JP2012044908A - Hollow fiber module for cell culture and method for cell culture - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prevent collection loss of cultured cells.SOLUTION: The hollow fiber module for cell culture is composed of a biodegradable material that includes more than one hollow fiber having a function as a semipermeable membrane. The module also includes: a hollow fiber bundle 30 for supplying the cells existing in an extracapillary space (ECS) with an essential component for growth, by supplying a culture solution to an internal space of hollow fiber; and a housing for storing the hollow fiber bundle 30. The module has a structure that an optional part of the housing 20 is detachable and at least a part of the hollow fiber bundle 30 is exposed to the outside. There is also provided a method for cell culture using the hollow fiber module.

Description

  The present invention relates to a hollow fiber module for cell culture and a cell culture method.

  Conventionally, organ transplantation and artificial organ implantation have been performed as useful treatment methods for chronic organ dysfunction diseases. However, organ transplantation involves medical problems such as rejection and administration of immunosuppressants, and social problems such as a serious shortage of donors. In addition, an artificial organ is not a satisfactory therapeutic means in terms of biological function substitutability such as biocompatibility, functionality, and 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, mass cell culture methods for producing various human-derived bioactive proteins such as interferon and erythropoietin have been developed with the technological development of genetic recombination or hybridoma. 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.

Thus, 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 (see Patent Documents 1 to 3). Among the cell culture techniques using these hollow fiber modules, the technique described in Patent Document 3 is more effective than the techniques described in Patent Documents 1 and 2 for recovering cultured cells than before. And the productivity of cultured cells can be increased.

JP 49-419579 A JP-A-6-169755 JP 2006-345778 A

  However, in order to recover the cultured cells after culturing the cells in the hollow fiber module using the technique described in Patent Document 3, it is necessary to separate the cells attached to the hollow fibers from the hollow fibers. is there. This is because the hollow fiber itself is unnecessary in transplanting cultured cells. For this reason, a recovery loss of the cultured cells occurs during the separation operation. In addition, since it is necessary to separate the cultured cells from the hollow fiber in order to collect the cultured cells, the cultured cells are easily damaged by the separation process, and the activity of the cultured cells is likely to be reduced. .

  The present invention has been made in view of the above circumstances, and provides a cell culture hollow fiber module capable of suppressing a recovery loss of cultured cells, and a cell culture method using the cell culture hollow fiber module. The issue is to provide.

The above-mentioned subject is achieved by the following present invention. That is,
The hollow fiber module for cell culture according to the present invention is made of a biodegradable material, includes at least one hollow fiber having a semipermeable membrane function, and supplies a culture solution to the internal space of the hollow fiber. It has at least a hollow fiber bundle for supplying a growth essential component to cells existing in the external space (ECS) and a housing for storing the hollow fiber bundle, and an arbitrary part of the housing is removable. In addition, at least a part of the hollow fiber bundle has a structure that can be exposed to the outside.

  In one embodiment of the cell culture hollow fiber module of the present invention, the biodegradable material is (i) polylactic acid, polyglycolic acid, polycaprolactone, aliphatic polyester, polyamide, (ii) a polymer material shown in (i) Polymerization of a mixed material containing at least two polymer materials selected from the group consisting of: and (iii) at least two polymer materials selected from the group consisting of polymer materials shown in (i) It is preferably a material selected from a copolymer obtained by copolymerizing each monomer used in the above.

  In another embodiment of the cell culture hollow fiber module of the present invention, the biodegradable material preferably has a weight average molecular weight in the range of 30,000 to 500,000.

  In the cell culture method of the present invention, after culturing cells using the hollow fiber module for cell culture of the present invention, an arbitrary part of the housing is separated, and at least a part of the hollow fiber bundle is exposed to the outside, The cells attached to the yarn are collected while attached to the hollow fiber.

  In one embodiment of the cell culture method of the present invention, the cell is preferably a chondrocyte.

  ADVANTAGE OF THE INVENTION According to this invention, the cell culture hollow fiber module which can suppress the collection | recovery loss of a cultured cell, and the cell culture method using the said hollow fiber module for cell culture can be provided.

It is a schematic diagram which shows an example of the cell culture hollow fiber module of this embodiment. 2 is a phase contrast microscopic image showing biodegradability in a living body of a porous body having the same weight average molecular weight as the hollow fiber used in the cell culture test of Example 1. FIG.

