JP2007110923A - Culturing module - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a culturing module capable of preventing the occlusion or entanglement of flow passages for the perfusion of a medium, also limiting the swinging space of the flow passage as minimum and suitable for a medium perfusion type culturing device capable of making it a small-size. <P>SOLUTION: This culturing module consisting of at least a culturing container as a proliferation space of cells, flow passages for medium perfusion and a member for holding or fixing these culturing container and flow passages for the perfusion of the medium is constituted so that the culturing module is freely rotating or reverse rotating by centering around a rotary shaft, and the medium-flowing in flow passage to the culturing module and medium-flowing out flow passage are gathered and arranged on the rotary shaft. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a culture module suitably used in a culture apparatus such as a medium perfusion culture apparatus for culturing cells. Specifically, the present invention relates to a culture module for a culture apparatus applicable to production of useful substances using human cells, animal cells, insect cells, and plant cells or amplification of useful cells.

  As a treatment method for leukemia and solid cancer, a cell therapy method has been studied in which a small amount of hematopoietic stem cells and autologous lymphocytes are proliferated in vitro and returned to the patient. In such a field, there is a demand for a culture technique that enables a large amount of target cells to be cultured at a high density in a closed state outside the living body while ensuring low cost and safety.

  In addition, methods for culturing cells that secrete useful physiologically active substances such as hybridomas and obtaining useful physiologically active substances from the culture supernatant have been studied. The problem is that there are few useful physiologically active substances secreted into the supernatant. In the case of producing an antibody as a physiologically active substance secreted by cells, a method of administering a hybridoma that produces an antibody to the abdominal cavity of a mouse, and collecting and purifying ascites is common. However, this method involves complicated work such as breeding of mice, and since antibodies are obtained by purification from ascites, various impurities such as infectious substances may be mixed in to ensure safety. In this state, a technique for culturing cells that secrete useful physiologically active substances such as hybridomas in high density and in large quantities is desired.

In general, cell culture techniques include flask culture, microcarrier culture, agitation culture using a spinner flask or culture tank, a method of culturing with cells adhered to a porous medium, a culture method using a bag and a roller. Examples include a culture method using a bottle. In the conventional flask culture method and culture method using a bag, nutritional deficiency in the culture solution and a decrease in pH value occur, and it is difficult to make the density higher than 1 × 10 ⁶ cells / ml at present. . In addition, the agitation culture method using a spinner flask or a culture tank is not suitable for culturing viable cells at high density for a long period of time because physical obstacles to cells due to agitation are large.

In recent years, a culture method using a hollow fiber or a porous material used for hemodialysis has been proposed as a means for performing high-density cell culture in a three-dimensional space (see Patent Documents 1 to 3). In the cell culture method using hollow fibers, mass exchange is performed between the culture solution passing through the hollow fiber lumen for the purpose of nutrient supply and waste removal, and the cells filled in the outer space and the surrounding culture solution. It is said that cells can be cultured at high density.
JP 10-33671 A JP 2004-248628 A JP 2004-121167 A

However, in these conventional culture methods, cells form an agglomeration soul in one place in the culture module due to the flow of gravity and medium perfusion, and it is difficult to supply nutrients and remove waste products in the cells. It is thought that cell growth is restricted or cells die. Therefore, the present inventors rotated and cultured the culture module in order to prevent cell aggregation, but it was found that rotational culture of the culture module has the following problems. .
(1) A tube or a pipe used as a medium perfusion channel may move in a direction unintended by the operator when the culture module is rotated, resulting in poor medium perfusion such as blockage or entanglement of the channel.
(2) It is necessary to increase the space of the culture module depending on the swirl diameter of the flow path, thereby increasing the size of the entire culture medium perfusion culture apparatus.
(3) In terms of operation, it is difficult to handle such as seeding of cells before culturing and transportation of culturing modules after culturing.
(4) Cell seeding and recovery operations in the culture apparatus are difficult.

  Accordingly, an object of the present invention is suitable for a medium-perfusion culture apparatus that can prevent the medium perfusion from being blocked or entangled, and can limit the swirling space of the flow path to a minimum. Is to provide a simple culture module.

