therebetween. It is configured to be rotatable.

A reaction liquid extraction nozzle 12 and a liquid supply nozzle 13 are hermetically sealed and fixed to the lid 11 of the reaction liquid tank 2, and each end is located in the reaction liquid and above the center of the disk 5. I do.

Further, a pump 14 for extracting the reaction liquid and supplying it to the center of the disk 5 is disposed between the reaction liquid extraction nozzle 12 and the liquid supply nozzle 13.

In the present embodiment, the disk 5 is rotated via the shaft 4 supported by the bearing 3, but the roller (not shown) which is brought into contact with the lower surface near the outer periphery of the disk 5 is rotated. Various mechanisms can be used, such as a configuration in which the disc is rotated.

Next, the biochemical reaction apparatus 1 having the above configuration
Will be described. First, the driving magnet 9 fixed to the disk 8 is rotated by the motor 10, whereby the disk 5 positioned inside the reaction liquid tank 2 is rotated. Moves toward the outer periphery of the disk 5 and the porous carrier 6, and the reaction solution 15 at the center of the porous carrier 6 is eliminated.

Next, when the pump 14 is driven to return the reaction solution moved in the outer peripheral direction of the disk 5 and the porous carrier 6 to the center of the disk 5, the reaction solution can be circulated. Here, if the amount of reflux of the reaction solution by the pump 14, the rotation speed of the disk 5, and the porous carrier 6 are appropriately selected, the amount of the reaction solution involved in the immersion, in other words, the amount of the reaction solution contained in the porous carrier 6 is adjusted. This allows the other portion to be immersed in the reaction liquid while keeping the upper surface of the porous carrier 6 and the outer peripheral wall in contact with the gas phase.

As a result, when the dissolved gas participates in the reaction, the dissolved gas can be supplied to the reaction solution via the reaction solution on the upper surface of the porous carrier 6 by adjusting the gas composition of the gas phase. . In addition, when gas is generated during the reaction, the gas easily diffuses into the gas phase, so that the supply of the reaction solution is not hindered. Here, the reflux system using the pump 14 is not always necessary, and the porous carrier 6 is not centrifuged by centrifugal force.
After the liquid inside the container is spun in the outer peripheral direction, the rotation may be slowed or stopped, and the reaction liquid may be impregnated again.

Next, an example in which the above-described biochemical reaction device of the present embodiment is applied to a system for culturing adherent animal cells will be described.

First, as a porous carrier 6 having open cells, porous cellulose was stuck on a disk-shaped glass plate, and the glass plate was further fixed to the disk 5, and a culture solution as a reaction solution was added to the porous carrier. 6 was supplied to the reaction solution tank 2 until the upper surface was immersed. Then, in a state where the porous carrier 6 was stationary, seed cells were seeded as uniformly as possible on the upper surface of the porous carrier 6.

At this time, the number of rotations is usually such that there is no influence of the centrifugal force, that is, the rotation speed is so slow that the liquid inside the porous carrier is not replaced, and the rotation speed is high only when the medium in the carrier is replaced. Was controlled as follows. When the pump 14 was not operated and the medium was replaced as needed, the number of cells per volume of the porous carrier 6 was increased to 10 ⁸ / cm ³ or more. In this case, if air in which oxygen is added to the gas phase is used, the lack of dissolved oxygen can be compensated.

In this embodiment, the reaction liquid is supplied to the vicinity of the center of the porous carrier 6 by using the pump 14. A disk 16 (indicated by a dashed line in FIG. 1) opposed to the porous carrier 6 and having a hole 17 at the center thereof is placed so that its outer periphery is immersed in the reaction solution and does not contact the porous carrier 6. May be arranged as follows. In this case, the reaction solution is refluxed to the vicinity of the center of the porous carrier 6 under negative pressure. In order to prevent the reaction solution from leaking, it is desirable that the lid 11 keeps the reaction solution tank 2 hermetically sealed.

