JPS63177780A - Cultivation apparatus using reversibly rotatable axial-flow impeller - Google Patents

Abstract

PURPOSE:To obtain a cultivation apparatus capable of stirring fragile cells without applying strong shearing force, by installing a cylindrical unit having many small holes near the center of a cultivation vessel, providing an axial-flow impeller therein and forming diffusion holes in the bottom thereof. CONSTITUTION:A cylindrical unit 12 consisting of porous bodies (12a) and (12b) having holes of a smaller diameter than that of cells or microcarriers (C) is fixed near the center of a cultivation vessel 11 and a reversibly rotatable axial- flow impeller 14 is provided therein. The part of the cylindrical unit 12 surrounding the impeller is constituted of a nonporous shroud part (12c). Many diffusion holes 15 are formed in the lower part of the cylindrical unit 12 and an oxygen- containing gas, e.g. air, etc., is fed through a germ-free filter 16 thereto. When the shaft 13 of the axial-=flow impeller 14 is rotated in the direction of solid arrow line, the state is as shown in the right half of the figure. When the shaft is rotated in the opposite direction, the state is as shown in the left half of the figure.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to an apparatus for culturing microorganisms such as animal and plant cells and prisoners, and particularly relates to a device for culturing microorganisms such as animal and plant cells and bacteria. The present invention relates to a culture device that can stir cells to promote mass transfer of metabolic products and supply oxygen to cells and the like.

[Conventional technology and problems]

Generally, in order to culture animal and plant cells and microorganisms such as bacteria, oxygen is supplied to the cells etc. through a culture solution, and
It is necessary to mix and agitate the culture solution and cells to promote mass transfer of metabolic products.

Conventionally, a force culture device was provided, for example as shown in FIG. 5, and air bubbles were discharged into the culture solution from an aeration pipe 1, and the culture solution t was stirred and mixed by a stirrer. In addition,
In the figure, 3 is a culture tank (container)≠ is a baffle plate protruding from the inner wall surface to enhance the stirring effect of the stirrer 2, and g is an oxygen-containing gas such as air sent into the aeration pipe 1.

However, in the above-mentioned conventional apparatus, the strong shear stress generated by the blades of the stirrer destroys the cells contained in the culture solution, which is undesirable.
Alternatively, stirring devices using ejectors, jet pumps, etc. have the problem of cells being destroyed by the strong shear stress of the liquid.

In addition, as shown in FIG. 6, an oxygen-containing gas (g) such as air is introduced from below the culture tank 3 to generate air bubbles in the tank, and the air bubbles cause the culture solution l to flow and stir. In the device, as the culture progresses and the viscosity of the culture solution t increases, it is not possible to obtain a sufficient stirring effect, and
There was a problem in that a large amount of bubbles were generated on the liquid surface, which was undesirable.

The present invention aims to treat animal and plant cells that are extremely weak and easily destroyed.
The technical challenge is to create a culture device that can supply oxygen while stirring without applying strong shear stress.

[Means for solving problems]

In order to solve the problems and technical problems of the prior art described above, the present invention has a porous hole near the center of the inside of a culture tank, which has a large number of holes so that only the culture solution passes through and cells etc. do not pass through. A cylinder formed at least in part by the body is provided, an aeration device is provided below the inside of the cylinder, and a diffuser is provided for aeration,
A feature is that a reversible axial flow impeller that can change the direction of circulation of the culture solution is installed above it.

In addition, the cylinder formed at least in part by the above-mentioned porous body is
It can be used stationary or rotated with a trickle impeller.

[For production]

Since the present invention is configured as described above, when the culture solution is poured into the culture tank to a predetermined level, the culture solution is at approximately the same level inside and outside the cylinder, at least a part of which is formed of a porous material. reach. Next, when the axial flow impeller is rotated in this state, the culture solution moves from top to bottom or from bottom to top according to the relationship between the blade angle of the cylindrical internal ζ axial flow impeller and the rotation direction. It flows out of the cylinder through the porous body provided in the cylinder, moves in the opposite direction to the flow inside the cylinder, that is, from bottom to top, or from top to bottom, and flows into the cylinder again. In this way, a circulating flow is formed inside and outside the cylinder, and the culture solution is stirred and mixed by the circulating flow.

On the other hand, oxygen-containing gas g such as air ejected from an air diffuser provided below inside the cylinder becomes bubbles and rises inside the cylinder, and the aeration effect by the bubbles is performed inside the cylinder,
A sufficiently oxygenated culture medium is supplied to the outside of the cylinder.