  The culture module of the present invention holds or fixes a culture container that is at least a cell growth space, a medium perfusion channel for supplying and discharging a culture medium to the culture container, and the culture container and the medium perfusion channel. A culture module comprising a member for performing culture, the culture module being rotatable or reversible around a rotation axis, and a medium inflow channel and a medium discharge channel to the culture module on the rotation axis The culture module is configured to be assembled and arranged.

  According to a preferred aspect of the culture module of the present invention, the culture container is detachable from a member for holding or fixing the culture container.

  According to a preferred embodiment of the culture module of the present invention, the culture container includes a port for seeding and collecting cells, and the direction of the port for seeding and collecting can be changed. ing.

  The culture module of the present invention is suitably used in a medium perfusion type culture apparatus for culturing cells. According to a preferred embodiment of the culture module of the present invention, the direction of the port for seeding and recovery is the medium It faces the operating side of the perfusion culture device.

By using the culture module of the present invention, in a culture apparatus such as a culture medium perfusion culture apparatus, the movement of the flow path is minimized by collecting the flow path on the rotating shaft during the module rotation culture for reducing cell aggregation. In addition, it is possible to prevent the channel perfusion or entanglement of the medium perfusion. In addition, since the culture module is rotated while holding the flow path, the flow path in the module can be fixed to the shortest and the flow path expansion can be minimized, so that the swirl space of the flow path can be limited to the minimum. Thereby, the size of the main body of the culture apparatus can be reduced.
Moreover, since it can isolate | separate from the member holding the culture container of a culture module, the conveyance and handling of the cell after proliferation can be made easy. Further, by directing the cell seeding (or recovery) port of the culture container to the culture device operation surface side, cell seeding and / or recovery to the culture module in the medium perfusion type culture device is easy.

(Medium perfusion culture device)
A culture medium perfusion type culture apparatus to which the culture module of the present invention is suitably applied is composed of at least a culture module, a culture medium reservoir, and a pump in an incubator capable of controlling temperature, and has a circuit that connects them. A culture apparatus capable of perfusing a culture medium into a culture module by a pump. In the culture module, nutrient substances can be supplied to the cells by the perfused medium, and waste substances from the cells can be discharged by the flow of the medium, so that the cells can be cultured at high density. Further, since the antibody produced by the hybridoma or the like is contained in the medium discharged from the culture module, the antibody can be recovered by purifying it. The culture medium discharged from the culture module may be returned to the culture medium reservoir, adjusted, and then supplied to the culture module again.

(Culture module)
The culture module of the present invention comprises at least a culture container, a medium perfusion channel, and a member for holding them. Specifically, a culture container that is at least a cell growth space, a medium perfusion channel for supplying and discharging the culture medium to and from the culture container, and for holding or fixing these culture container and medium perfusion channel The culture module is rotatable or reversible about a rotation axis fixed to the culture module, and the medium inflow channel and the medium discharge channel to the culture module are the culture module. It is a culture module configured to be collectively arranged on a rotating shaft.

(Culture module culture vessel)
The culture vessel constituting the culture module of the present invention is a cell growth space that maintains the culture environment. The cell culture part and the medium perfusion part are separated by a semi-permeable membrane, etc. so that the space for cell growth is always secured and the nutrients and waste of the medium pass through the perfusion medium flow path without passing the cells. It is preferable that

  The semipermeable membrane used as a separation material for the cell culture portion and the medium perfusion portion is preferably a hollow fiber-like semipermeable membrane having a large specific surface area in order to enhance substance exchange. Examples of semipermeable membrane materials include high thermoplasticity such as polysulfone, polyethylene, polystyrene, polypropylene, polyethersulfone, polyvinyl alcohol, polymethyl methacrylate, cellulose acetate, polyacrylonitrile, fluororesin, regenerated cellulose, and polyamide. Molecular compounds or derivatives thereof are preferably used. Further, these materials may be chemically modified such as fixing an adhesion factor such as collagen or fibronectin, and a membrane material having excellent biocompatibility is desirable.

