JP6469287B1 - Hollow fiber cell culture device, cell culture method, culture supernatant production method - Google Patents

Abstract

[PROBLEMS] To provide a culture apparatus that has high robustness when it is assumed that a culture supernatant becomes a pharmaceutical product, and can be used for mass production that is efficient and reduces production costs. SOLUTION: A filter housing 1 and a hollow fiber 3 existing inside the filter housing 1 and having a pore size of 5 nm or more and 1 μm or less, in which cells are introduced into the inside by a cell introduction part 5, 1 end 4a, a first end cap 6a for sealing the filter housing 1, a culture solution introduction part 7 for introducing a culture solution into the filter housing, and a culture solution passed through the inside of the filter housing. A hollow fiber cell culturing apparatus for producing a culture supernatant, comprising a culture solution discharge part 9 for discharging. [Selection] Figure 1

Description

The present invention relates to a hollow fiber cell culture device, a cell culture method using the device, and a method for producing a culture supernatant.

Japanese Unexamined Patent Application Publication No. 2017-176043 describes a cell culture method using a hollow fiber. In this application, after mesenchymal stem cells are seeded in hollow fibers, cells and culture medium are circulated in the hollow fibers, and useful components having physiological activity secreted from the cells are contained in the hollow fibers. Keep the concentration and collect the culture supernatant from the hollow fiber. In this document, it is intended to limit as much as possible that the secretions from the cells in the hollow fiber flow out into the culture fluid flowing outside the hollow fiber.

JP 2017-176043 A

  An object of the present invention is to provide a technique that has high robustness when it is assumed that the culture supernatant becomes a pharmaceutical product and can be used for mass production that is efficient and reduces the production cost.

  The solution described in this specification is basically that cells are cultured in the hollow fiber, and the culture solution is circulated outside the hollow fiber, so that the cells can be stably cultured and the culture has been completed. Is based on the knowledge that high-quality cells and culture supernatants can be obtained at low cost and in large quantities.

The apparatus disclosed in this specification is a hollow fiber cell culture apparatus for producing a culture supernatant. And this device
A filter housing 1;
A hollow fiber 3 existing inside the filter housing, having a pore size of 5 nm to 1 μm, and the first and second ends 4 a and 4 b of the hollow fiber 3 are first and second ends of the filter housing 1. 1a and 1b are connected, and the hollow fiber 3 has a cell introduction part 5 into which cells are introduced,
A first end 4a of the hollow fiber 3, a first end cap 6a for sealing the filter housing 1,
A culture medium introduction part 7 connected to the filter housing 1 and for introducing a culture medium into the filter housing, and connected to a culture medium supply path 8 for supplying the culture medium into the filter housing When,
A culture medium discharge part 9 connected to the filter housing 1 and for discharging the culture medium passing through the inside of the filter housing, and connected to a culture discharge path 10 for discharging the culture medium inside the filter housing And a hollow fiber cell culture device.

This device comprises a culture solution supply channel (8),
Preferably, the apparatus further includes a liquid feeding device (11) provided in the culture solution supply path (8) and for sending the culture solution into the filter housing (1) via the culture solution introduction part (7). .

  In this device, the hollow fiber (3) preferably has a diameter of 0.1 mm or more and 1.6 mm or less when the lumen is circular.

This device further comprises a cell introduction part (5),
The cell introduction part (5) is a device that is connected to a site corresponding to the hollow fiber (3) that connects the first end cap (6a), and the cell is introduced into the lumen of the hollow fiber filter. preferable.

This device further has a cell introduction part 5,
The cell introduction part 5 is a cylindrical volume part having a spatial volume provided at a site corresponding to the hollow fiber filter connecting the first end cap 6a, and at one end of the cylindrical volume part, A pusher with a gasket is inserted, and the other end of the cylindrical volume is connected to the hollow fiber filter. By pushing the pusher in the direction of the hollow fiber filter, the cells enter the hollow fiber filter lumen. The device to be introduced is preferable.

  This device is preferably a device further comprising a second end 4 b of the hollow fiber 3 and a second end cap 6 b for sealing the filter housing 1.

