JP2006345778A - Hollow fiber module for culturing cell and method for culturing the cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a hollow fiber module capable of increasing productivity of cell culture, by efficiently recovering cultured cells, when the cell culture is conducted by using the hollow fiber module, and to provide a method for culturing cells using the same. <P>SOLUTION: This hollow fiber module is used for culturing the cells and has a hollow fiber bundle which is made of a material having a function as a semipermeable membrane and is used for supplying the cells existing in an extracapillary space (ECS) with an essential component for growth, by circulating a culture solution through an intracapillary space, and a housing for receiving the hollow fiber bundle. Further, the hollow fiber module for culturing the cells has such a structure that an optional part of the housing is detachable and attachable and at least a part of the hollow fiber bundle is exposed to the outside. The method for culturing the cells uses the module. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a hollow fiber module used for culturing cells, and a cell culturing method using the hollow fiber module for cell culture.

  Conventionally, organ transplantation and artificial organ implantation have been performed as a useful treatment method for chronic organ dysfunction disease, but organ transplantation is a serious medical problem such as rejection and administration of immunosuppressive agents, and serious problems. Accompanying social problems such as lack of donors. In addition, the artificial organ is not a satisfactory therapeutic means in terms of biocompatibility and biofunction substitutability such as functionality or convenience.

  Regenerative medicine has attracted attention as a method for solving such problems. Regenerative medicine is useful as a method for regenerating biological tissues such as organs that have become dysfunctional, and has already been put to practical use in the field of skin and bone, and has been applied to clinical applications worldwide. However, regarding organs, clinical application has not yet been achieved. This is because organ formation requires a large amount of biological tissue cells, but at present, a technique for mass culture of biological tissue cells has not been established. Therefore, in order for regenerative medicine to be widely applicable to clinical practice, it is necessary to develop a mass culture technique for cells.

  In recent years, with the technological development of genetic recombination or hybridomas, mass cell culture methods for producing various human-derived bioactive proteins such as interferon and erythropoietin have been developed. Such a cell culture method uses animal cells that proliferate under aerobic conditions, and is generally a technique for performing cell culture by the jar fermenter method. However, such a technique is a cell culture technique as a means for producing a pharmaceutical raw material, and is not a method of using cells that express and maintain the physiological characteristics or functions unique to the cells.

  On the other hand, a three-dimensional culture method has been proposed as a biological tissue cell culture method for expressing cell-specific functions. Such a technique is to proliferate cells three-dimensionally using a cell adhesion matrix made of collagen or the like as a nucleus. For this purpose, a space for the cells to grow and a cell attached through the matrix to grow It is necessary to provide a carrier.

  It is also conceivable to apply the jar fermenter method to such a culture method. However, in the jar fermenter method, in addition to the possibility of physical damage of cells due to agitation, it is difficult to adequately prevent cross-contamination, so individual cell culture for each individual is required. It is not a suitable method for regenerative medicine.

  Therefore, a cell culture technique using a hollow fiber module has been proposed as a large-scale cell culture method having a cell adhesion carrier and suppressing physical damage and cross contamination of cultured cells.

  For example, in Patent Document 1, a semipermeable tubular membrane (capillary) bundle is enclosed in a glass shell to form a cell culture unit, and a nutrient medium is circulated through the capillary to proliferate cells attached to the capillary surface. Technology is disclosed.

  However, in the example of Patent Document 1, the cells are not separated from the capillary bundle, and the growth of the cells is mainly confirmed by a microscope or the naked eye. In order to confirm cell growth by staining, an example is described in which the medium is replaced with agarose and then the agarose with solidified cells is removed, but such removal is performed by breaking both ends of the cell culture unit. Has been done.

Further, in Patent Document 2, in cell culture using a hollow fiber membrane module, a polymer that undergoes a phase transition at a constant temperature is held on the surface of the hollow fiber membrane, and the cultured cells are easily detached from the surface of the hollow fiber membrane. Thus, a technique for facilitating the recovery of cultured cells is disclosed. However, this technique requires a treatment process on the surface of the hollow fiber. In addition, since the technology peels off cells attached to the surface of the hollow fiber due to temperature changes, it is difficult to uniformly change the temperature of all the hollow fibers in the module in a short time. May fluctuate.
JP-A-49-41579 JP-A-6-169755

  The present invention has been made in view of the above circumstances, and in cell culture using a hollow fiber module, it is possible to recover the cultured cells more efficiently than before and increase the productivity of the cultured cells. An object of the present invention is to provide a cell culture hollow fiber module and a cell culture method using the cell culture hollow fiber module, and particularly to provide means for obtaining a large amount of cells in the field of regenerative medicine.