The nutrients that need to be supplied to cells are mainly proteins, amino acids, glucose, oxygen, albumin and vitamins, and the main waste products are low-molecular substances such as organic acids, urea, and carbon dioxide. The semipermeable membrane is preferably a membrane that allows a substance having a substance diameter of about 0.1 μm to pass through.
As cells used in this culture vessel, suspension cells are usually cultured, but adherent cells can also be cultured. In this case, it is possible to culture at a higher density by supplying the cells to the cell culture side and culturing them preferably in a state where the cells are adhered to microcarriers, nonwoven fabrics, and fine particles such as ultrafine fibers. .

  The shape of the culture vessel used in the present invention is not particularly limited, but is preferably a cylindrical shape so as not to retain the medium in the culture vessel, and has a shape structure including a port for seeding and collecting cells. Preferably there is. The port may have a structure having two separate ports, a port for seeding cells (seeding port) and a port for recovering cells (collection port). One port) may have both functions of cell seeding and recovery.

  The material of the culture vessel may be any of inorganic materials such as glass and metal, or organic materials such as polypropylene, polystyrene, polyvinyl chloride, polyacryl and polycarbonate, and preferably sterilized glass and organic materials. Is used. It is also possible to use different materials on the surface of these materials. More preferably, a coating for suppressing the adsorption of protein to the container surface can be applied.

(Flow path for culture medium perfusion)
The culture medium perfusion flow path constituting the culture module in the present invention is a component for supplying and discharging the culture medium from the culture medium reservoir to the culture container.

  The material for the medium perfusion channel may be an inorganic material such as silicon, glass and metal, or an organic material such as polypropylene, polystyrene, polyvinyl chloride, polyacryl and polycarbonate. In addition, since it is not necessary to remove the culture medium perfusion channel from the culture module, when the holding parts are made of polypropylene, polystyrene, polyvinyl chloride, polyacryl, polycarbonate, etc., they are integrated with the same material. May be.

(Culture module holding member)
The holding member constituting the culture module in the present invention is a skeletal component of the culture module, and has a function of holding or fixing the culture container and the medium perfusion channel. It is preferable to have an attachment mechanism for the culture apparatus so that the attachment to the culture medium perfusion type culture apparatus becomes easy.

  The holding member has a structure in which the medium perfusion channel can be fixed with a guide or the like, and by this structure, the channel can be changed and fixed in the right-angled direction in the immediate vicinity of the culture vessel, so that the size of the culture module is minimized. This makes it possible to reduce the turning diameter of the culture module. As a result, the size of the culture medium perfusion culture apparatus using the rotating module can be reduced.

  In addition, the medium inflow channel for inflow of the medium and the medium discharge channel for the medium discharge extending from the medium perfusion channel of the culture module are configured to be collectively arranged on the rotation axis of the culture module in the culture module. It is important that By configuring the culture medium inflow channel and the culture medium discharge channel in this way, the moving distance of the channel that moves when the module rotates can be reduced, and blockage and entanglement of the channel can be prevented. On the rotation axis of the culture module, for example, it is assumed that the rotation axis is fitted to one end of the culture module and the rotation axis extends toward the opposite side of the culture module, and preferably It is preferable that the radius is within 10 mm centering on the extension line, and may be within 50% of the culture module length.

  The material of the holding member may be either an inorganic material such as metal or an organic material such as polypropylene, polystyrene, polyvinyl chloride, polyacryl and polycarbonate.

(Rotation mechanism of medium module)
The proliferated cells become aggregated souls due to sedimentation by gravity and the flow of the medium in the culture vessel. Since the nutrients do not reach the cells inside the agglomerated soul, the growth is inhibited. Therefore, the culture perfusion culture apparatus is provided with a horizontal drive rotation axis, and the cells near the center of the culture module are connected to the rotation axis of the culture apparatus (culture module rotation axis) and rotated to rotate the cells aggregated in the culture vessel To prevent the formation of agglomerated souls. The culture module may be rotated in one direction, but it is necessary to connect the medium perfusion channel with a rotary connector. For this reason, by reciprocatingly rotating at an angle of 360 ° or less, the same effect as 360 ° rotation can be obtained without requiring a rotary connector. The rotation angle at this time is preferably reciprocating between 90 ° and 270 ° in terms of cell agitation efficiency. Moreover, in order to reduce the stress to a cell, it is preferable that it is within 1-20 rpm, More preferably, it is 4-10 rpm.