On the other hand, the cells are sealed outside the cylinder and separated from the inside of the cylinder by a porous material. In addition, the cells are
In addition to floating the cells alone in the culture medium, in some cases they may also be attached to microcarriers. Therefore 1
The pores of the porous body formed in a part of the cylinder are formed to be as small as cells or microcarriers.

As mentioned above, the cells are confined outside the cylinder,
Since it does not enter the inside of the cylinder, there is no direct contact with the impeller or air bubbles. Therefore, a large amount of bubbles will not be generated due to cell damage.

In addition, changing the rotational direction of the axial impeller changes the circulation direction of the culture solution, so the flow passing through the cylindrical porous body is in the opposite direction, thus preventing clogging of the porous body. It will be resolved.

Note that by rotating the cylinder together with the axial flow impeller, clogging of the porous portion of the cylinder can be further prevented, and since the culture solution is in contact with the rotating cylinder, some of the culture solution may also be A turning force is applied to give a turning effect.

〔Example〕

Next, embodiments of the present invention will be described with reference to the drawings.

FIG. 1 is a longitudinal sectional view of a cell culture apparatus showing a first embodiment of the present invention.

In the figure, a porous body such as a wire mesh or a porous resin /2a, which has pores with a smaller diameter (for example, 7≠μm or less) than cells or microcarriers C, near the center of the culture tank (vessel) //;
A cylinder lλ consisting of 12b is fixedly attached,
Inside the cylinder 12, there is provided an axial flow impeller l≠ attached to a reversible shaft 13. The axial flow impeller/4' is constructed by installing blades having a flat plate blade or a symmetrical airfoil shape in cross section at an angle to the rotation direction (direction perpendicular to the axis).

The portion of the cylinder /2 surrounding the impeller 1 is constituted by a shroud portion /2c without holes. In addition, an aeration device consisting of a large number of aeration holes /J is provided in the lower part of the interior of the cylinder lλ. The containing gas g is supplied. In addition, in the figure, 17 is the cylinder 12
A level sensor detects the liquid level on the inside of the cylinder 12, and /su is a level sensor that similarly detects the liquid level on the outside of the cylinder 12.

Next, the effect will be explained.

After pouring the culture solution t into the culture tank ll to a predetermined level,
For example, when the trickle impeller/Hiro is rotated in the direction of the solid arrow,
As shown in the right half of FIG. 1, the culture solution t inside the cylinder/2 moves from top to bottom, passes through the lower cylindrical porous body part/2b, and flows from the bottom to the top outside of the cylinder/2. Move to. Therefore, the liquid level on the inside and outside of the cylinder 12 is lower on the inside and higher on the outside, as shown in the right half, so that
The culture solution 2 on the outside of 2 flows into the inside of the cylinder 12 through the upper porous body part 2a. In this way, the culture solution t forms a force circulation flow as shown by the double arrow in the figure, and is mixed with stirring.

On the other hand, after the oxygen-containing gas g such as air is sterilized by the air filter B, it is ejected into the cylinder from the air diffuser hole J' provided in the lower part of the cylinder 7.2, forming bubbles. 1 cylinder/2, the aeration effect by the bubbles is performed inside the cylinder, and the culture solution rich in oxygen passes through the lower porous body part 2b to the outside (outside) of the cylinder/λ. supplied to

Further, the cells C are sealed on the outside of the cylinder/2, and are isolated from the inside of the cylinder by the porous parts/, 2a, /2b and the shroud/Jc.

As mentioned above, the cells are sealed outside the cylinder and do not enter the inside of the cylinder, so they do not come into direct contact with the air bubbles ejected from the impeller or the air diffuser hole /j. Therefore, cells are not damaged and bubbles are not generated in large quantities.

Furthermore, if the axial flow impeller continues to rotate in one direction as shown by the solid arrow, cells and microcarriers C.
is likely to adhere to the outside of the upper porous body/2a and cause clogging.

In such a case, by reversing the rotational direction of the axial impeller 1H in the direction of the dotted arrow, the liquid level inside the cylinder 12 is high and the liquid level outside is low, as shown in the left half of Fig. 1. ,
Since the circulation direction of the culture solution t is changed as shown by the double arrow on the left half, the porous body part /2 above the cylinder /2 mentioned above
Since the flow passing through a is in the opposite direction, clogging of the porous body portion is eliminated in this way.

The rotation direction of the axial flow impeller/Hiro as described above may be changed at regular intervals using a timer, etc., but the increase in the liquid level difference between the inside and outside of the cylinder caused by clogging can be detected by using both level sensors/Hiro.
7. This may be done by detecting with l♂ or the like.

