JP2004248628A - Efficient bioreactor system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a bioreactor having a system to proliferate bacteria, cells, tissues, etc., develop the function of the bacteria, etc., perform the object metabolism and secretion by a biochemical reaction and finally recover the product in high efficiency. <P>SOLUTION: The bioreactor system has a flow path composed of a hollow fiber to flow the reaction solution. A porous sheet such as a nonwoven fabric holding cells or bacteria reacting or metabolizing outside of the hollow fiber. The system can perform a radial flow-type medium circulation to keep the bioactivity or survival rate of the bacteria, etc., in high efficiency. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a system for incorporating a microscopic organism or a part of an organism, such as bacteria, cells, tissues, organs, and organs, into a bioreactor to efficiently express functions.
[0002]
[Prior art]
Hitherto, many methods for making a bioreactor using bacteria, cells, tissues, and the like have been proposed. However, almost all use beads as a carrier to fix bacteria, cells, and tissues, so cells and the like can easily become clogged between beads, and can be blocked by secretions, resulting in stable It was difficult to secure the flow path.
[0003]
In addition, it is difficult to sufficiently supply nutrients and oxygen when a large amount of bacteria, cells, and tissues are fixed because the medium is not a radial flow type medium supply, and it is difficult to avoid the necrosis of bacteria, cells, and tissues. .
[0004]
[Problems to be solved by the invention]
The present invention has been made in view of the above-mentioned problems of the prior art, and has been effective in immobilizing bacteria, cells, tissues, organs, organs, and the like on a porous sheet capable of adhering cells and maintaining its biological activity. The objective of the present invention is to provide a system for supplying a good medium, and to be able to efficiently collect and separate products (secretions) and treatment solutions by hollow fibers.
[0005]
[Means for Solving the Problems]
The present invention relates to the following bioreactors and bioreactor systems.
Item 1. A culture space containing a medium outlet tube (7), a plurality of hollow fibers (4), and a porous sheet material (3) allowing cells to pass between the hollow fibers is provided in a tubular reactor body (8). Bioreactor,
(I) At least one first inlet port (1) formed in the reactor body (8), a first outlet port (2) in which the medium supplied from the (1) communicates with one end of the medium outlet tube (7). A first flow path flowing out of the
(Ii) a second outlet port through which the processing liquid supplied from the second inlet port (5) formed at one end of the reactor body passes through the plurality of hollow fibers (4) and communicates with the other end of the reactor body ( 6) a second flow path flowing out of
And the porous sheet-like material (3) is capable of adhering cells and passing cells, and the medium outlet tube (7) and the hollow fiber (4) do not allow cells to pass, but allow liquids and substances to pass. A possible radial flow type bioreactor.
Item 2. Item 2. The bioreactor according to item 1, wherein a cell-adhesive porous sheet and a porous nonwoven fabric are overlapped and wound around a central axis between a plurality of hollow fibers.
Item 3. Item 3. The bioreactor according to Item 1 or 2, wherein the culture space is provided with bacteria, cells or tissues.
Item 4. Item 1. The bioreactor according to Item 1, wherein the first inlet port is connected to a medium tank via a pump, the first outlet port is connected to the medium tank, and the second inlet port is connected to the processing liquid via a pump. A bioreactor system connected to a tank and a second outlet port connected to the processing liquid tank.
[0006]
The main body of the bioreactor of the present invention has a medium outlet tube for outflow of the medium, a plurality of hollow fibers serving as a flow path of the processing solution in the reactor, the medium and the cells can pass therethrough, and the cells alone on the surface thereof Or a group of porous sheets that can adhere and grow together.
[0007]
The reactor main body preferably has a cylindrical shape such as a cylindrical shape or a rectangular tube shape. FIG. 1 shows a cylindrical reactor body.
[0008]
The culture space contains a porous sheet, and cells are usually adhered to the surface of the sheet and cultured. The sheet-like material is present between the hollow fibers, preferably wound around a medium outlet tube (7) existing on the central axis of the reactor main body, and a plurality of, preferably a plurality of, preferably sandwiched between the porous sheet-like materials (3). Has a structure in which a number of hollow fibers (4) are provided.
[0009]
A plurality of hollow fibers are used in one reactor body, usually a large number, for example, about 10 to 500, and preferably about 20 to 300. The hollow fiber is preferably fixed with a support (9) through which the hollow fiber passes, for example, so that the processing liquid can pass through both ends. The hollow fiber is capable of transferring a substance having a molecular weight of 1,000,000 or less, preferably about 500,000 or less, more preferably about 200,000 or less. When the treatment solution is passed through the hollow fiber, the useful fiber produced in the culture space is useful. The substance diffuses into the hollow fiber and can be recovered.