  The hollow fiber module for cell culture of the present invention is a hollow fiber module for culturing cells, and is made of a material having a semipermeable membrane function, and a culture solution is placed in a hollow fiber internal space (hereinafter referred to as “lumen”). A hollow fiber bundle for supplying growth essential components to cells existing in a hollow fiber external space (hereinafter referred to as “ECS” (Extracapillary Space)) by circulation and a housing for storing the hollow fiber bundle are provided. In addition, an arbitrary part of the housing can be detached and at least a part of the hollow fiber bundle can be exposed to the outside. After cell culture, the hollow fiber bundle to which the cultured cells are attached can be easily exposed to the external environment without destroying the housing, so that it is easy to collect the cultured cells from the hollow fiber surface and gaps and other ECS, In addition, the cells can be recovered at a high recovery rate without causing physical damage to the cultured cells or loss such as dropping.

  The hollow fiber module has a sleeve inserted into a housing and a core rod, the sleeve is for holding the hollow fiber bundle by covering the hollow fiber bundle, and the core rod is a hollow fiber It is preferable to hold the hollow fiber bundle by fixing both ends together with the bundle in the sleeve. After cell culture, the hollow fiber bundle to which the cultured cells adhere can be taken out while being held by the sleeve and the core rod, so that physical damage and loss of the cultured cells can be further suppressed.

  The hollow fiber module is inserted into the housing to store the hollow fiber bundle, can be separated from the housing, and is an opening for directly collecting the cells attached to the hollow fiber. It is preferable to further have an inner housing having the inside of the housing. After the cell culture, the hollow fiber bundle to which the cultured cells adhere can be taken out in a state protected by the inner housing, so that physical damage and loss of the cultured cells can be further suppressed. Further, since the hollow fiber bundle is protected by the inner housing, damage to the hollow fiber bundle can be avoided when the hollow fiber bundle is housed in the housing for forming a module. Furthermore, since almost all of the ECS cultured cells can be reliably taken out of the housing as a lump, the cells can be easily and reliably collected.

  The housing preferably has removable caps at both ends. This is because the hollow fiber bundle in the hollow fiber module can be easily taken out after the cell culture.

  It is also a preferable aspect of the present invention that the housing has a window portion that can be opened and closed or removed for directly collecting cells attached to the hollow fiber in the body portion. After cell culture, the cultured cells can be collected without removing the hollow fiber bundle, so that physical damage and loss of the cultured cells can be further avoided.

  As a preferable aspect of the present invention, the housing may have a structure that can be separated into two or more at an arbitrary position of the body portion and at least a part of the hollow fiber bundle can be exposed to the outside. After cell culture, the cultured cells can be collected without removing the hollow fiber bundle, so that physical damage and loss of the cultured cells can be further avoided.

  The hollow fiber module of the present invention preferably has a volume ratio of ECS to hollow fiber of 1 or more and 20 or less. This is because by making the volume of the space portion appropriate, cell culture can be made efficient, and recovery from ECS after culture can be made easier.

  The hollow fiber used in the hollow fiber module of the present invention is one selected from the group consisting of regenerated cellulose, cellulose ester, polysulfone, polyethersulfone, polyacrylonitrile, polyvinyl alcohol, polystyrene, polymethyl methacrylate or polycarbonate, or 2 What consists of a mixture of a seed | species or more is preferable.

  Moreover, it is preferable that the hollow fiber has a surface subjected to a hydrophilic treatment. This is because cell affinity increases and membrane permeability of the culture medium also improves.

  The cell culturing method of the present invention is a method for cultivating cells using the above-described cell culture hollow fiber module, separating an arbitrary part of the housing, exposing at least a part of the hollow fiber bundle to the outside, It is characterized by directly collecting the cells attached to the yarn.

  With the hollow fiber module for cell culture and the cell culture method of the present invention, the hollow fiber bundle to which the cells cultured in the hollow fiber module are attached can be easily exposed to the external environment while avoiding physical damage or loss. Therefore, it becomes possible to collect cultured cells extremely efficiently. Accordingly, the productivity of cultured cells is improved, and useful cells such as biological tissue cells can be provided to regenerative medicine and the like, so that the present invention is extremely useful industrially.

  FIG. 1 shows a conventional hollow fiber module used for cell culture. As described above, the conventional hollow fiber module has a hollow fiber bundle in which a hollow fiber bundle is inseparably fixed using a sealing material inside a cylindrical housing, and caps are applied to both end portions of the housing. in use. Thus, by sealing the housing with the sealing material and the cap, the space portion (lumen) in the hollow fiber and the space portion (ECS) formed by the outer side of the hollow fiber and the housing are isolated from each other. . The lumen is connected to a tube connection tube for circulating the culture solution (hereinafter referred to as “culture solution conduit”), and the ECS is connected to the tube connection tube for supplying cells (hereinafter referred to as “cell input tube”). The cell culture can be performed by introducing cells into the module and circulating the culture solution. Since these spaces are isolated from each other, the cells do not flow out into the circulating medium. On the other hand, since the cells are supplied with sufficient nutrients and oxygen through the hollow fiber membrane, they can grow at a high density. Cultured cells densified in ECS adhere directly to the surface of the hollow fiber or indirectly to the surface of the hollow fiber via a cell attachment carrier.