(Cell culture hollow fiber module)
The hollow fiber module for cell culture of the present embodiment (hereinafter sometimes abbreviated as “module”) is made of a biodegradable material, includes at least one hollow fiber having a semipermeable membrane function, and the hollow fiber. Grows into cells existing in the external space of the hollow fiber (hereinafter referred to as “ECS”) by supplying a culture solution to the internal space (hereinafter sometimes referred to as “lumen”) It has at least a hollow fiber bundle for supplying essential components and a housing for storing the hollow fiber bundle, and any part of the housing is removable, and at least a part of the hollow fiber bundle is external. It has the structure which can be exposed to.

  Cell culture using the module of the present embodiment is performed by supplying a culture solution into the lumen while the cells are arranged in the module. In addition, since the lumen and ECS are isolated from each other, the cells do not flow out into the culture solution. On the other hand, since cells can be supplied with sufficient nutrients and oxygen through the hollow fiber, they can proliferate at high density. And the cultured cell densified in ECS adheres to a hollow fiber. “Cultivated cells adhere to the hollow fiber” means that the cultured cells adhere directly to the surface of the hollow fiber or indirectly through a cell attachment carrier, and the gap between the hollow fibers or the hollow fiber. It also exists in the gap between the housing and the housing.

-Hollow fiber and hollow fiber bundle-
In the module of this embodiment, the hollow fiber is made of a biodegradable material. For this reason, even if this hollow fiber is disposed in the living body, the hollow fiber is decomposed in the living body and absorbed in the body, so there is no possibility of any adverse effects on the living body. Therefore, when cells are cultured using the module of this embodiment, the cultured cells can be collected in a state where they are attached to the hollow fiber. In this case, the cultured cells can be transplanted into the living body together with the hollow fiber in a state of being attached to the hollow fiber. Therefore, when cells are cultured using the hollow fiber module for cell culture of the present embodiment, compared to the technique described in Patent Document 3, the utilization efficiency of cultured cells that can be used for various treatments such as cell transplantation is further increased. Moreover, it can suppress that the activity of a cultured cell falls at the time of collection | recovery of a cultured cell.

The hollow fiber is made of a biodegradable material. A known biodegradable material can be used as this biodegradable material. However, considering the affinity with cells and the like, the biodegradable material is preferably selected from the materials shown in the following (i) to (iii).
(I) A mixed material containing at least two kinds of polymer materials selected from the group consisting of polylactic acid, polyglycolic acid, polycaprolactone, aliphatic polyester, polyamide (ii) (i) (iii) (iii) ) A copolymer obtained by copolymerizing each monomer used for polymerization of at least two kinds of polymer materials selected from the group consisting of polymer materials shown in (i)

  The hollow fiber is required to have excellent biocompatibility when the hollow fiber is transplanted into the living body together with the cultured cells, and the burden on the living body accompanying the decomposition of the material is small. In addition, in culturing cells, it is necessary to sterilize the hollow fiber module in advance. Therefore, the biodegradable material constituting the hollow fiber is excellent in biocompatibility, has a low burden on the living body due to material decomposition, and withstands high-pressure steam sterilization that enables effective and simple sterilization. It is necessary to have high heat resistance. From the viewpoint of satisfying these properties in a well-balanced manner, among the biodegradable materials listed above, each of the monomers used for polymerization of polylactic acid, polyglycolic acid, polycaprolactone, or these polymer materials is 2 A copolymer obtained by polymerizing more than one species is particularly preferable.

  Moreover, as a weight average molecular weight of a biodegradable material, the inside of the range of 30000-500000 is preferable, and the inside of the range of 70000-180000 is more preferable. By setting the weight average molecular weight to 30000 or more, the viscosity of the spinning raw material used for spinning the hollow fiber increases, and spinning becomes easy. In addition, by setting the weight average molecular weight to 500,000 or less, during the period required for culturing cells in the module (usually 6 months at the maximum), the hollow fiber significantly increases with the progress of degradation of the biodegradable material. It is suitable for transplantation treatment because rapid degradation is possible after transplantation into a living body while preventing a decrease in strength. Furthermore, when the hollow fiber is made of a cylindrical membrane and a semipermeable membrane function is exhibited by providing a hole penetrating in the thickness direction of the membrane, it is possible to prevent a significant increase in the viscosity of the spinning raw material. The formation of holes is facilitated.