A culture supernatant can be produced using this apparatus. This manufacturing method is
A cell introduction step of introducing cells into the hollow fiber 3 via the cell introduction portion 5;
A culture solution supply step of supplying the culture solution to the inside of the filter housing via the culture solution introduction part 7;
A culture solution discharging step of discharging the culture solution inside the filter housing from the culture solution discharge unit 9; a step of culturing cells inside the hollow fiber 3;
A culture supernatant recovery step for recovering the culture supernatant from the effluent obtained by the culture solution discharge step;
Is a method for producing a culture supernatant.

  According to the present invention, it is possible to provide a technique that has high robustness when it is assumed that the culture supernatant becomes a pharmaceutical product, and can be used for mass production that is efficient and reduces the production cost.

  The present invention is characterized in that cells are sealed in the lumen of the hollow fiber, and the culture medium in the lumen moves mainly by diffusion with the outside of the hollow fiber through the hollow fiber filter. A large effect is produced in cells in which each other's cells strongly aggregate, such as leaf stem cells. When we consider culturing mesenchymal stem cells over a long period of time using conventional hollow fiber culture methods, the mesenchymal stem cells seeded in the lumen of the hollow fiber easily form aggregates and proliferate there. If culture conditions that can be applied are provided, the circulating culture fluid that blocks the lumen of the hollow fiber is blocked. Even if the entire hollow fiber is not occluded by cells, it is difficult to ensure that the culture conditions, cell properties, and quality are the same between the hollow fibers that are embolized and those that are not. Adopting it as a robust and robust manufacturing method for drug development etc. is expected to be risky. In addition, when a hollow fiber that is so thick that the hollow fiber lumen is not shielded by cells is used, cell death may occur due to nutrient depletion in the deep layer of the cell aggregate after cell proliferation.

  In addition, in the past, in the cell culture using a hollow fiber filter, the industry only has a method of circulating the culture medium on the side where the cells exist. The reason for the efficiency of the product from the cells in the hollow fiber lumen is that For recovery, it is necessary to recover through the filter not as the culture medium in the opposite space but as the culture medium in the space where the cells are present. If the cells are present in the hollow fiber lumen, This is because it was thought that it was difficult to efficiently collect secretory components outside the hollow fiber.

  The present inventor has developed a method in which the hollow fiber lumen with an appropriate inner diameter is blocked with cells and the culture medium is not actively fed in the hollow fiber lumen, using a flexible idea that is not confined to existing methods. . Instead, by properly exchanging and circulating the culture solution outside the hollow fiber, the cells inside the hollow fiber were successfully maintained for a long period of time, and a culture supernatant with sufficient biological activity was obtained. , It was found that it can be recovered outside the hollow fiber continuously during that period.

  This completes a culture supernatant production method that has high robustness and can reduce production costs, and can be provided as a technology that can be scaled up to a culture scale of several hundred to several thousand liters. It became.

FIG. 1 is a conceptual diagram showing a configuration example of a culture apparatus. FIG. 2 is a conceptual diagram showing an example of a ferrule. FIG. 3 is a conceptual diagram illustrating an example of a cell introduction unit. FIG. 4 is a photograph replacing a drawing showing the cells taken out from the hollow fiber lumen. FIG. 5 is a graph instead of a drawing showing the results of quantitative analysis of TIMP2 protein (FIG. 5 (a) and HGF protein (FIG. 5 (b)) measured by ELISA. FIG. 6 is a graph instead of a drawing showing the results of quantitative analysis of dsDNA. FIG. 7 is a graph instead of a drawing showing the results of quantitative analysis of glucose.

  The 1st side surface indicated by this specification is related with the hollow fiber cell culture device for manufacturing a culture supernatant. This device basically cultivates cells (for example, stem cells) in the hollow fiber and circulates the culture solution outside the hollow fiber, so that the cells can be stably cultured and the culture supernatant can be easily obtained. It can be recovered. This device is also called a hollow fiber filter module.

  FIG. 1 is a conceptual diagram showing a configuration example of a culture apparatus. As shown in FIG. 1, this apparatus includes a filter housing 1, a hollow fiber 3 existing inside the filter housing, a first end cap 6 a, a culture solution introduction unit 7, a culture solution discharge unit 9, and Have

  The filter housing 1 is a housing for accommodating hollow fibers and circulating a culture solution therein. A housing in a known hollow fiber filter can be used as the filter housing. The size of the filter housing may be appropriately adjusted according to the amount of supernatant to be obtained.