  However, the conventional hollow fiber module can cultivate cells in the module, but the main purpose is to recover the compounds produced by the cells, and it is not a structure considering the recovery of the cells themselves. In both cases, the recovery rate of the cultured cells having adhesion was low, and it was difficult to effectively use the obtained cultured cells. For example, in order to recover cells from a conventional hollow fiber module, a reagent for peeling cells such as trypsin from the surface of the hollow fiber or the cell adhesion carrier is introduced from the cell loading conduit, and then the detached cells are used for cell loading. A method of washing out with a culture solution from a conduit was used. However, such a method requires the use of a detachment reagent that is a heterogeneous protein, and the cells remain on the hollow fiber surface or in the gaps, so that the cell recovery rate is not completely satisfactory. If the concentration of trypsin or the like is increased to increase the recovery rate or the treatment time is extended, the cells will be damaged. In addition, if the cells are directly collected from the surface of the hollow fiber in the conventional hollow fiber module, it is necessary to destroy the housing. However, if the destruction operation is performed, there is a risk of impacting the cells. There is also the risk of debris mixing into the cell mass and the risk of compromising sterility.

  Therefore, the present inventors have intensively studied the recovery of cultured cells from the hollow fiber module and found the present invention.

  That is, the hollow fiber module for cell culture of the present invention is a hollow fiber module for culturing cells, which is made of a material having a semipermeable membrane function, and is present in ECS by circulating a culture solution through a lumen. A hollow fiber bundle for supplying essential components for growth and a housing for storing the hollow fiber, and any part of the housing can be detached and at least a part of the hollow fiber bundle is exposed to the outside It is a structure that can be made.

  The cell culture method of the present invention is a method for embodying the cell culture method using the hollow fiber module of the present invention, and after culturing cells using the hollow fiber module for cell culture, a hollow fiber bundle And the housing are separated, and the cells attached to the hollow fiber are directly collected.

  Hereinafter, the hollow fiber module for cell culture and the cell culture method of the present invention will be described.

  In the present invention, “adhering to the hollow fiber” means that the cultured cell is directly attached to the surface of the hollow fiber or indirectly attached via a cell attachment carrier, and the hollow fiber gap or hollow fiber is attached. It also exists in the gap between the housing and the housing.

  The hollow fiber bundle of the present invention means one or two or more hollow fibers fixed in the length direction in the housing.

  The hollow fiber bundle used in the present invention is made of a material having a semipermeable membrane function, and is intended to provide a growth essential component to cells existing in ECS by circulating a culture solution through the lumen. Any hollow fiber bundle known to those skilled in the art can be used as long as it is such a hollow fiber bundle, and is not particularly limited.

  The semipermeable membrane function means a material having a function as a membrane that allows some components in the solution and the dispersion system to pass but does not allow other components to pass. For example, a porous membrane capable of changing the component to be passed depending on the pore diameter, or a membrane having functions similar to those of a biological membrane having various membrane passage functions as well as passage due to a concentration gradient can be used. Can be appropriately selected and used according to the type of cells or the culture medium.

  Such hollow fibers are considered from the group consisting of regenerated cellulose, cellulose ester, polysulfone, polyethersulfone, polyacrylonitrile, polyvinyl alcohol, polystyrene, polymethyl methacrylate, or polycarbonate in consideration of affinity with cells. It is preferable to consist of 1 type or the mixture of 2 or more types selected. In this illustration, the hollow fiber is made of one polymer means that all the hollow fibers are made of the polymer. Moreover, what consists of a mixture should just have a some polymer as a result of a hollow fiber. That is, each hollow fiber may be made of the mixture, or a part of the hollow fibers may be made of any one polymer of the mixture, and the other hollow fiber may be made of another arbitrary polymer. In some cases, a plurality of polymers are contained when the is formed, and is not particularly limited. Of these polymers, the hollow fiber of the present invention is more preferably composed of regenerated cellulose, cellulose ester, polysulfone, or polyethersulfone. In culturing cells, it is necessary to sterilize the hollow fiber module in advance. Therefore, by using regenerated cellulose, cellulose ester, polysulfone, or polyethersulfone having high durability against heat, high-pressure steam sterilization is possible, and sterilization can be performed efficiently.