  Moreover, it is more preferable that the hollow fiber has a hydrophilic surface. This is because the hydrophilic treatment increases the affinity for cells and also improves the membrane permeability of the culture solution. 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. Among these hydrophilization treatment methods, polyvinylpyrrolidone treatment is preferable. This is because stable hydrophilicity can be imparted to the surface of the hollow fiber.

  The hollow fiber bundle used in the cell culture hollow fiber module of the present embodiment includes one or more hollow fibers. In addition, when a hollow fiber bundle is comprised from two or more hollow fibers, each hollow fiber is normally arrange | positioned in parallel mutually. Further, both ends of the hollow fiber are fixed in the housing so as to form a straight line. For example, when the housing is a cylindrical body, both ends of the hollow fiber can be fixed in the housing so that the hollow fiber is parallel to the axial direction of the housing.

  The hollow fibers constituting the hollow fiber bundle have a semipermeable membrane function. Here, the semipermeable membrane function means a function that allows some components contained in the solution to pass from the lumen side to the ESC side or from the ESC side to the lumen side but not other components. . A hollow fiber having a semipermeable membrane function has the same function as, for example, a porous membrane that can change the component that can be passed depending on the pore diameter, and a biological membrane that has various membrane passage functions as well as passage due to a concentration gradient. A member constituting the hollow fiber can be appropriately selected and used according to the type of cell to be cultured, the culture solution, and the like. In the following description, a case will be described in which the hollow fiber is made of a cylindrical membrane and a semipermeable membrane function is exhibited by providing a hole penetrating in the thickness direction of the membrane.

  Here, the pore diameter (hereinafter sometimes referred to as “membrane pore diameter”) provided in the membrane constituting the mesoporous yarn is such that the culture solution can be passed from the lumen side to the ESC side. There is no particular limitation as long as the cells to be passed cannot pass from the ESC side to the lumen side. It should be noted that 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. Therefore, in order to achieve a balance between membrane strength, that is, the strength of the hollow fiber and a large mass exchange rate, the average membrane pore diameter of the hollow fiber is preferably in the range of 0.01 μm to 0.8 μm. More preferably within the range of 1 μm to 0.4 μm.

  The inner diameter of the hollow fiber is not particularly limited as long as it is thick enough to supply the culture solution, but is preferably in the range of 0.1 mm to 3 mm from the viewpoint of ensuring high membrane permeability. In view of ease of processing and suppression of clogging due to insoluble components generated in the culture solution, the inner diameter is more preferably in the range of 0.5 mm to 1.5 mm. When the inner diameter is in the range of 0.5 mm to 1.5 mm, the outer diameter is preferably in the range of 0.2 mm to 3.0 mm.

  In addition, the number of hollow fibers constituting the hollow fiber bundle and the film thickness are not particularly limited as long as cells can be cultured. However, as will be described later, the number of cells required and the amount of nutrients required by the cultured cells. It is preferable to appropriately adjust the amount of oxygen, the amount of oxygen, the volume of ECS in the module, or the relationship with one or more.

  When the hollow fiber bundle is composed of two or more hollow fibers, both end portions of the hollow fiber bundle should be sealed with a generally used sealing material and, for example, glued Can be fixed to the inner wall of the housing. As the sealing material, for example, a known resin such as urethane resin or epoxy resin can be used.

-Culture solution-
Further, the hollow fiber supplies a growth essential component to cells existing in ECS by supplying a culture solution to the lumen, and diffuses and removes metabolic components of the cells. Here, as long as the culture solution contains an essential component for growth of cells to be cultured, any conventionally known culture solution can be used and is not particularly limited. Here, the “growth essential component” means a component indispensable for the cell to be cultured to proliferate, and is usually composed of various organic and inorganic substances for supplying oxygen and the like to the cell. Dissolved gas is also included. As the essential growth component, for example, a medium prepared by adding serum or various growth factors and differentiation-inducing factors to a general-purpose medium such as Dulbecco MEM, RPMI 1640, and Ham F12 is used.

  The supply of the culture solution into the lumen is usually performed by repeatedly circulating the culture solution stored in the container through the lumen with a pump, but the culture solution supplied into the lumen is not circulated. It may be a temporary supply.