  The hollow fiber (3) is a hollow fiber (a filter having a void inside) present inside the filter housing. An example of the pore size (nominal pore size) of the hollow fiber is 5 nm or more and 1 μm or less. For example, the diameter of the hollow fiber 3 when the lumen is circular is 0.1 mm or more and 1.6 mm or less. Regarding the hollow fiber constituting the hollow fiber filter module, the diameter of the hollow fiber lumen is selected in the range of 0.02 mm to 1.6 mm, and may be 0.1 mm to 1.6 mm, or 0.1 mm to 0 mm It may be 5 mm or less. Hollow fiber materials such as polyethersulfone (PES), cellulose acetate (CE), polysulfone (PS), polyvinylpyrrolidone (PVDF) polyacrylonitrile (PAN), etc. It is preferable to be selected, but it has characteristics as a filter and is resistant to the substantial performance as a filter in any sterilization process such as gamma ray sterilization, autoclave sterilization, ethylene oxide gas sterilization, etc. As long as it shows, the material is not limited. An example of the nominal pore diameter of the hollow fiber membrane is an ultrafiltration membrane or a microfiltration membrane from 5 nm (corresponding to 3 kDa) to 1 μm, preferably a microfiltration membrane in the range of 0.1 μm to 0.65 μm. Yes, it can be arbitrarily selected according to the actual transmittance as a result of the molecular weight, three-dimensional structure, hydrophobic, ionic interaction, etc. of the recovered component in the target culture supernatant. Moreover, the tube length on the long diameter side of the hollow fiber is preferably 20 cm to 100 cm because it is easy to handle, but is not limited within the allowable range for designing the hollow fiber cell culture apparatus constituting the present invention. Absent.

  The first and second ends 4 a and 4 b of the hollow fiber 3 are preferably connected to the first and second ends 1 a and 1 b of the filter housing 1. However, after the first and second ends 4a and 4b of the hollow fiber 3 on the same side and the first and second ends 1a and 1b of the filter housing 1 are closed with the end caps 6a and b, respectively, The lumen of the hollow fiber 3 and the internal space of the filter housing 1 are preferably configured so as not to have a spatial connection.

  The hollow fiber 3 is a cell into which cells are introduced by the cell introduction part 5. An example of a cell is a stem cell. An example of a stem cell is a mesenchymal stem cell. The type of cell is not particularly limited. Any cell type that secretes or produces the target component outside the cell and may be used for the purpose of recovering the target component. In particular, suitable cells that can enjoy the effects of the present invention are adhesive cells, and more preferably, mesenchymal stem cells are selected. As mesenchymal stem cells, those isolated and cultured from humans, dogs, horses, cats, pigs, mice, rats, etc. are used. Examples of tissues from which mesenchymal stem cells are isolated include umbilical cord, umbilical cord blood, bone marrow, amniotic membrane, placenta, dental pulp, fat, cartilage, menstrual blood, breast milk, and the tissue and animal species are particularly limited It is not a thing. In addition, mesenchymal stem cells are those that have undergone gene transfer or gene modification in order to establish an immortalized cell line, to provide cell characteristics that can withstand long-term culture, or to improve the effectiveness of the culture supernatant. It may be used.

  The composition and type of the culture solution are not particularly limited. You may use the culture solution similar to the adhesion culture method by the conventional flask etc. When mesenchymal stem cells are taken as an example, they are generally grown in αMEM medium containing 10% serum. For superior cell growth and process shortening, media sold by each company or proprietary media may be used. In addition, considering that the industrial value of the culture supernatant is an agent used for living bodies such as therapeutic uses and cosmetic raw materials, the composition of the culture solution should not use human or animal-derived components. Desirable (eg, cell culture kit Procul AD®; Rohto Pharmaceutical). In addition, techniques for enhancing the action of mesenchymal stem cells by various stimuli have been reported, and in preparing cells and culture supernatants having more effective therapeutic effects, a culture medium utilizing these techniques is used. Can also be used. As described above, there are various options for the medium for proliferating mesenchymal stem cells, and a medium having performance suitable for the origin of the tissue and the species is selected. Any culture medium that can be used to prepare a liquid can be used.