FIG. 2 is a longitudinal sectional view showing a second embodiment of the present invention, and in the figure, the same reference numerals as those shown in FIG. 1 indicate the same or similar parts.

In this embodiment, a cylinder 22 having porous body parts 22a, 22b and a shroud part 2.20 is attached to the shaft 13 via a support disk λ2d and rotated in unison with the trickle impeller lhiro. If necessary, as shown in the left half of the diagram, a one-rotation disc may be placed below the cylinder 22.
This embodiment differs from the first embodiment (FIG. 1) in that the components are mounted so that they can rotate as one, but there is no difference in other respects.

According to this embodiment, when the cylinder 22 is rotated together with the axial flow impeller/g, for example in the direction of the solid line arrow, the culture solution t inside the cylinder 22 flows from the bottom to the top as shown in the figure. It becomes higher than the facing bell, flows out of the cylinder 2.2 from the upper porous body part 22a, is sucked in from the lower porous body part 22b, and is cultured in the direction of the double arrow shown in the figure. Lt circulates and 'i! If the five-axis flow impeller/g is rotated in the opposite direction, the circulation flow will be reversed and the clogging will be eliminated by backwashing. There is no difference from the first embodiment (FIG. 1) in that the aeration is performed by rising inside the cylinder 22.

However, in this embodiment, since the cylinder 2.2 is rotated together with the axial flow impeller /+, the cylindrical porous body part 2.
．． Since clogging in 2a and 22b is further prevented, and a slight swirling force is applied to the culture solution t, the stirring effect can be enhanced.

In addition, as shown in the left half of Fig. 2, in the case where the rotary disk kota is rotated together with the cylinder 22, the centrifugal force effect caused by the disk 2 causes the outside of the cylinder 2-2 to Cells that tend to accumulate near the bottom can be gently agitated.

FIG. 3 is a longitudinal sectional view showing a third embodiment of the present invention, and in the figure, the same reference numerals as those shown in FIGS. 1, 2, and 2 indicate the same or similar parts. .

This embodiment solves the problem of the above-mentioned λth embodiment (FIG. 2), that is, if cells and microcarriers have a lower specific gravity than the culture medium, it is difficult for cells etc. to accumulate near the bottom surface.
The circulation direction as shown by the double arrow in Figure 2 is the normal rotation direction, and there is no problem even if you operate for a long time, but if the specific gravity of cells or microcarriers is higher than that of the culture solution, Circulation direction as in Figure 1, one! In a circulating flow that flows downward outside the cylinder, cells and the like tend to accumulate on the bottom surface. (On the other hand, in the λth embodiment, stirring is performed using the rotating disk 2.) Therefore, it is preferable to operate the pump for a long time with the pump rotating in the forward direction so that an upward flow is generated outside the cylinder. , in this way, the second embodiment (
The configuration shown in Fig. 2) deals with the problem that a downward flow occurs inside the cylinder and prevents the bubbles from rising. Inside the rotating cylinder 22, an inner cylinder 3/ whose lower part is fixed to the culture tank // is provided protrudingly, and bubbles of oxygen-containing gas g are ejected from an air diffusion hole 3j formed below the inner cylinder 3/. Suo,
It is adapted to be diffused into the interior of the inner cylinder 3/.

Holes 3/a and 31b, through which the culture solution t flows in and out, are formed in the upper and lower parts of the inner cylinder 3/, respectively, and an aeration flow rate regulating valve 32 is provided in the lower hole 31b. ing. In the figure, 22e is a rotating liner ring attached to the inner circumference of the bottom of the rotating cylinder 22, and 3/c is a fixed liner ring attached to the outer circumference of the bottom of the fixed inner cylinder 3/.

According to this embodiment, during normal rotation as shown by the solid line arrow, due to the pressure difference caused by the axial flow impeller,
The culture solution t bubbles and rises inside the inner cylinder 3/ from bottom to top as shown by the double arrow in the figure, and also on the outside (outside) of the cylinder 22 that rotates with the shaft 13, the culture YLt rises with double arrows. As shown by the arrow, the upward flow and the sedimentation of the cells C can be prevented. On the other hand, the aerated culture solution t rising inside the inner cylinder 3/ flows down between the inner cylinder 31 and the cylinder 22 by the axial flow impeller/≠ as shown by the double arrow, and flows down into the lower part. After passing through the cylindrical porous body part 22b, it flows along with the cells C along the outside of the cylinder from bottom to top, and returns to the inside of the cylinder 22 again. During this time, by adjusting the flow rate of the culture solution t passing through the inner cylinder 31 with the aeration flow rate adjustment valve 32, an optimal oxygen supply state can be obtained.