-Housing-
The housing stores the hollow fiber bundle. Further, in a state where the module is assembled, the housing forms a sealed space, that is, an ECS, between the outer peripheral surface of the hollow fiber disposed therein and the inner wall of the housing. For this reason, cell culture can be performed in this ECS. Also, the housing is usually a tube connection conduit (lumen conduit) for supplying the culture solution to the lumen of the hollow fiber fixed in the housing and discharging the culture solution supplied in the lumen to the outside again, In addition, a tube connection conduit (ECS conduit) for discharging cells into the ECS and discharging other components in the ECS corresponding to the volume of the cells input with the cells into the ECS. Is provided. Then, after the cells are introduced into the ECS from the ECS conduit, the cells can be cultured by supplying a culture solution into the lumen using the lumen conduit. Here, since the ECS and the lumen are isolated from each other, the culture solution and the cells are not in direct contact with each other, and only predetermined components in the culture solution that have passed through the hollow fiber hole having a semipermeable membrane function are cells. Contact with. Two lumen conduits are usually provided in the housing, and two ECS conduits are usually provided in the housing.

  Although the material which comprises a housing is not specifically limited, For example, resin materials, such as a polycarbonate resin, a polysulfone resin, a polypropylene resin, a polystyrene resin, a polyethylene resin, an acrylic resin, can be utilized. Among these resin materials, polycarbonate resin and polysulfone resin are preferable. Since the polycarbonate resin and the polysulfone resin are excellent in heat resistance, the high-pressure steam sterilization of the housing becomes possible, and an efficient sterilization process can be performed. In addition, since the polycarbonate resin and the polysulfone resin are highly transparent, the cultured state of the cells can be easily confirmed with the naked eye.

  Moreover, in the module of this embodiment, the arbitrary parts of a housing are removable. Here, “any part of the housing is removable” means that any part of the housing (removable part) can be separated from the other part (main body part) of the housing, and the removable part is the main body It means that it can be attached to the part. “Removable” means that the removable part can be opened and closed while being in contact with the main body part, or the removable part can be separated so as to be completely separated from the main body part. The aspect which can attach a detachable part to a main-body part again is meant.

  Furthermore, the module of this embodiment has a structure in which at least a part of the hollow fiber bundle can be exposed to the outside. Here, “at least a part of the hollow fiber can be exposed to the outside” means that at least a part of the hollow fiber bundle that is sealed in the module and shielded from the external environment has the removable part of the housing as the main body part. It means that it can be exposed to the external environment. Here, when the detachable part of the housing is separated from the main body part, the degree of exposure of the hollow fiber bundle to the external environment is not particularly limited, but the larger the degree of exposure, the better. It is particularly preferred that it can be exposed to the environment. In this case, it is because it becomes easier to collect | recover the cell cultured in the state attached to the hollow fiber.

  The shape and size of the housing is not particularly limited, and can be set as necessary. However, from a practical point of view such as reduction of dead space in the module and ease of cell culture, the housing preferably has a cylindrical member, and the cylindrical member is preferably a cylindrical member. More preferred. When the housing has a cylindrical member, the hollow fiber bundle is disposed in the cylindrical member so that the axial direction of the hollow fiber and the axial direction of the housing coincide, for example. And as for a hollow fiber bundle, the both ends of a hollow fiber bundle are fixed to the inner wall of a cylindrical member via a sealing material. Moreover, the opening part of a cylindrical member both ends is sealed using a cap. That is, in this case, the main part of the housing is composed of a cylindrical member and a cap.

  Next, the case where the main part of the housing is composed of a cylindrical member and a cap will be described in more detail. In this case, after cell culture, the hollow fiber bundle with the cultured cells attached can be removed from the housing by removing at least one of the two caps sealing both ends of the cylindrical member from the cylindrical member. Thus, the hollow fiber bundle can be completely exposed to the external environment. As a result, the cultured cells can be collected while attached to the hollow fiber bundle. Here, as the cap, any known cap can be used as long as it can be attached to and removed from both ends of the cylindrical member. Although not particularly limited, for example, a fitting cap can be used. Moreover, when a cylindrical member is a cylindrical member, a screw type cap can be used. In addition, as a material which comprises the cylindrical member and cap which comprise a housing, the resin material etc. which were mentioned above can be utilized. The same material may be used for each material which comprises a cylindrical member and a cap, and a different material may be used for it.