  The hollow fiber is more preferably one whose surface has been subjected to a hydrophilic treatment. This is because by performing the hydrophilization treatment, the cell affinity is increased and the membrane permeability of the culture solution is also improved. Examples of the hydrophilization treatment method include treatment with polyvinylpyrrolidone, ethylene vinyl alcohol copolymer, polyvinyl acetal diethylaminoacetate, alcohol, etc. Examples of alcohol include ethanol, isopropanol, and the like. Of these, polyvinylpyrrolidone treatment is preferred. This is because stable hydrophilicity can be imparted to the hollow fiber surface. In addition, by performing such a hydrophilic treatment, it becomes possible to use hydrophobic hollow fibers such as polypropylene and polyethylene.

  The pore diameter of the hollow fiber as a semipermeable membrane (hereinafter referred to as “membrane pore diameter”) is particularly limited as long as the culture solution can be passed from the hollow to the outside and the cells to be cultured cannot be passed. It is not a thing. As the membrane pore size decreases, the membrane strength increases but the material exchange rate decreases. On the other hand, as the membrane pore size increases, the membrane strength decreases but the material exchange rate increases. Considering these, the average membrane pore diameter of the hollow fiber is preferably 0.001 to 2 μm, and more preferably 0.005 to 0.2 μm. In addition, when it is necessary to add various cell growth factors and differentiation inducing factors, these factors can be encapsulated in ECS, and considering that the cost can be greatly reduced, the pore size is 0.005. More preferably, it is -0.01 micrometer.

  The inner diameter of the hollow fiber is not particularly limited as long as the culture medium can be circulated, but a thin hollow fiber is preferable from the viewpoint of ensuring high membrane permeability, and is 0.1 to 3 mm. be able to. In consideration of ease of processing and suppression of clogging caused by insoluble components generated in the culture solution, 0.5 to 1 mm is more preferable.

  In addition, the number of hollow fibers and the film thickness are not particularly limited as long as the cell culture of the present invention is possible, but as will be described later, the required number of cells, nutrients of cultured cells, oxygen requirement, It is preferable to adjust appropriately according to the relationship with the volume of ECS in the hollow fiber module.

  The hollow fiber bundle can be fixed by gluing and fixing the gap between the hollow fibers using a generally used sealing material. The sealing material to which the hollow fiber bundle is fixed is called a sealing material block. Examples of the sealing material include resins known to those skilled in the art, such as urethane resins and epoxy resins.

  The hollow fiber of the present invention having such a semipermeable membrane function is for supplying a growth essential component to cells existing in ECS by circulating a culture solution through a lumen and diffusing and removing metabolic components.

  As long as the said culture solution contains the growth essential component of the cell to culture | cultivate, any conventionally well-known culture solution can be used and it does not specifically limit. The essential component for cell growth is a component indispensable for the cells to be cultured to proliferate, and is composed of various organic and inorganic substances, including dissolved gas for supplying oxygen and the like. As the essential growth component, for example, a medium prepared by adding serum, various growth factors, or differentiation-inducing factors to a general-purpose medium such as Dulbecco MEM, RPMI 1640, and Ham F12 is used.

  Note that the culture solution is stored in a suitable container and is circulated through the lumen repeatedly with a pump, but this does not exclude the temporary supply of the culture solution. In the present invention, the “circulation” of the culture solution includes a transient supply.

<Housing>
The hollow fiber module of the present invention has the above hollow fiber bundle and a housing for storing the hollow fiber bundle, and an arbitrary part of the housing can be detached and at least a part of the hollow fiber bundle is externally provided. It has a structure that can be exposed.

  The housing is for storing a hollow fiber bundle, and can perform cell culture in a space formed by the hollow fiber and the housing. That is, the housing can be sealed, and the lumen and ECS are formed by sealing. The lumen is provided with a culture fluid conduit, and the ECS is provided with a cell introduction conduit. Cell culture can be performed by introducing cells into the ECS of the module and circulating the culture fluid. Since these spaces are isolated from each other, the culture solution and the cells are not in direct contact with each other, and only the components in the culture solution that have passed through the membrane hole of the hollow fiber having a semipermeable membrane function are in contact with the cells.

  The material of the housing is not particularly limited, and examples thereof include polycarbonate, polysulfone, polypropylene, polystyrene, polyethylene, and acrylic resin. Of these, polycarbonate and polysulfone are preferred. Since these materials have high heat resistance, high-pressure steam sterilization is possible, and efficient sterilization can be performed. Moreover, since the transparency is high, the cultured state of the cells can be easily confirmed by the naked eye.

  The phrase “any part of the housing can be detached” means that any part of the housing can be separated from the housing, and any part of the separated part can be attached to the housing. “Desorption” includes opening and closing the optional portion while keeping it in contact with the other portion, or removing the arbitrary portion so as to be completely separated from the other portion.