(Embodiment 2) FIG. 2 is a sectional view showing another embodiment of the biochemical reaction apparatus of the present invention. In FIG. 1, a housing 21 of the biochemical reaction device 20 is formed in a bottomed cylindrical dish shape. A reaction liquid collecting gutter 22 is formed at the outermost peripheral portion (edge) of the inner bottom surface of the housing 21.

The glass disk 23 is housed inside the housing 21. On the upper surface thereof, a donut-shaped open-cell urethane as a porous carrier 24 which is an example of a porous fixing plate is fixed. And in the center of this disk 23,
A rotating shaft 25 extending perpendicularly to the bottom surface of the disk 23 is fixed, and the rotating shaft 25 passes through the bottom surface of the housing 21 in a sealed manner.

The housing 21 is covered with a transparent cover 26, through which a reaction liquid supply nozzle 27 hermetically penetrates. The housing 21, the disk 23, the cover 26, and the like are inclined by 30 degrees with respect to the horizontal plane,
At the lowest position 22a, a liquid collection nozzle 28 is attached so as to pass through the housing 21 in a sealed manner.

The reaction liquid extracted by a pump 29 through a liquid collecting nozzle 28 is stored outside the housing 21.
An adjustment tank 30 is provided for adjusting the components, and a pump 31 is also provided for returning a part of the reaction solution to the vicinity of the center of the disk 23 again via the reaction solution supply nozzle 27.

Next, the operation of the biochemical reaction device 20 of the present embodiment having the above-described configuration will be described. First, an enzyme as an example of a physiologically active substance is immobilized on the porous carrier 24. When the rotating shaft 25 is rotated by a motor (not shown), the porous carrier 24 fixed to the disk 23 rotates.

Then, the reaction solution is supplied from the adjusting tank 30 to the pump 3.
1 and continuously supplied to the central portion of the porous carrier 24, the supplied reaction solution reaches the inner peripheral portion of the porous carrier 24, and then flows through the porous carrier 24 in the outer peripheral direction by centrifugal force. And eventually released to the outer edge.

The reaction liquid collects at the lowest portion 22 a through the inclined reaction liquid collecting gutter 22 formed at the inner bottom edge of the housing 21, and the collected reaction liquid is drawn out by the pump 29. The pH is returned to the adjustment tank 30, and appropriate pH and component adjustment can be performed here. The inclination of the reaction liquid collecting trough 22 is preferably not more than 60 degrees from horizontal in consideration of the convenience in manufacturing the reaction liquid collecting trough 22.

In the biochemical reactor 20 having the above configuration, the reaction solution moves in the porous carrier 24 in the outer circumferential direction by centrifugal force. Further, since the upper surface and the outer peripheral wall of the porous carrier 24 are in contact with the gaseous phase, gas exchange with the gaseous phase is possible, and gas can be supplied. Can be discharged from the upper surface of the porous carrier 24.

Here, it is sometimes preferable to adjust the supply amount of the reaction liquid from the pump 31 so that the reaction liquid flows not only inside the porous carrier 24 but also on the upper surface. That is, since a larger centrifugal force acts on the porous carrier 24 toward the outer peripheral portion, a sufficient reaction liquid is not supplied at the outer peripheral portion by the supply of the reaction liquid from the inside, and a negative pressure region is generated. This is because a liquid can be supplied.

In the above embodiment, the porous carriers 6 and 24 are formed in a donut shape, but may be formed in a disk shape or a disk shape. Also, if a hole is provided in the porous carrier by making a hole in a part of the porous carrier, or a donut-shaped concentric circle with a gap, etc., a fresh reaction flowing from the upper surface of the porous carrier into the cavity is provided. Since liquid can be supplied,
There is also an advantage that the reaction channel can be shortened. This is convenient to avoid the effects of waste products caused by the reaction.

The discs 5 and 23 are not limited to porous discs formed of ceramics, but may be glass discs having no holes. It is better to use one having no properties,
24 can be reinforced structurally, which is convenient when supplying the reaction solution.