-Specific examples of modules-
Next, a specific example of the cell culture hollow fiber module of the present embodiment will be described with reference to the drawings. FIG. 1 is a schematic diagram showing an example of the cell culture hollow fiber module of the present embodiment. Here, the upper side of the central axis C extending in the horizontal direction in FIG. 1 is a cross-sectional view showing the internal structure of the module, and the lower side of the center line is a side view showing the appearance of the module.

  The main part of the module 10 shown in FIG. 1 includes a housing 20 and a hollow fiber bundle 30 arranged in the housing 20. Here, the housing 20 includes a cylindrical member 22 disposed in the center of the drawing, and caps 24A and 24B that seal both end openings of the cylindrical member 22. On the outer peripheral surface side of the cylindrical member 22, a first ECS conduit 26A is provided on the side where the cap 24A is disposed, and a second ECS conduit 26B is provided on the side where the cap 24B is disposed. ing. A first lumen conduit 28A is provided on the outer surface side of the cap 24A in the axial direction, and a second lumen conduit 28B is provided on the outer surface side of the cap 24B in the axial direction.

  The hollow fiber bundle 30 is composed of a plurality of hollow fibers arranged in parallel to each other, and is arranged in the housing 20 so that the longitudinal direction thereof is parallel to the axial direction C of the cylindrical member 22. Further, both end portions of the hollow fiber bundle 30 are filled with a sealing material so as to fill the gaps between the individual hollow fibers constituting the hollow fiber bundle 30 and cover the outer peripheral portion of the hollow fiber bundle 30, Cylindrical sealing material blocks 40A and 40B made of this sealing material are formed. And while holding the outer peripheral surface of sealing material block 40A, 40B, cylindrical sleeve 50A, 50B so that the clearance gap between sealing material block 40A, 40B and the internal peripheral surface of the cylindrical member 22 may be plugged up. Is arranged. For this reason, both ends of the hollow fiber bundle 30 are fixed to the inner peripheral side of the cylindrical member 22 constituting the housing 20 via the sleeves 50A and 50B. Further, a cylindrical core rod 60 is provided in the hollow fiber bundle 30 so as to penetrate the central axis. The sleeves 50 </ b> A and 50 </ b> B and the core rod 60 provided in the hollow fiber bundle 30 are provided to facilitate the holding and handling of the hollow fiber bundle 30. Therefore, an adhesive such as glue is provided between the inner peripheral surface of the sleeve 50A and the outer peripheral surface of the sealing material block 40A and between the inner peripheral surface of the sleeve 50B and the outer peripheral surface of the sealing material block 40B. Glued through.

  Furthermore, between the outer peripheral surface of the cylindrical sleeve 50A on the side where the cap 24A is disposed and the inner peripheral surface of the cylindrical member 22, the outer peripheral surface on the side where the cap 24B of the cylindrical sleeve 50B is disposed and the cylindrical member 22 are disposed. In order to ensure airtightness between the inner peripheral surface of the cylindrical member 22, the end surface of the cylindrical member 22 and the inner surface of the cap 24A, and between the end surface of the cylindrical member 22 and the inner surface of the cap 24B, An O-ring 70 is disposed.

  The length of the hollow fiber bundle 30 is set to be approximately the same as the length of the cylindrical member 22. Further, the diameters of the sleeves 50 </ b> A and 50 </ b> B that hold both ends of the hollow fiber bundle 30 are set slightly smaller than the inner diameter of the cylindrical member 22 in order to facilitate the insertion and removal of the hollow fiber bundle 30 with respect to the cylindrical member 22. It is preferable. Here, the positions of the respective holes of the ECS conduits 26A and 26B provided on the outer peripheral surface side of the cylindrical member 22 in the axial direction C are obtained by adding the lengths of the sleeves 50A and 50B from the end side of the cylindrical member 22. It is set at a position that does not overlap with the selected area. That is, the positions of the holes of the ECS conduits 26A and 26B are not overlapped with the sleeves 50A and 50B.