  Further, "at least a part of the hollow fiber can be exposed to the outside" means that the hollow fiber bundle sealed in the hollow fiber module and cut off from the external environment separates an arbitrary part of the housing, It means that at least a part can be exposed to the external environment. The hollow fiber bundle is not particularly limited as long as it is at least partially exposed to the external environment, and the entire hollow fiber bundle may be exposed. Such an aspect should be appropriately set as necessary.

  Thus, by allowing the hollow fiber bundle to be exposed to the external environment, it is possible to facilitate the collection of the cultured cells.

  In the present invention, recovering cultured cells means recovering cells directly attached to the surface of the hollow fiber or indirectly through a cell attachment carrier, and the cells or hollow fibers existing in the hollow fiber gap and the housing Recovery of cells present in the interstitial space.

  That is, since the hollow fiber module of the present invention has the housing structure described above, the cultured cells attached to the hollow fiber are exposed to the external environment to an extent sufficient for physical recovery such as scraping, so that the cultured cells are damaged. It can be recovered very easily by physical means without inducing. Further, such a housing structure allows such cells to flow out and be recovered very easily even when cells that can be recovered from the housing are included.

  In addition, the recovery rate can be further improved by adding chemical treatment such as enzyme treatment during cell collection. However, the enzyme usually used for such chemical treatment is an enzyme derived from animals such as pigs and is a foreign substance for a living body. Therefore, when it is applied to regenerative medicine, it is dangerous if it is mixed in cultured cells and transplanted into a living body, and we want to avoid it as much as possible. In the case of the present invention, such chemical treatment is only an auxiliary means, and does not require strong treatment conditions as in the case of conventional hollow fiber modules, and thus light treatment that does not damage cultured cells. The cell recovery rate can be improved.

  The housing is not particularly limited in shape and size as long as it satisfies the above requirements, and can be appropriately set as necessary, but the dead space in the module, cell culture, In consideration of ease of operation, a cylindrical housing is preferable.

  Hereinafter, as preferred embodiments of the housing, embodiments in which the hollow fiber bundle is separated from the housing are exemplified in (1) and (2), and embodiments in which the hollow fiber bundle is not separated from the housing are exemplified in (3) and (4).

  In the aspects (1) and (2), it is preferable to have caps at both ends of the housing. By attaching removable caps to both ends of the housing, a lumen can be formed. By removing the cap after cell culture, it becomes possible to take out the hollow fiber bundle to which the cultured cells are attached from the housing. The yarn bundle can be completely exposed to the external environment. As a result, cultured cells can be easily recovered from the hollow fiber bundle. Any cap known to those skilled in the art can be used as the cap as long as it can be attached to and removed from both ends of the housing, and examples thereof include a screw cap and a fitting cap. Although the material of the said cap is not specifically limited, For example, the material similar to the said housing can be mentioned.

(1) It has a sleeve and a core rod which are inserted into the housing, and the sleeve is for holding the hollow fiber bundle by covering the hollow fiber bundle. This is an embodiment for holding the hollow fiber bundle by fixing the portion in the sleeve (FIG. 2).

  The “holding” means that the hollow fiber bundle is maintained in a certain shape. The lumen and ECS can be easily formed by inserting a hollow fiber bundle held by a sleeve and a core rod into the housing and capping the hollow fiber bundle. In addition, after cell culture, after removing the cap, the hollow fiber bundle with the cultured cells attached can be taken out from the housing while being held by the sleeve and the core rod, and the hollow fiber bundle can be exposed to the outside. The cultured cells attached to the hollow fiber can be easily recovered. As a result, the hollow fiber bundle can be taken out while being protected by the sleeve and the core rod and the shape stability of the hollow fiber bundle is maintained, and loss due to physical damage or dropping of cultured cells can be avoided. .

  As shown in FIG. 2, the sleeve has a shape corresponding to the shape of the housing, and one end of both ends can be flush with the sealing material and the hollow fiber bundle, and the other side can cover the sealing material. It is cut to an arbitrary length in size. At both ends of the sleeve, both ends of the hollow fiber bundle are glued and fixed together with the sealing material, and are integrally formed. In this way, the hollow fiber bundle is held by the sleeve. The outer diameter of the sleeve is slightly smaller than the inner diameter of the housing so that it can be taken in and out. The gap between the sleeve and the housing is closed via a packing such as an O-ring or a gasket to form an ECS.