  In addition, depending on the production method of the culture supernatant, a method of maintaining the culture medium close to the basic medium that does not aim for cell growth at the collection stage of the culture supernatant has been reported. The reason for this is to eliminate as much as possible the introduction of serum or the like when cell growth is performed in a serum-containing medium.

  If you want to avoid the possibility that cells seeded in the hollow fiber form uneven aggregates inside the hollow fiber, the cellulosic water-soluble polymer such as methylcellulose (MC) or carboxymethylcellulose (CMC) ), Other cellulose derivatives, or other water-soluble polymers such as polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP), or sodium alginate, and their gelling agents. May be. The concentration of these water-soluble polymers in that case can be set with reference to the previous report. Thereby, in the cell culture space in the hollow fiber, it becomes possible to form spheroids of uniform size and to arrange them uniformly.

  The first end cap 6a is for sealing the first end 4a of the hollow fiber 3 and the filter housing end 1a. End caps are used to seal both ends of the hollow fiber filter module. It is preferable to seal the hollow fiber ends 4a and b so that the cells and the culture medium do not move through the openings at both ends of the hollow fiber. For this purpose, a disc-shaped silicone gasket is most suitable as an end cap that contacts the hollow fiber ends 4a, b, and is made of a resin for fixing to the hollow fiber filter module on the outside with a ferrule connection clamp or the like. Parts may be attached. Two end caps are prepared for each hollow fiber filter module, and one is preferably attached before introducing cells into the hollow fiber lumen. The other is installed after cell introduction to seal the cell introduction side. The end cap to be mounted after the introduction of the cell may be replaced with the cell introduction portion 5 configured to have the function of 4a and the end cap. Neither side end cap is preferably opened during the culture period for collecting the culture supernatant after cell introduction.

  The example of the cell introduction part 5 has a ferrule connection port provided at a portion corresponding to the hollow fiber 3 of the first end cap 6a, and the cell passes through the ferrule connection port so that the cells can pass through the lumen of the hollow fiber filter. Is to be introduced. Another example of the cell introduction part 5 is a cylindrical volume part having a spatial volume provided in a part corresponding to the filter housing end 1a and the hollow fiber end 4a to which the first end cap 6a is connected. A pusher with a gasket is inserted into one end of the cylindrical volume part, the other end of the cylindrical volume part is connected to the hollow fiber 3, and the pusher is pushed in the direction of the hollow fiber 3. Therefore, it is preferable that the device is such that cells are introduced into the lumen of the hollow fiber 3.

  The cell introduction part 5 is used as an element for uniformly introducing cells into the hollow fiber lumen. The form is appropriately selected according to the size of the hollow fiber filter module to be used and the amount of the introduced cell suspension.

  For example, in the industrialization stage such as pharmaceutical manufacturing, it is assumed that a cell suspension of several liters or more is introduced per one hollow fiber filter module. In that case, a tube with a ferrule connection port corresponding to the shape of the ferrule connection port at the end of the hollow fiber filter module is prepared separately, and a process bag or container containing a cell suspension is connected to the opposite side, and a peristatic pump The cell can be fed to the ferrule connection port by using the drive fluid. FIG. 2 is a conceptual diagram showing an example of a ferrule. FIG. 2 is quoted from the website of Nitto Metal Industry Co., Ltd. The cell introduction part 5 is connected to the filter housing end 1a and the hollow fiber end 4a via the ferrule so that cells can be introduced aseptically into the hollow fiber filter. The hollow fiber filter can be sealed after the cells are introduced into the hollow fiber. Ferrule connection is also called sanitary piping or sanitary joint. Ferrule connection can be achieved using, for example, ferrules, gaskets, and clamp bands. The ferrule has a groove in the connecting portion, and is fixed by tightening with a clamp band with a cross-shaped (or O-ring) gasket in between.

Also, in the small scale culture stage, the filter housing ends 1a, 1b are male or female type luer lock-shaped ports and commercially available injections with luer lock-shaped tips (eg, Nipro syringe lock type, medical device notification) No. 27B1X00045000033: Nipro) etc. can also be used as the cell introduction part.