In the above two embodiments, the apparatus for supplying the reaction solution to the physiologically active substance fixed on the porous carrier 6 having open cells has been described. It is also possible to add a configuration that can be supplied into the liquid tank 2 or the housing 21 and a configuration that removes gas generated by this reaction.

It is known that in a tissue such as a liver, a plurality of types of cells are affected and function, but it is necessary to culture a plurality of types of cells simultaneously to have the same ratio as the original tissue. Therefore, it is preferable to adhere a plurality of types of cells to a plurality of porous carriers and culture them, and to use them afterwards.

(Embodiment 3) FIG. 3 is a schematic sectional view showing still another embodiment of the biochemical reaction apparatus of the present invention. In this biochemical reaction apparatus, a medium (culture solution) 47 and a disc-shaped fixing plate 43 are accommodated in a bottomed cylindrical container 42 having a detachable upper lid 41. Has a rotating shaft 44 attached thereto.
Is sealed through the bottom of the container 43 and is connected to rotating means (not shown).

Further, on the upper surface of the disk-shaped fixing plate 43,
A plurality of living tissue sections 45 are spread, and further pressed and fixed to the upper surface by a net 46. In addition, the tissue section 45
Is mostly immersed in the culture medium 47, but its upper surface may be exposed to the gas phase. A circumferential eave 42a extending toward the center is provided on the inner upper edge of the container 42. Further, the culture medium 47 is sent to a culture medium storage tank 50 as an example of a reaction liquid adjustment tank via a culture medium outlet chisel 48 and a pump 49, and can be returned to the container 42 via a pump 51 and a culture medium inlet 52. ing.

Next, the operation of the biochemical reaction device of this embodiment will be described. In this biochemical reaction apparatus, the rotation shaft 44 is controlled by a motor (not shown) and its control device so as to automatically stop and repeat the low-speed rotation and the high-speed rotation. Therefore, the living tissue section 45 fixed to the fixing plate 43, particularly the liquid such as the culture medium 47 contained therein, receives an external force according to the stop, the low-speed rotation, and the high-speed rotation. In the present embodiment, substantially no centrifugal force is exerted on the liquid contained in the tissue section, and such liquid stays in the same position without moving inside the tissue on the outer peripheral side of the fixing plate 43, that is, in the radial direction. The rotating motion in which the liquid moves in the radial direction of the fixed plate 43 and is discharged from the inside of the tissue without damaging the tissue is controlled as "high-speed rotation".

In the rotation control, the culture medium 47 is discharged from the tissue section 45 during high-speed rotation, moves in the radial direction of the fixing plate 43, and is discharged to the vicinity of the inner peripheral wall of the container 42. At this time, the released medium 4 is contained in the tissue section 45.
There is a void for seven, and when the rotation is stopped or rotated at a low speed, a fresh medium is sufficiently supplied. In this case, if the high-speed rotation is continued too much, the tissue section 45 may be damaged due to long-term exposure to the centrifugal force and the flow of the culture medium. To protect the tissue section 45. During low-speed rotation, the medium inlet 52
Is uniformly supplied to the tissue section 45. The eaves 42a prevent the medium released as described above from leaking from the gap between the container 42 and the upper lid 41.

(Embodiment 4) FIG. 4 is a schematic sectional view showing another embodiment of the biochemical reaction apparatus of the present invention. In this biochemical reaction apparatus, a plurality of disks 61a fixed to a rotating shaft 62 having a bottom portion hermetically sealed therein are provided in a housing 60.
And 61b, on which a donut-shaped spacer 63a-63d formed of a coarse material (liquid-permeable material) through which a liquid easily flows, and a donut-shaped porous carrier on which a physiologically active substance is fixed A plurality of sets in which the layers 64a to 64d are stacked are fixed and immersed in the culture solution 67. Also,
The housing 60 is provided with a reaction liquid drawing nozzle 65 and a reaction liquid supply nozzle 66.