  Further, as shown in FIG. 2, the core rod is inserted between the hollow fibers, glued and fixed to the sleeve using a sealing material together with both ends of the hollow fiber bundle, and both ends of the hollow fiber bundle formed integrally with the sleeve. Is for fixing. Thus, the hollow fiber bundle is held by the core rod. Examples of the material of the core rod include the same material as the housing or a metal such as stainless steel. The number and the number of core rods may be one at the center, and a plurality of core rods may be inserted between the hollow fiber bundles.

(2) An internal housing that is inserted into the housing for storing a hollow fiber bundle, can be separated from the housing, and has an opening for directly collecting cells attached to the hollow fiber In the housing (FIGS. 3 and 4).

  Lumen and ECS can be easily formed by inserting the internal housing into the housing (hereinafter referred to as “external housing” in the description of this embodiment) and capping the housing. Further, after the cell culture, after removing the cap, the hollow fiber bundle to which the cultured cells are attached can be taken out from the outer housing while being protected by the inner housing. As a result, the hollow fiber bundle can be exposed to the outside through the opening of the inner housing, and the cultured cells attached to the hollow fiber can be easily recovered. Furthermore, since the hollow fiber bundle to which the cultured cells are attached can be taken out after the cell culture while being protected by the inner housing, physical damage and loss of the cultured cells can be further suppressed. Further, since the hollow fiber bundle is protected by the internal housing, damage to the hollow fiber can be avoided when the hollow fiber bundle is housed in the housing for forming a module. Since almost all of the ECS cultured cells can be reliably taken out of the housing as a lump, the cells can be easily and reliably recovered.

  The inner housing has an opening in the body side surface, has the same shape as the outer housing, and may be any one that can be inserted into the outer housing to form a lumen and ECS.

  The length of the inner housing is the same as or shorter than the length of the outer housing to form a lumen and ECS. Examples of the material of the inner housing include the same materials as the outer housing. Further, the outer diameter of the inner housing is preferably slightly smaller than the inner diameter of the outer housing so that the inner housing can be taken in and out. A gap between the inner housing and the outer housing is closed through a packing such as an O-ring or a gasket.

  The side opening of the inner housing is preferably wider for collecting cells, but the minimum window frame frame is required to sufficiently hold the structure of the inner housing when the hollow fiber bundle is taken in and out of the outer housing. It is necessary to leave the structure. From such a viewpoint, it is preferable that the inner housing side surface area be set to 1/2 to 3/4.

  The shape and number of the openings are not particularly limited and can be appropriately set as necessary. However, in consideration of operability and structural strength, the openings are divided into two or four surfaces. Is desirable.

  The inner housing is more preferably one in which hooks are attached to the sealing material blocks at both ends (FIG. 4). This is because it is easier to remove the inner housing. The hook may be made of any material as long as it can withstand the force of taking out the inner housing. The size may be any size as long as it does not prevent the outer housing from being sealed. In addition, it is possible to provide a depression opening on a part of the sealing material block so that a drawer key bar can be hooked.

(3) The above-mentioned housing is an embodiment in which the body portion has an openable / closable or removable window portion for directly collecting cells attached to the hollow fiber (FIG. 5).

  That is, a window hole is formed at an arbitrary position of the body portion of the housing, and a window portion for closing the window hole is provided. By closing or mounting the window portion, the lumen and ECS can be easily formed. Moreover, the hollow fiber bundle can be easily exposed to the external space by opening or separating the window portion. As a result, since the hollow fiber bundle is not moved from the housing when the cells are taken out, loss due to physical damage or dropping of the cultured cells attached to the hollow fibers can be further avoided.

  Although the material of the said window part is not specifically limited, For example, the material similar to the said housing can be mentioned.

  Examples of the window portion that can be attached include those attached to the housing by bolts, screws, hooks, belt tightening, and the like. Moreover, as what can be opened and closed, what was attached to the said housing using the hinge etc. can be mentioned.

  The size of the window portion may be any size as long as cells can be collected, and is not particularly limited. However, in order to maintain the structural strength and hermeticity of the housing, it is ½ of the side surface area of the housing. In view of operability, it is preferably 1/5 or more.

(4) It is an aspect in which the housing has a structure that can be separated into two or more at an arbitrary position of the body portion and at least a part of the hollow fiber bundle can be exposed to the outside (FIGS. 6 and 6). 7).

  The housing is divided into two or more at an arbitrary position of the body part, and the two or more housings are combined and fixed to form a lumen and an ECS. In addition, after the cell culture, by releasing the fixation, the housing can be divided into two or more, and a part of the hollow fiber bundle can be exposed to the external environment. As a result, the cultured cells attached to the hollow fiber in each housing can be recovered from the space formed by the division. Further, since the hollow fiber bundle is not moved from within the housing, loss due to physical damage or dropping of cultured cells attached to the hollow fiber can be further avoided.