  Furthermore, it is possible to select a part that combines the purpose of the cell introduction part and the aforementioned end cap. FIG. 3 is a conceptual diagram illustrating an example of a cell introduction unit. The cell introduction part 5 having a structure such as injection may have a connection port adapted to the connection type of the filter housing ends 1a and 1b, and more preferably, ferrule connection ports are selected for both. Moreover, depending on another system, the hollow fiber filter module and the cell introduction part 5 may be connected in advance at the time of manufacturing them. In the example of FIG. 3, a cell suspension is enclosed inside the cell introduction part 5. And it connects with the filter housing terminal 1a using a ferrule connection. Then, a cell suspension is injected into the hollow fiber 3 by pushing the piston pusher. In this way, cells can be introduced into the hollow fiber. After the cells are injected into the hollow fiber, a gasket seals the hollow fiber end 4a. In addition, the cell introduction part 5 includes at least an outer cylinder, a gasket, a pusher, and a lock mechanism. When this is used, when cells are pushed into the hollow fiber lumen by the gasket, the hollow fiber end 4a on the cell introduction side is sealed with the gasket, and the gasket is fixed at that position by the lock mechanism. This eliminates the need to open the filter housing end 1a on the cell introduction side after cell introduction, thereby reducing the risk of contamination. Further, a dedicated end cap 6a may be used to block the filter housing end 1a and the hollow fiber end 4a on the cell introduction side.

  The culture medium introduction part 7 is connected to the filter housing 1. The culture solution introduction part 7 may be formed integrally with the filter housing 1. The culture medium introduction part 7 is an element for introducing the culture medium into the filter housing 1. On the other hand, the culture solution introduction part 7 is not connected to the inside of the hollow fiber 3 but for introducing the culture solution into a region inside the filter housing existing around the hollow fiber. The culture medium introduction unit 7 is connected to a culture medium supply path 8 for supplying the culture medium to the inside of the filter housing. The culture solution supply path 8 exists outside the filter housing 1. Thereby, the culture solution is introduced into the filter housing from the outside of the filter housing 1. The culture solution supply path 8 is connected to, for example, a culture solution tank that contains the culture solution, and is supplied with the culture solution.

  That is, this apparatus includes a flow path culture liquid supply path 8 for introducing a culture liquid into the filter housing 1 and a flow path culture liquid discharge path 10 for discharging the culture liquid outside the hollow fiber filter housing. Is preferred. These are connected to the culture medium introduction part 7 and the culture liquid discharge part 9 of the hollow fiber filter module. As a material of the tube, a silicon material, a thermoplastic elastomer material, a polyvinyl chloride material, or the like is selected as a suitable material. However, the present invention is not necessarily limited to these, and it does not adversely affect the results of cell culture, and can sterilize materials. There is no denaturation or unintentional adsorption of target products or components, and the effluents of cells If there is no toxicity such as toxicity, irritation, carcinogenicity, and fever, it can be selected arbitrarily.

  In addition, what has further the liquid feeding apparatus 11 is preferable. The liquid feeding device 11 may be provided in the culture solution supply path 8 or may be provided in the culture solution discharge path 10. The liquid feeding device 11 is an element for feeding the culture solution into the filter housing 1 through the culture solution introduction unit 7 and discharging the culture solution through the culture solution discharge unit 9. An example of the liquid delivery device 11 is a pump. As a method for supplying or circulating the culture solution into the filter housing 1, it is preferable to use a peristatic pump as a method that can be used at any production scale. However, if the culture scale is large, a single-use pump head can be used. A floating magnet pump (eg PuraLev: LEVITRONIX) consisting of a pump driver can also be used as an ideal liquid delivery method. However, the liquid feeding drive system is not limited to these, and can be selected.

  The culture solution storage container is connected to, for example, a culture solution supply path 8 for introducing the culture solution into the filter housing 1. Examples of the shape of the culture solution storage container may be a cylindrical bottle shape, a spinner flask shape, a 2D process back shape, or a 3D process back shape. Alternatively, in the case of a large-scale culture scale, when the culture solution is refluxed through the filter housing 1 which may be a stainless tank container, the culture solution for discharging the culture solution in the filter housing 1 It is preferable that the discharge path 10 is also connected. This apparatus preferably has a culture solution sampling port for monitoring the culture state, and has various centers such as temperature, pH, dissolved oxygen concentration, dissolved carbon dioxide concentration and the like. In addition, in the case of a culture system that does not reflux, in addition to a culture solution storage container that contains a culture solution to be introduced into the filter housing 1, a culture solution that has been discharged from the filter housing 1 is used. Those having another culture solution storage container are preferable.