Next, the operation of the present embodiment will be described.
A physiologically active substance is fixed on the porous carrier 64 in advance, and as shown in FIG.
When disposed above, the spacer 63 is formed of a material through which liquid flows more easily than the inside of the porous carrier 64, so that there is an advantage that the reaction liquid is easily supplied from both surfaces (upper surface and lower surface) of the porous carrier. .

When the rotating shaft 62 is rotated by a motor (not shown) at a speed at which an appropriate centrifugal force is generated, the reaction solution collects on the inner peripheral wall of the housing 60, and the reaction solution inside the porous carrier is also discharged. Then, when the rotation is stopped or set to a low speed, the reaction liquid can be supplied again. At this time, if the spacer 63 is arranged, the reaction solution can be supplied from both sides of the porous carrier. Therefore, even if the density of the porous carrier becomes high, the reaction solution is quickly supplied to the inside, and as a result, The quality carrier can be thickened. Furthermore, if the configuration as in the present embodiment is adopted, a plurality of porous carriers can be easily accommodated in one housing, so that production of a large amount of reaction products generated from a plurality of types of physiologically active substances, It is effective to carry out the reaction of

As described above, according to the biochemical reaction device of the present embodiment, a sufficient amount of medium can be supplied to the inside of a living tissue without damaging the living tissue, so that the living tissue can be maintained and grown. Also, particularly at high speed rotation, the tissue section 45
Since the upper surface or the like is partially exposed to the gas phase, it is also possible to absorb gas into the living tissue or discharge gas from the living tissue. At the time of low-speed rotation or stop, there is often no problem even if the whole living tissue is immersed in the medium.

As described above, the present invention has been described in detail with reference to preferred embodiments. However, the present invention is not limited to these embodiments, and various modifications can be made within the scope of the present disclosure. For example, in the above embodiment, the fixed plate is rotated, but an external force can be applied to the physiologically active substance,
It is sufficient that the size and direction can be appropriately changed. For example, the present invention can be applied to a movement related to a rotation, a swing, a reciprocation such as up and down, left and right, or a combination thereof. In addition, such a change of the external force can be realized by adding various acceleration mechanisms.

If the porous fixing plate is formed of a rigid body such as porous ceramics, the disks 5 and 23 can be omitted. In this case, the “porous material” means a material capable of maintaining or culturing a physiologically active substance, cells and tissues inside the pores, and includes porous ceramics, porous cellulose, porous plastics, and physiologically active substances. Materials coated or modified with components compatible with the active substance can be exemplified. Further, as biologically active substances such as biological tissues, substances derived from living organisms such as enzymes and antibodies can also be used, and not only one kind but also a plurality of kinds can be fixed at the same time. It is possible to provide a difference in the applied centrifugal force by appropriately changing the installation position of, and it is also possible to satisfy the discharge / supply of the reaction solution such as the culture solution described above between a plurality of types of physiologically active substances. It is. In other words, the reaction product of one physiologically active substance can be supplied to another physiologically active substance.

[0066]

As described above, according to the present invention, the bioactive substance is fixed and rotated, and the reaction solution is moved to the outside of the fixing plate by centrifugal force, whereby the reaction near the bioactive substance is achieved. By excluding the liquid and thus excluding or excluding the reaction liquid, by supplying a fresh reaction liquid, and by changing the rotation speed and the rotation direction, the biologically active substance, particularly the biological tissue, is removed. Adjusting the force applied to the reaction solution contained therein and discharging it to the outside, and supplying a fresh reaction solution to it, etc., appropriately supply the reaction solution to the entire fixed physiologically active substance, Further, it is possible to provide a biochemical reaction apparatus and method capable of supplying a dissolved gas. Particularly, in the case of culturing animal cells, it is difficult to supply a substrate, a reaction solution and dissolved oxygen to each cell when the cell concentration is increased or the cells are dense as in a living tissue. If this can be overcome, high-density culturing of animal cells and maintenance / proliferation of living tissues can be achieved.

[Brief description of the drawings]

FIG. 1 is a schematic sectional view showing one embodiment of a biochemical reaction device of the present invention.