  Examples of the two or more housings include those divided perpendicularly to the length direction of the housing and those divided obliquely. FIGS. 6 and 7 show a mode in which it is divided into two parts and perpendicular to the housing length direction.

  Any method known to those skilled in the art can be used as the fixture for fixing the two or more housings, and is not particularly limited. For example, a clamp band (FIG. 6), a union nut (FIG. 7), etc. Can be mentioned. Moreover, it is preferable that both housings are fixed through packing such as an O-ring and a sanitary gasket. It is because it can seal more completely.

<Hollow fiber module>
The hollow fiber module of the present invention is formed from the above-described hollow fiber bundle and a housing, and is used for culturing cells in ECS.

  The volume ratio of ECS with respect to the hollow fiber bundle can be appropriately set as required, such as cell type and cultured cell density, but is preferably 1 or more and 20 or less. By setting the number to 1 or more, it is possible to increase the amount of cells put into the module and to sufficiently supply a space in which cells grow. Moreover, by setting it to 20 or less, it becomes possible to avoid the shortage of supply of the essential component for growth to the cells in the module. In order to perform cell culture most suitably, 2 or more and 6 or less are preferable. The volume of the hollow fiber bundle in the above volume ratio is the volume of each hollow fiber that contacts the ECS in the module, and the cross-sectional area (including the hollow part) formed by the outer periphery of the hollow fiber cross section and the hollow It can be expressed as the sum of products of yarn lengths.

  It is preferable to appropriately set the number of the hollow fiber bundles so as to achieve such a volume ratio.

<Cell culture method>
As the cell culture method using the hollow fiber module of the present invention, any cell culture method known to those skilled in the art can be used, and is not particularly limited. From the viewpoint of culturing and applying to regenerative medicine, a three-dimensional culture method is preferable.

  Examples of the three-dimensional culture method include a microcarrier method and a gel embedding method. The microcarrier method is a method of culturing by attaching cells to beads made of glass, gelatin, cellulose or the like. The gel embedding culture method is a method of culturing cells three-dimensionally in collagen or agarose.

  In these cell culture methods, cells can be cultured by introducing a microcarrier or gel together with the cells from the cell introduction conduit and circulating the above-mentioned culture solution through the lumen. In addition, culture conditions such as culture medium circulation rate, presence / absence of medium exchange, and oxygen addition method are appropriately set according to the cell type, cell concentration, and the like. After cell culture, the cultured cells can be collected by the method described above.

  The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to the following examples, and may be appropriately modified and implemented within a range that can meet the purpose described above and below. All of which are within the scope of the present invention.

Experimental example 1
Uses mixed material cellulose ester, hollow fiber bundle with inner diameter of 0.6mm, pore diameter of 0.1μm (Spectrum), polycarbonate cylindrical housing and inner housing, and polycarbonate screw cap, with urethane resin as sealing material A hollow fiber module shown in FIG. 3 was prepared.

  The lumen of the hollow fiber module includes 5% FBS (Fetal Bovine Serum), FGF-2 (Fibroblast Growth Factor-2), IGF-I (Insulin-like Growth Factor-I), insulin, transferrin, selenic acid, and the like. 300 mL of chondrocyte growth medium (Bullet kit Chronocyte Growth Medium, manufactured by Cambrex) was refluxed through a culture medium conduit.

  Next, chondrocytes (derived from human auricular cartilage, 200,000 cells) were embedded in 10 mL of 0.3% siatelopeptide collagen and added to ECS through a cell input conduit. After the addition, the conduit was sealed and cultured.

  The culture was performed in a thermostatic chamber set at 37 ° C., maintaining the pH of the culture solution at 7 and the oxygen partial pressure at 20%.

  Six weeks later, collagenase was added from the cell input conduit, and collagenase treatment was performed to weaken the adhesion to the hollow fiber. After the treatment, the module cap was removed, the inner housing was taken out, and the cultured cells adhering to the hollow fiber were scraped and collected from the opening. After collection, few cells remained in the module.

Experimental example 2
Using a hollow fiber bundle similar to Experimental Example 1 and using a conventional hollow fiber module (CellMax, manufactured by Spectrum) as shown in FIG. Cell culture was performed under the same conditions.

  After culturing for 6 weeks, collagenase was added from the cell input conduit, collagenase treatment was performed, and recovery of free cells from the cell input conduit was attempted, but it was not possible to recover much. Thereafter, the housing body was smashed with a hammer, and the cultured cells were collected by scraping from the destroyed part. However, it was difficult to destroy the housing in such a way that the cells could be easily collected, and cultured cells remained in the module. In addition, the housing fragments were mixed into the remaining cells by the destruction operation. Thereafter, trypsin treatment was performed on the remaining cultured cells to further weaken the adhesion of the cultured cells and recovery was attempted, but the remaining cells could hardly be recovered.