[0010]
In the culture space, it is preferable to use a porous non-woven fabric used as a spacer by overlapping it with the porous sheet material (3). It is not necessary for the porous nonwoven fabric to allow cells to pass through, and the pore size is usually 1 to 200 μm, preferably about 1 to 50 μm. The porous non-woven fabric preferably fills the gaps between the hollow fibers and secures the hollow fibers.
[0011]
The material of the porous sheet material and the porous nonwoven fabric is not particularly limited, and known organic polymers can be widely used. Preferably, a fluorine-containing polymer such as PTFE, polyolefin, polyester, polyurethane, cellulose fiber, regenerated cellulose fiber, Polyamide, polyimide and the like can be used.
[0012]
The porous sheet is preferably treated with a polyamino acid / urethane copolymer, optionally crosslinked protein such as collagen, albumin, fibronectin, hydrogel or the like in order to promote cell adhesion.
[0013]
In the medium outlet tube (7) of the present invention, cells do not enter inside, but the medium supplied from the first inlet port (1) flows out from the first outlet port communicating with one end of the medium outlet tube (7). Have enough holes to It is preferable that the medium lead-out tube (7) is formed along the central axis in the tubular reactor body (8), but a plurality of the medium lead-out tubes may be provided at positions symmetrical with respect to the central axis.
[0014]
In the case of the bioreactor shown in FIG. 1, the culture medium is supplied from the first inlet port (1), and the medium is supplied to the space between the reactor body and the porous sheet (3) or the hollow fiber (4). There is a first flow from (1) along the circumferential direction, divided into two sides, and a second flow from the space near the cylindrical reactor body toward the center culture medium outlet tube (7). The medium supplied from one inlet port (1) collects in the central medium outlet tube (7) and flows out from the first outlet port (2). The other end (7a) of the medium outlet tube (7) is sealed, and the medium flowing into the medium outlet tube (7) is directed to the first outlet port (2).
[0015]
The medium lead-out tube (7) has a pore size (usually 1 to 50 µm, preferably 1 to 30 µm) that allows passage of a liquid such as a medium but does not allow cells to pass through.
[0016]
The reactor body preferably has an elongated cylindrical shape, and the hollow fibers are preferably fixed parallel to the central axis of the reactor body.
[0017]
The number of the first inlet ports (1) formed in the reactor body may be one or two or more, but is usually one to six, preferably two to four, and particularly two. When a plurality of first inlet ports are formed, the positions are preferably formed at positions symmetrical with respect to the axial center of the reactor body (see FIG. 1).
[0018]
The culture solution supplied from the first inlet port (1) passes through the porous sheet material (3) accommodated in the culture space and the porous nonwoven fabric as an optional element, and further enters the medium outlet tube (7). The fluid flows out of the first outlet port (2) communicating with the outlet tube (7) to form a first flow path.
[0019]
On the other hand, the processing liquid is supplied into the hollow fiber from the second inlet port (5) opposite to the first outlet port. Since the hollow fiber is capable of transferring a substance, it is possible to diffuse a high molecular or low molecular useful substance such as a protein into the hollow fiber and to guide the substance from the inside of the reactor to the outside. The treatment liquid flows out of the second outlet port (6) through the hollow fiber (4) to form a second flow path.
[0020]
The treatment liquid is not particularly limited, and examples thereof include blood, plasma, serum, a medium, and a liquid to be subjected to an enzyme treatment (beer, food, chemical solution, and the like).
[0021]
As the medium, a liquid capable of growing cells, microorganisms, tissues, organs, and organs can be widely used.
[0022]
Since the porous sheet-shaped material of the present invention has a pore size sufficient to allow cells to pass through, the cells can move, and even if a large amount of cells, microorganisms, and tissues are held, the porous sheet-like material can be used. Clogging is unlikely. The pore size is 200 μm or more, preferably about 200 to 500 μm.
[0023]
The reactor of the present invention has a radial flow system capable of efficiently supplying and recovering a medium, and forms a flow path independent of this system and the flow path of the recovery hollow fiber.
[0024]
BEST MODE FOR CARRYING OUT THE INVENTION
FIG. 1 is a bioreactor according to a first embodiment of the present invention, and FIG. 2 is a schematic diagram showing a bioreactor system. In FIG. 1, the culture medium entered from the first inlet port (1) passes through the cell-adhesive porous sheet (3), enters the culture medium outlet tube (7), and enters the first exit port (2). Spilled out of. The liquid to be treated enters through the second inlet port (5), passes through the hollow fiber (4), and exits through the second outlet port (6).