  As a material constituting the core rod 60 and the sleeves 50A and 50B, the same material as that constituting the housing, or a material having an appropriate rigidity such as a metal such as stainless steel can be used. In the example shown in FIG. 1, one core rod 60 is disposed on the central axis of the hollow fiber bundle 30, but a plurality of core bars 60 may be provided in the hollow fiber bundle 30. The core rod 60 may have a prismatic shape or may be omitted.

  When cell culture is performed using the module 10 illustrated in FIG. 1, first, the module 10 is assembled. In this case, the hollow fiber bundle 30 provided with the core rod 60 so as to penetrate the central axis, and the sleeves 50A and 50B respectively covering the sealing material blocks 40A and 40B located at the ends of the hollow fiber bundle 30, A hollow fiber bundle unit composed of is inserted into the inside of the cylindrical member 22. Thereafter, both ends of the cylindrical member 22 are sealed with caps 24A and 24B. In this assembling operation, the O-ring 70 is disposed at the position described above. In this case, the space formed between the hollow fiber bundle unit and the inner peripheral surface of the cylindrical member 22 is ECS.

  Subsequently, cells to be cultured are introduced into the ECS from the first ECS conduit 26A, the second ECS conduit 26B, or both of the ECS conduits 26A and 26B. Next, for example, the culture solution is supplied into the housing 20 from the first lumen conduit 28A side. First, the culture fluid flows into a space formed between the cap 24A and the end surface of the hollow fiber bundle 30 on the side where the cap 24A is disposed. Then, the culture fluid flows through the internal space (lumen) of the hollow fiber from the opening of each hollow fiber exposed at the end face of the hollow fiber bundle 30. Furthermore, the culture fluid flowing in the lumen passes from the opening of each hollow fiber exposed at the end surface on the cap 24B side of the hollow fiber bundle 30 to the end surface on the side where the cap 24B and the cap 24B of the hollow fiber bundle 30 are disposed. And is discharged from the space to the outside of the module 10 through the second lumen conduit 28B.

  Here, the essential growth component contained in the culture fluid flowing in the lumen is supplied to the cells present in the ECS through the holes provided in the outer peripheral surface of the hollow fiber. For this reason, cell culture is promoted. After the cell culture is completed, the cap 24A and / or the cap 24B is removed from the cylindrical member 22, and then the hollow fiber unit is taken out from the cylindrical member 22. For this reason, the hollow fiber bundle 30 in a state where the cultured cells are attached to the individual hollow fibers can be collected. In addition, when transplanting into a living body, the core rod 30 is pulled out from the hollow fiber unit, and the sealing material blocks 40A and 40B are removed by cutting together with the sleeves 50A and 50B. A hollow fiber bundle 30 composed only of the hollow fibers and a portion composed only of cultured cells can be used.

  The ratio of the volume X of ECS to the volume W of the hollow fiber bundle 30 (X / W) should be appropriately set as required, such as the type of cells to be cultured and the density of cells cultured in the ECS. However, it is preferably in the range of 1-20, more preferably in the range of 2-6. By setting the volume ratio to 1 or more, the amount of cells put into the module 10 can be increased, and a sufficient space for cell proliferation can be ensured. In addition, by setting the volume ratio to 20 or less, it becomes possible to avoid insufficient supply of essential growth components to the cells in the module 10. For this reason, it is preferable to set the number and size of the hollow fibers included in the hollow fiber bundle 30 so that the above-described volume ratio can be realized. The “volume of the hollow fiber bundle 30” means the volume of the portion of the hollow fiber constituting the hollow fiber bundle 30 that contacts the ECS in the module 10, and is formed by the outer periphery of the cross section of the hollow fiber. It can be represented by the sum of the product of the cross-sectional area including the hollow portion and the hollow fiber length.

(Cell culture method)
As described above, when the cells are cultured using the module 10 of the present embodiment, the cells attached to the hollow fiber can be collected while attached to the hollow fiber. Here, “the cell adheres to the hollow fiber” means that the cultured cell adheres directly to the surface of the hollow fiber or indirectly via a cell attachment carrier. In this case, if the cells are held so as not to fall off from the outer peripheral surface of the hollow fiber, the cells existing in the gap between the hollow fibers or the gap between the hollow fiber and the housing also correspond to those attached to the hollow fiber. To do.