FIG. 3 is a longitudinal sectional view showing a fourth embodiment of the present invention, and in the drawing, the same reference numerals as those shown in FIGS. 1 to 3 indicate the same or similar parts.

In this embodiment, a cylinder 7.2 is fixed to the culture tank //, and a rotating shaft /3 is made hollow inside the culture tank to form an inner cylinder (inner cylinder shaft) 3. An axial flow impeller/l is attached to the inner cylinder≠3. Further, above and below the inner cylinder 4L3, there are holes 33 and '13 through which the culture solution flows in and out.
An aeration flow rate adjusting valve t-2 is provided on the outside of the opening of the lower hole≠3b so that the opening area can be adjusted by vertical movement, and an aeration hole 1 is provided on the inside.
1K has been formed. Moreover, above the diffuser hole≠j,
A fixed shaft extends through the inner tube 3,
Its upper end is supported by a shaft 13.

According to this embodiment, as in the third embodiment, the culture solution t has an upward flow outside the cylinder 12, so that it is possible to prevent the cells C from settling, and the culture solution t can be prevented from settling in the inner cylinder 4t3. Since the direction of flow is also upward, it does not prevent the bubbles from rising. Further, by controlling the flow rate of the culture solution t passing through the inner cylinder t3 with the aeration flow rate adjustment valve≠2, an optimum oxygen supply state can be achieved.

〔Effect of the invention〕

As explained above, according to the present invention, a cylinder formed at least in part with a porous material is provided near the center of the inside of the culture tank so that only the culture solution permeates and cells etc. do not permeate. By installing a diffuser for aeration at the bottom and a reversible axial flow impeller above it, the cells are always confined to the outside of the cylinder and isolated from the inside of the cylinder by a porous material. Since a sufficiently oxygen-rich culture solution is supplied to the outside of the cylinder by the aeration effect of air bubbles inside the cylinder, strong shear stress is not applied to the extremely weak and easily destroyed cells of animals and plants. Oxygen can be supplied while stirring.

In addition, since the trickle impeller is reversible, the direction of the circulating flow of the culture solution can be changed.
This can prevent clogging of the cylindrical porous body portion.

[Brief explanation of the drawing]

FIGS. 1 to 4 are vertical cross-sectional views showing first to first embodiments of the culture apparatus according to the present invention, and FIGS. 5 and 2 are vertical cross-sectional views showing conventional examples. / /-...Culture tank, / +2.22-...Cylinder, /2
a, /, 21) 22a, 22b... Porous body part, /3.
...Axle, l hiro...Axial flow impeller, / j , , 3 J
−, ≠! -... Diffusion hole, 31... Inner cylinder, 32, 11.
2...Aeration flow rate adjustment valve, ≠3...Inner cylinder, b...Bubble, c--Cell, microcarrier, g...Oxygen-containing gas, t...Culture solution.

Claims (1)

[Scope of Claims] 1. A cylinder formed at least in part by a porous material through which only the culture solution permeates and cells etc. do not permeate is provided near the center of the inside of the culture tank, and a cylinder for aeration is provided at the lower part of the inside of the cylinder. What is claimed is: 1. A culture device using a reversible axial flow impeller, characterized in that a reversible axial flow impeller is provided above the aeration device, and a reversible axial flow impeller capable of changing the circulation direction of the culture solution. 2. A culture device using a reversible axial flow impeller according to claim 1, in which the cylinder is fixed. 3. The reversible axial flow impeller according to claim 2, wherein the rotating shaft of the axial flow impeller is formed as a hollow inner cylinder, and the culture solution is aerated in the inner cylinder. Culture device used. 4. A culture apparatus using a reversible axial flow impeller according to claim 1, wherein the cylinder is rotatable together with the axial flow impeller. 5. A culture device using a reversible axial flow impeller according to claim 4, wherein an inner cylinder is provided fixed at the center of the cylinder, and the culture solution is aerated in the inner cylinder. . 6. A culture apparatus using a reversible axial flow impeller according to claim 4, wherein a device for stirring the culture solution rotating together with the cylinder is provided at the outer lower part of the cylinder. 7. A culture device using a reversible axial flow impeller according to claim 3, further comprising a valve for adjusting the flow rate of the culture solution passing through the inner cylinder. 8. A culture apparatus using a reversible axial flow impeller according to claim 5, which is provided with a valve for adjusting the flow rate of the culture solution passing through the inner cylinder.