[0025]
In FIG. 2, 1, 2, 3, and 4 are for adjusting the pH, the dissolved oxygen concentration, and the like of the culture medium adjusting tank. Data is recorded by the computer 5. The culture medium that has passed through the culture medium adjustment tank 7 enters the bioreactor 10 after passing through the pump 9, enters from outside the hollow fiber, passes through the central hollow fiber (shown in FIG. 1), and passes through the oxygenation membrane 11. Then, the process returns to the adjustment tank 7 again. It has a channel independent of the hollow fiber. The liquid to be treated passes through the pump 9 from the plasma bottle 14 as a reservoir, enters the hollow fiber, exits from the second outlet port separate from the medium flow path, and returns to the plasma bottle 14 again.
[0026]
As shown in FIG. 1 and FIG. 2, the system can pack cells, tissues, and bacteria with high efficiency and maintain their activity, and can be easily incorporated into a commercially available artificial dialysis machine.
[0027]
【Example】
Example 1 Hereinafter, the present invention will be described in more detail with reference to Examples.
In a bioreactor system shown in Figure 2, the medium and the treatment liquid as a medium hepatocytes contains no viable albumin, ammonia 200μ mol / 10 ⁹ cultured culture space while supplying under the conditions of hepatocytes / h Were cultured, and the metabolic rate of ammonia and the secretion rate of albumin into the treatment solution were measured. The results are shown in FIGS.
[0028]
In addition, a porous sheet made of PTFE treated with a polyamino acid / urethane copolymer (FIGS. 5A and 5B) and a porous sheet made of rayon fabric (FIGS. 5C and 5D) 3 shows the state of adhesion of hepatocytes when a sheet is used.
[0029]
As shown in FIG. 5, it was revealed that the use of a porous sheet surface-treated with a substance for improving cell adhesion results in good adhesion and proliferation of hepatocytes.
[0030]
【The invention's effect】
As a result, a large amount of bacteria, cells and tissues can be fixed, and functions such as high metabolism and secretory activity can be continuously maintained. As this application, the present invention can be applied to artificial organs such as an artificial liver and an artificial pancreas, or a scale-up, a water treatment system, and a chemical reaction system such as fermentation.
[Brief description of the drawings]
FIG. 1 illustrates one embodiment of a bioreactor.
FIG. 2 is a schematic diagram of a bioreactor system.
FIG. 3 shows the results of measuring the metabolic rate of ammonia.
FIG. 4 shows the results of measuring the metabolic rate of albumin.
FIG. 5 is a view showing a state of hepatocyte adhesion to a porous sheet-like material.
[Explanation of symbols]
(1) First inlet port (2) First outlet port (3) Porous sheet material (4) Hollow fiber (5) Second inlet port (6) Second outlet port (7) Medium outlet tube (7a) The other end of the culture medium outlet tube (8) Reactor body (9) Support 1 Carbon dioxide cylinder 2 Oxygen cylinder 3 Air cylinder 4 Controller 5 Computer 6 Filter 7 Medium adjustment tank 8 Stirrer 9 Pump 10 Bioreactor 11 Oxygenation membrane 12 Medium bottle 13 Waste liquid bottle 14 Plasma bottle 15 Incubator

Claims (4)

A culture space containing a medium outlet tube (7), a plurality of hollow fibers (4), and a porous sheet material (3) allowing cells to pass between the hollow fibers is provided in a tubular reactor body (8). Bioreactor,
(I) At least one first inlet port (1) formed in the reactor body (8), a first outlet port (2) in which the medium supplied from the (1) communicates with one end of the medium outlet tube (7). A first flow path flowing out of the
(Ii) a second outlet port through which the processing liquid supplied from the second inlet port (5) formed at one end of the reactor body passes through the plurality of hollow fibers (4) and communicates with the other end of the reactor body ( 6) a second flow path flowing out of
And the porous sheet-like material (3) is capable of adhering cells and passing cells, and the medium outlet tube (7) and the hollow fiber (4) do not allow cells to pass, but allow liquids and substances to pass. Possible radial flow type bioreactor. The bioreactor according to claim 1, wherein a cell-adhesive porous sheet and a porous nonwoven fabric are superposed and wound around a central axis between a plurality of hollow fibers. 3. The bioreactor according to claim 1, wherein bacteria, cells or tissues are provided in the culture space. The bioreactor according to claim 1, wherein the first inlet port is connected to a medium tank via a pump, the first outlet port is connected to the medium tank, and the second inlet port is processed via a pump. A bioreactor system connected to a liquid tank and a second outlet port connected to the processing liquid tank.

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