  In the cell culture method using the module 10 of the present embodiment, any cell may be cultured as long as it constitutes a living tissue, but the cartilage that has the greatest medical needs as the cell to be cultured. Particularly preferred are cells.

  In the cell culture method using the module 10 of the present embodiment, as a cell culture method that can be used biologically, a known cell culture method can be used. From the viewpoint of application, 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, microcarriers or gel can be introduced together with cells from ECS conduit 26A (or ECS conduit 26B), and the cells can be cultured by supplying the aforementioned culture solution to the lumen. In addition, culture conditions such as the supply rate of the culture solution, the presence / absence of medium exchange, and the oxygen addition method can be appropriately set according to the cell type, cell concentration and the like.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the following examples.

Example 1
-Cell culture test-
Using the module 10 shown in FIG. 1, the cells were cultured according to the following procedure. First, as individual hollow fibers constituting the hollow fiber bundle 30, a hollow fiber made of polylactic acid having a weight average molecular weight of 145800 (manufactured by Duct, inner diameter 0.7 mm, outer diameter 1.3 mm, average membrane pore diameter: 0.1 μm) ) Was used. The hollow fiber bundle 30 is composed of 70 hollow fibers, and its length is 64 mm. Further, urethane resin is used as the sealing material constituting the sealing material blocks 40A and 40B, and the cylindrical member 22, caps 24A and 24B, sleeves 50A and 50B, and the core rod 60 are made of polycarbonate resin. It was. The cylindrical member 22 has a size of an outer diameter of 32 mm, an inner diameter of 26 mm, and a length of 100 mm. The caps 24A and 24B are screw-type caps.

  After assembling the module 10 shown in FIG. 1, the lumen is filled with chondrocyte growth medium containing 5% human serum, FGF-2 (Fibroblast Growth Factor-2) and insulin through a lumen conduit 28A, 28B. Circulated at / min.

  In this state, chondrocytes (derived from human auricular cartilage, the number of cells: about 4000 cells) were embedded in 10 mL of 0.3% siatelopeptide collagen and introduced into ECS through ECS conduit 26A. Thereafter, the ECS conduits 26A and 26B were sealed, and cell culture was started. The cells were cultured in a state where the module 10 was placed in a thermostat set at 37 ° C., and the culture solution was maintained at a pH of about 7 and an oxygen partial pressure of about 20% by volume.

In order to confirm the proliferation of the cells, two weeks after the start of cell culture, the module 10 was disassembled, and the hollow fiber unit with the cultured cells attached to the hollow fibers was taken out. The number of cells at the time of removal was about 8000 when the water permeability of the culture solution in the hollow fiber was 400 (L / m ² · h · atm), and the water permeability of the culture solution in the hollow fiber was 1000. In the case of (L / m ² · h · atm), the number was about 18000, and it was confirmed that the cells were proliferating.

  Next, the core rod 60 was pulled out and removed from the hollow fiber unit in which cells cultured on the hollow fiber were adhered, and the sealing material blocks 40A and 40B were cut and removed together with the sleeves 50A and 50B. During this removal operation, there was almost no loss of cells attached to the hollow fiber. In addition, the hollow fiber bundle 30 does not lose its shape and has sufficient strength when the hollow fiber unit is taken out or removed from the hollow fiber unit except for the cells and the biodegradable material. It was confirmed that

-Biodegradability test-
The biodegradability was evaluated by implanting a porous body made of polylactic acid having the same weight average molecular weight as the hollow fiber used in the cell culture test, along with the auricular chondrocytes, subcutaneously in the back of a beagle dog (8 months old male). The frozen sections of the transplanted samples at 1 month, 2 months and 6 months after the transplantation were evaluated by evaluating the crystallinity of polylactic acid constituting the porous body with a phase contrast microscope. The results are shown in FIG.

  As shown in FIG. 2, the white portion indicating the high degree of crystallinity decreases as time passes, such as the first, second, and sixth months after transplantation. From this, it was found that polylactic acid degrades in vivo over time. In the figure, the left phase contrast microscope image shows the state of the first month after transplantation, the center phase contrast microscope image shows the state of the second month after transplantation, and the right phase contrast microscope image after the transplantation. The state of the sixth month from is shown. Moreover, the length of the bar shown at the lower right in the three phase contrast micrographs means 50 μm.