(Medium reservoir)
The medium reservoir in the present invention is a part for temporarily storing a medium to be supplied to the culture module and adjusting the medium properties. The material of the medium reservoir may be either an inorganic material such as glass or metal, or an organic material such as polypropylene, polystyrene, polyvinyl chloride, polyacryl and polycarbonate, preferably using a sterilizable glass or organic material. It is also possible to coat different materials on the surface of these materials, but more preferably, surface smoothing or hydrophilic treatment is performed to suppress protein adsorption to the reservoir surface. Use material. The capacity of the medium reservoir is not particularly limited.

(Desorption / separation mechanism of culture vessel)
The detachment / separation mechanism of the culture vessel used in the present invention can sterilize the culture medium perfusion type culture apparatus when collecting the cells after culturing, and can also collect the cells as they are in the apparatus. As in the present invention, the mechanism for holding or fixing the culture container is made detachable and the culture container can be separated from the culture module. It is possible to transport the cells to a space where the cells can be operated, such as in a clean bench, and collect the cells.

  Further, as a cell operation, it is possible to collect more cells from the culture container by collecting the culture container after shaking it with a vortex mixer or the like.

(Port for cell seeding and recovery)
The port for seeding and collecting cells in the present invention is a connector for taking cells into and out of the cell growth space in the culture vessel. The culture vessel is separated into a cell culture part and a medium perfusion part, but this seeding and port or recovery port is connected to the cell culture part. Seeding and collection can be performed without leaking cells. In the sterilized medium perfusion type culture apparatus, it is preferable to set the port for seeding and collecting the culture container so as to face the operation side (or the opening direction) of the medium perfusion type culture apparatus. It becomes easy to seed and collect cells in the culture apparatus.

  As described above, the port may include two types of ports, a seeding port for seeding cells and a recovery port for recovering cells. One port may have both functions of seeding and collecting cells. That is, the same port can be used for both cell seeding and collection.

  In addition, when inserting cells from a syringe used for injection or the like, the shape of the port portion may be a connector that fits the syringe needle insertion portion. The use of a stabable stopper is a preferred embodiment.

(Types of cells to be cultured)
In the present invention, any of adherent cells and suspension cells can be used as the cells cultured in the culture module. For example, NIH3T3 cells, Hela cells, COS cells, HEK cells, L929 cells are used as animal cells. , Daudi cells, Jurkat cells, cell lines such as KG-1a cells, various hybridoma cell lines or animal-derived blood cells and nerve cells, hepatocytes, keratinocytes, fibroblasts, epithelial cells, hepatocytes, etc. Examples thereof include cultured cells or insect cells. Furthermore, stem cells and progenitor cells such as ES cells, hematopoietic stem cells, hepatic stem cells and mesenchymal stem cells may be used. In addition, these cells may be cells into which a foreign gene has been introduced before culturing, or may be cells that have been previously stimulated and processed with stimulating factors such as antibodies and ligands.

(Conditions for culture medium)
The medium for cell culture using the culture module of the present invention is appropriately used depending on the type of cells used, but MEM medium, BME medium, DME medium, α-MEM medium, IMEM medium, ES medium, DM-160. Examples include, but are not limited to, a medium, a Fisher medium, an F12 medium, a WE medium, an RPMI medium, a StemSpan medium, a StemPro medium, a Hybridoma SFM medium, and a mixture thereof. In addition, during culture, for example, serum such as bovine serum, fetal bovine serum, horse serum, human serum, cytokines such as interleukin, interferon, insulin, transferrin, and selenium, and growth factors can be added. . Moreover, when using it for the use which manufactures the useful substance secreted from a cell, since these additives become impurities, it is preferable to add as little as possible.

(Method for producing cell preparation)
The culture apparatus using the culture module of the present invention has a very low possibility of mixing foreign substances from the outside, and can perform cell culture at low cost. Therefore, it is advantageous for culturing medical cells, and it is also possible to produce cell preparations by culturing cells that are effective against diseases and diseases. Cell preparations refer to drugs and medical devices processed from tissues and cells. Cell preparation manufacturing methods include cell isolation, cell proliferation, cell stimulation, cell differentiation induction, cell apoptosis induction, etc. It includes all processes for processing cells into cell preparations that are effective for diseases and diseases.