Evaluation Example FIG. 8 shows the results of measuring the number of cultured cells collected in Experimental Examples 1 and 2 above. In addition, the number of cells at the start of culture is indicated by a dotted line.

  As shown in FIG. 8, the number of cells after recovery was about 1.5 million in Experimental Example 2 using the conventional module, whereas the number of cells was significant at 2.2 million in Experimental Example 1 using the module of the present invention. It was found that the recovery rate of the module of the present invention was significantly higher.

  In fact, considering that only cultured cells collected from the module can be used as useful cells, the number of cells after culturing compared to before culturing is substantially the growth rate of the entire cell culture system. Can be considered. From the above experimental example, the growth rate with respect to the number of cells of 200,000 at the start of culture was about 7 times in Experimental Example 2, whereas it was about 11 times in Experimental Example 1 of the present invention. Therefore, it was found that the substantial cell growth rate was significantly higher when the hollow fiber module of the present invention was used.

  As described above, since the hollow fiber module of the present invention has a significantly higher culture cell recovery rate than the conventional hollow fiber module, the substantial cell growth rate of the entire cell culture system is also significantly high. Therefore, it is confirmed that the cell culture method using the hollow fiber module for cell culture of the present invention can cultivate cells useful as a substrate for various body tissues required for regenerative medicine more efficiently than conventional methods. It was done.

It is a schematic cross section which shows an example of the conventional hollow fiber module for cell cultures. It is a schematic cross section which shows an example of the hollow fiber module for cell cultures of this invention. It is a schematic cross section which shows an example of the hollow fiber module for cell cultures of this invention. It is a schematic cross section which shows an example of the hollow fiber module for cell cultures of this invention. It is a schematic cross section which shows an example of the hollow fiber module for cell cultures of this invention. It is a schematic cross section which shows an example of the hollow fiber module for cell cultures of this invention. It is a schematic cross section which shows an example of the hollow fiber module for cell cultures of this invention. It is a graph which shows the cell number of an Example.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Hollow fiber 2 Housing 3 Sealing material 4 Cap 5 Culture solution conduit 6 Cell input conduit 7 O-ring 8 Core rod 9 Sleeve 10 Inner housing 11 Opening portion 12 Hook 13 Window portion 14 Bolt 15 Clamp band 16 Union nut

Claims (10)

A hollow fiber module for culturing cells,
A hollow fiber bundle made of a material having a semipermeable membrane function, for supplying a growth essential component to cells existing in the hollow fiber outer space (hereinafter referred to as “ECS”) by circulating a culture solution in the hollow fiber inner space. When,
A housing for storing the hollow fiber bundle, and
A hollow fiber module for cell culture, characterized in that an arbitrary part of a housing can be detached and at least a part of a hollow fiber bundle can be exposed to the outside. Having a sleeve and core rod inserted into the housing;
The sleeve is for holding the hollow fiber bundle by covering the hollow fiber bundle,
The hollow fiber module for cell culture according to claim 1, wherein the core rod is used for holding the hollow fiber bundle by fixing both ends thereof together with the hollow fiber bundle in the sleeve.   An internal housing which is inserted into the housing and for storing a bundle of hollow fibers, can be separated from the housing, and has an opening for directly collecting cells attached to the hollow fibers. The hollow fiber module for cell culture according to claim 1, further comprising:   The hollow fiber module for cell culture according to any one of claims 1 to 3, wherein the housing has caps that can be removed at both ends.   2. The hollow fiber module for cell culture according to claim 1, wherein the housing has a window part that can be opened / closed or removed to directly collect cells attached to the hollow fiber in the body part. 3.   The hollow fiber module for cell culture according to claim 1, wherein the housing has a structure that can be separated into two or more at an arbitrary position of the body part and at least a part of the hollow fiber bundle can be exposed to the outside.   The hollow fiber module for cell culture according to any one of claims 1 to 6, wherein the volume ratio of ECS to the hollow fiber bundle is 1 or more and 20 or less.   The hollow fiber is composed of one or a mixture of two or more selected from the group consisting of regenerated cellulose, cellulose ester, polysulfone, polyethersulfone, polyacrylonitrile, polyvinyl alcohol, polystyrene, polymethyl methacrylate or polycarbonate. Item 8. A hollow fiber module for cell culture according to any one of Items 1 to 7.   The hollow fiber module for cell culture according to claim 1, wherein the hollow fiber has a surface hydrophilized.   After culturing cells using the hollow fiber module for cell culture according to any one of claims 1 to 9, an arbitrary part of the housing is separated, and at least a part of the hollow fiber bundle is exposed to the outside and attached to the hollow fiber A method for culturing cells, wherein the collected cells are directly collected.

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