(Example 2)
-Cell culture test-
As individual hollow fibers constituting the hollow fiber bundle 30, a polylactic acid hollow fiber having a weight average molecular weight of 133000 (manufactured by Duct, inner diameter 0.7 mm, outer diameter 1.3 mm, average membrane pore diameter: 0.1 μm) is used. A cell culture test was carried out in the same manner as in Example 1 except that. As a result, it was confirmed that the cells were growing to the same extent as in Example 1.

  Next, the core rod 60 was pulled out and removed from the hollow fiber unit in which cells cultured on the hollow fiber were adhered, and the sealing material blocks 40A and 40B were cut and removed together with the sleeves 50A and 50B. During this removal operation, there was almost no loss of cells attached to the hollow fiber. In addition, the hollow fiber bundle 30 was slightly out of shape as compared with Example 1 during the removal operation of the hollow fiber unit and the removal operation of removing portions other than the cells and the biodegradable material from the hollow fiber unit. However, it was confirmed to have sufficient strength.

(Example 3)
-Cell culture test-
As each hollow fiber constituting the hollow fiber bundle 30, a polylactic acid hollow fiber having a weight average molecular weight of 145800 (manufactured by Duct, inner diameter 0.7 mm, outer diameter 1.3 mm, average membrane pore diameter: 0.05 μm) is used. A cell culture test was carried out in the same manner as in Example 1 except that. As a result, it was confirmed that the cells were growing to the same extent as in Example 1.

  Next, the core rod 60 was pulled out and removed from the hollow fiber unit in which cells cultured on the hollow fiber were adhered, and the sealing material blocks 40A and 40B were cut and removed together with the sleeves 50A and 50B. During this removal operation, there was almost no loss of cells attached to the hollow fiber. In addition, it was confirmed that the hollow fiber bundle 30 has sufficient strength when the hollow fiber unit is taken out or removed from the hollow fiber unit except for the cells and the biodegradable material. .

(Evaluation of hollow fiber strength)
The tensile strength of the hollow fiber when the hollow fiber used in Examples 1 to 3 was immersed in physiological saline at 37 ° C. was evaluated under the following conditions. The results are shown in Table 1 below.
・ Immersion time: 0, January, February, May ・ Hollow fiber sample length: 60 mm
-Number of measurement samples: 8
-Tensile strength measurement device and measurement conditions: Measured at a tensile speed of 60 mm / min using an Instron model 4443

10 Module (Cell culture hollow fiber module)
20 Housing 22 Cylindrical members 24A, 24B Cap 26A First ECS conduit 26B Second ECS conduit 28A First lumen conduit 28B Second lumen conduit 30 Hollow fiber bundles 40A, 40B Sealant block 50A , 50B Sleeve 60 Core rod 70 O-ring

Claims (5)

It is made of biodegradable material and contains at least one hollow fiber having a semipermeable membrane function, and it is essential to grow in cells existing in the outer space of the hollow fiber by supplying a culture solution to the inner space of the hollow fiber. A hollow fiber bundle for supplying the components;
A housing for storing the hollow fiber bundle, and
A hollow fiber module for cell culture, wherein an arbitrary part of the housing is detachable and at least a part of the hollow fiber bundle can be exposed to the outside. In the cell culture hollow fiber module according to claim 1,
The biodegradable material is
(I) polylactic acid, polyglycolic acid, polycaprolactone, aliphatic polyester, polyamide,
(Ii) a mixed material containing at least two or more kinds of polymer materials selected from the group consisting of the polymer materials shown in (i) above, and
(Iii) a copolymer obtained by copolymerizing each monomer used for polymerization of at least two kinds of polymer materials selected from the group consisting of the polymer materials shown in (i) above;
A hollow fiber module for cell culture, which is a material selected from: In the cell culture hollow fiber module according to claim 1 or 2,
The cell culture hollow fiber module, wherein the biodegradable material has a weight average molecular weight in the range of 30,000 to 500,000. After culturing a cell using the hollow fiber module for cell culture according to any one of claims 1 to 3, an arbitrary part of the housing is separated, and at least a part of the hollow fiber bundle is exposed to the outside. Exposed,
A cell culture method comprising collecting the cells attached to the hollow fiber in a state of being attached to the hollow fiber. The cell culture method according to claim 4, wherein
A cell culture method, wherein the cell is a chondrocyte.