  In order to produce a cell preparation, it is first necessary to collect a tissue, a body fluid, or the like that is a supply source of a cell group. The source of these cell groups is preferably human, but is not limited thereto. In addition, a cell group including stem cells and immune cells is preferable as a source, and as a source of such a cell group, umbilical cord blood, bone marrow fluid, amniotic tissue, placental tissue, gonad, fetal tissue, peripheral blood and G-CSF mobilization Although peripheral blood etc. are mentioned, it is not limited to these. When using body fluids, etc. as a supply source, it is common to obtain a uniform cell group that excludes extra components in cell culture by centrifugation, unit gravity sedimentation, and centrifugal sorting prior to culture. is there. Furthermore, it is preferable to make a subset of high-purity stem cells and certain immune cells using cell separation methods such as flow cytometry, magnetic bead method, and affinity column method before culturing.

  After performing such various processes, by performing cell culture using the culture module of the present invention, cells necessary as a cell preparation can be obtained with high purity.

(Method for producing physiologically active substance)
The culture apparatus using the culture module of the present invention can also produce a useful physiologically active substance by culturing cells that secrete a physiologically active substance useful for diseases and disorders. A physiologically active substance is a chemical substance that has some specific action on the physiology and behavior of a living organism in a small amount. -There are cytokines such as CSF, and growth factors such as FGF and EGF.

  For these physiologically active substances, cells that secrete physiologically active substances useful for diseases and disorders are cultured using the culture module of the present invention, and the physiologically active substances secreted from the cells are purified from the culture supernatant. Can be manufactured. Examples of cells that secrete such useful physiologically active substances include primary cells such as fibroblasts, neurons and hepatocytes, and hybridomas obtained by fusing cancer cells with cells having specific differentiation traits. . In particular, a B cell hybridoma producing a monoclonal antibody can be preferably used.

  By culturing the above cells using the culturing module of the present invention, useful physiologically active substances secreted from the cells are accumulated in the culture supernatant. During this culture, a substance that promotes secretion of a useful physiologically active substance from the cells may be added to the culture solution. After culturing, the culture supernatant is useful by purifying the physiologically active substance using ammonium sulfate salting out, dialysis, ultrafiltration, or chromatographic techniques such as ion exchange chromatography, gel filtration chromatography, affinity chromatography, etc. A physiologically active substance can be produced. Further, when the secreted physiologically active substance is an antibody or the like, it can be purified by a method using an affinity carrier having affinity for the Fc part of the antibody such as protein A or protein G.

  Hereinafter, the present invention will be described in more detail with reference to the drawings, but the present invention is not limited to these examples. FIG. 1 is a schematic perspective view illustrating a culture module of the present invention, and FIG. 2 is an overall schematic perspective view illustrating a medium perfusion culture apparatus according to an embodiment of the present invention. In FIG. 2, the door on the front side of the culture medium perfusion culture apparatus is omitted.

(Culture container)
In a cylindrical polycarbonate culture container case having a diameter of 10 mm and a length of 120 mm, 95 polysulfone hollow fibers (diameter of 460 μm, film thickness of 80 μm, pore diameter of 0.1 μm) were filled to a filling rate of 20%, 1. A shape as shown in FIG. 1, wherein both ends are fixed to a culture vessel case with a polyurethane potting agent so as not to block the hollow portion of the polysulfone hollow fiber, and a seeding port 5a is provided on the upper side and a recovery port 5b is provided on the lower side. The culture container 1 was prepared.

(Culture container fixing part)
The culture vessel fixing holder 3 was fixed to the culture module frame 2 made of SUS304 for housing the culture vessel 1 with a bolt, and the culture medium channel clasp 4 was fixed by welding to prepare a culture vessel fixing portion. Further, as shown in FIG. 1, a culture module rotating shaft 8 is attached to the middle of one side of the culture module frame 2, and a culture having a diameter of 8 mm is placed on the opposite opening side frame 6a to which the rotating shaft 8 of the culture module is attached. A module frame opening 6b was provided. The culture module frame opening 6b is formed on the extension of the rotation shaft 8 of the culture module.