FIG. 2 is a schematic sectional view showing another embodiment of the biochemical reaction device of the present invention.

FIG. 3 is a schematic sectional view showing still another embodiment of the biochemical reaction device of the present invention.

FIG. 4 is a schematic sectional view showing another embodiment of the biochemical reaction device of the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Biochemical reaction apparatus 2 Reaction liquid tank 3 Bearing 4 shaft 5 Disk 6 Porous carrier 7 Magnet 8 Disk 10 Motor 11 Lid 12 Reaction liquid extraction nozzle 13 Liquid supply nozzle 14 Pump 15 Reaction liquid 16 Disk 17 Hole 20 Raw Chemical reaction device 21 Housing 22 Reaction liquid collecting gutter 23 Disk 24 Porous carrier 27 Reaction liquid supply nozzle 29 Pump 30 Adjustment tank 31 Pump 41 Top lid 42 Bottomed cylindrical container 42a Eaves 43 Disk-shaped fixing plate 44 Rotating shaft 45 Living body Tissue section 46 Mesh 47 Medium 48 Medium outlet 49 Pump 50 Medium storage tank 51 Pump 52 Medium inlet 60 Housing 62 Rotary shaft 63 Spacer 64 Porous carrier 65 Reaction liquid extraction nozzle 66 Reaction liquid supply nozzle 67 Reaction liquid

Claims (14)

[Claims] 1. A fixing plate to which a physiologically active substance is fixed, a housing for accommodating a reaction solution supplied to the physiologically active substance and the fixing plate, and a pivoting or swinging movement of the fixing plate for the physiological A biochemical reaction device comprising: a rotating means for generating a centrifugal force capable of removing a reaction solution near an active substance. 2. The biochemical reaction apparatus according to claim 1, further comprising a reaction liquid supply means for supplying the reaction liquid near a rotation axis of the fixed plate. 3. The biochemical reaction device according to claim 1, further comprising a variable mechanism capable of periodically changing a speed and / or a direction of the turning or swinging movement of the turning means. 4. The biochemical reaction apparatus according to claim 1, wherein the fixing plate is a porous body having open cells. 5. The biochemical reaction device according to claim 4, wherein said porous fixing plate is fixed on an upper surface of another disk. 6. The biochemical reaction device according to claim 4, further comprising a liquid-permeable spacer that contacts the physiologically active substance or the porous fixing plate. 7. The porous fixing plate according to claim 4, wherein the porous fixing plate is immersed in the reaction solution in a state where an upper surface and an outer wall portion are exposed to a gas phase. Biochemical reactor. 8. The method according to claim 1, wherein the physiologically active substance fixed on the fixing plate is immersed in the reaction solution with a part thereof exposed to a gas phase.
Biochemical reactor according to one of the paragraphs. 9. The biochemical reaction apparatus according to claim 1, further comprising a reaction liquid collecting gutter at an inner bottom edge of the housing. 10. The biochemical reaction apparatus according to claim 9, wherein the reaction liquid collecting gutter communicates with the reaction liquid supply means via a reaction liquid adjustment tank provided outside the housing. . 11. The biochemical reaction apparatus according to claim 1, wherein an installation angle of the fixed plate with respect to a horizontal plane is variable. 12. The biologically active substance is a living tissue,
The reaction solution is a culture solution.
Item 12. The biochemical reaction device according to any one of items 11. 13. A centrifugal force is generated by rotating or oscillating a fixing plate on which a physiologically active substance is fixed, whereby a reaction solution near the physiologically active substance is eliminated, and a reaction liquid is added to the physiologically active substance. A biochemical reaction method, characterized in that: 14. The physiological activity fixed to the fixed plate by adjusting at least one of the rotation speed of the fixed plate, the supply speed and / or the withdrawal speed of the reaction solution, and the usage amount of the reaction solution. 14. The biochemical reaction method according to claim 13, wherein the substance is immersed in the reaction solution with a part thereof being exposed to a gas phase.