(Culture module)
As shown in FIG. 1, after attaching a silicon tube having an inner diameter of 2 mm and an outer diameter of 4 mm as the culture medium perfusion channel 7 to the culture container 1 produced as described above, the orientation of the seeding port 5a and the recovery port 5b of the culture container 1 Was attached from the opening side frame 6a of the culture module frame 2 so that the angle θ could be changed to 10 ° to 55, and the culture vessel 1 was sandwiched and fixed by the culture vessel fixing holder 3. The medium perfusion channel 7 is fixed by a medium channel clasp 4, and the medium inflow channel 7 a and the medium discharge channel 7 b extending from the medium perfusion channel 7 are connected to the extension of the culture module rotation shaft 8. It was taken out of the culture module frame 2 from the opposite culture module frame opening 6b. As a result, the medium inflow channel 7a and the medium discharge channel 7b were gathered and arranged on the rotating shaft 8 of the culture module.

(Culture circuit)
As shown in FIG. 2, the medium inflow channel 7a and the medium discharge channel 7b of the culture module prepared above were connected to the medium reservoir 9 and sterilized by an autoclave before use.

(Preparation before culture)
The culture medium perfusion culture apparatus 10 accommodates a culture module including the culture container 1, a culture medium reservoir 9, and a pump 11. The culture module rotating shaft 8 was connected to the culture module rotating mechanism of the sterilized culture medium perfusion culture apparatus 10, and the culture medium inflow channel 7 a was connected to the pump 11 of the culture medium perfusion culture apparatus 10.

(Culture method)
The temperature of the culture medium perfusion culture apparatus 10 is set to 37 ° C., and 100 mL of RPMI-1640 with 10% fetal bovine serum is placed in the culture medium reservoir 9 and suspended at 1 × 10 ⁶ cells / mL from the seeding port 5a of the culture container 1. 10 ml of the hybridoma was seeded and cultured for 7 days. The culture module was rotated by 90 ° every 5 minutes, and after rotating 270 °, the rotation direction was reversed.

After 3 days, 5 days and 6 days from the start of the culture, the medium in the medium reservoir 9 was changed. After 7 days, the culture vessel 1 was removed from the culture module, and the recovery port 5b and the cells were recovered in a clean bench. Cells could be grown to a cell density of 10 ⁸ cells / mL. In addition, the survival rate was 80% or more, and good results could be obtained.

The culture module of the present invention is suitably used in a culture apparatus such as a culture medium perfusion culture apparatus, can minimize the movement of the flow path, can prevent the flow path blockage or entanglement of the culture medium perfusion, The swirl space can be limited to a minimum, thereby reducing the size of the main body of the culture apparatus, which is useful for cell culture.

FIG. 1 is a schematic perspective view illustrating a culture module of the present invention. FIG. 2 is an overall schematic perspective view illustrating a culture medium perfusion culture apparatus according to an embodiment of the present invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Culture container 2 Culture module frame 3 Culture container fixed holder 4 Medium flow path clasp 5a Seeding port 5b Collection port 6a Opening side frame 6b Culture module frame opening 7 Medium perfusion flow path 7a Medium inflow flow path 7b Medium discharge flow Path 8 Rotation axis of culture module 9 Medium reservoir 10 Medium perfusion culture apparatus 11 Pump

Claims (6)

  A culture vessel comprising at least a culture vessel that is a cell growth space, a medium perfusion channel for supplying and discharging a culture medium to the culture vessel, and a member for holding or fixing these culture vessel and the medium perfusion channel The culture module is configured such that the culture module is rotatable or reversible around a rotation axis, and a medium inflow channel and a medium discharge channel to the culture module are collectively arranged on the rotation axis. A culture module characterized by being made.   The culture module according to claim 1, wherein the culture container is detachable from a member for holding or fixing the culture container.   The culture module according to claim 1 or 2, wherein the culture container comprises a port for seeding and collecting cells.   The culture module according to claim 3, wherein the orientation of the port for seeding and collection is configured to be changeable.   The culture module according to any one of claims 1 to 4, which is used in a medium perfusion culture apparatus for culturing cells.   6. The culture module according to claim 5, wherein the direction of the port for seeding and recovery is directed to the operation side of the culture medium perfusion type culture apparatus.

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