  The culture medium discharge part 9 is connected to the filter housing 1. The culture medium discharge part 9 may be formed integrally with the filter housing 1. The culture solution discharge part 9 is an element for discharging the culture solution that has passed through the inside of the filter housing. The culture solution discharge part 9 is not connected to the inside of the hollow fiber, but is for discharging the culture solution from a region inside the filter housing existing around the hollow fiber. The culture solution discharge unit 9 is connected to a culture discharge path 10 for discharging the culture solution inside the filter housing. The culture discharge path 10 exists outside the filter housing 1. Thereby, the culture solution is discharged out of the filter housing 1.

  Each element such as filter housing, housing terminal connection, hollow fiber fixing material, sealing material, and flow path forming port can be designed with reference to existing technologies and products. For example, the housing end connection portion is preferably designed to support a versatile ferrule sanitary connection. For these, the shape and design can be freely selected. In addition, regarding these materials, it is preferable to select from the general materials conventionally used in biotechnology-related fields, but it is not necessarily limited to these and adversely affects the results of cell culture. The material can be sterilized, there is no denaturation or unintentional adsorption of the target product or components, and the eluate is cytotoxic, irritant, carcinogenic, pyrogenic, etc. Selectable. For example, urethane resin or epoxy resin is allowed as a hollow fiber fixing material for bundling and fixing the hollow fiber at the end of the filter housing. Moreover, a silicon material is suitable for the seal material used for joining the parts to form a complete hermetic seal. For the filter housing, it is recommended to select polysulfone that is strong, rigid, transparent, and excellent in resistance to various chemicals. Similarly, the flow path forming port can be selected from polysulfone, polypropylene, and the like.

    In order to grow or maintain the cells, an apparatus for controlling the culture solution in the range of 30 ° C. to 42 ° C. is preferable. More preferably, 37 ° C. is often selected. As long as the hollow fiber filter module containing the cells is controlled to the target temperature, the temperature control mode is not limited. Specifically, the entire apparatus of the present invention may be installed in a chamber of a constant temperature apparatus, or may be controlled by a jacket type heating apparatus for heating. Alternatively, heating is performed in the vicinity of the flow path on the side where the medium is fed to the filter module 1 and the flow path forming port, and control is performed so as to reach the optimum temperature immediately before the culture liquid is fed to the filter module 1. In addition, the filter module 1 also has a mechanism for maintaining the optimum temperature, and the channel forming port and the channel on the side from which the culture solution is discharged from the filter module 1 are cooled to 4 to 10 ° C. The liquid container may also be cooled to 4 to 10 ° C. to take measures to suppress the denaturation and decomposition of medium components and cell secretory components during long-term culture.

A culture supernatant can be produced using this apparatus. This manufacturing method is
A cell introduction step of introducing cells into the hollow fiber 3 via the cell introduction portion 5;
A culture solution supply step of supplying the culture solution into the filter housing 1 via the culture solution introduction part 7;
A culture solution discharging step for discharging the culture solution in the filter housing 1 from the culture solution discharge unit 9;
Culturing cells inside the hollow fiber 3,
A culture supernatant recovery step for recovering the culture supernatant from the effluent obtained by the culture solution discharge step;
Is a method for producing a culture supernatant.

<Preparation of seeded cells>
In the preparation of cells required for seeding into the hollow fiber 3, the above-mentioned culture solution and cells are used. The cells to be seeded are preferably cells prepared by normal adhesion plane culture and easily dispersible with a release agent. In addition, cells prepared by culture using microcarriers or spheroid culture are used. It may be used, as long as it is of a size that fits into the inner hole of the hollow fiber filter, it may contain microcarriers that remain adhered without detaching the cells, or the cells may form aggregates. .

<Assembly of equipment>
At least the end caps 6a and 6b are arranged in the hollow fiber filter module including the filter housing 1, the hollow fiber 3 disposed therein, the filter housing ends 4a and 4b, the culture medium introduction part 7 and the culture liquid discharge part 9. A gasket or a seal material may be included in the hollow fiber filter housing ends 4a and 4b, and the culture medium supply path 8 and the culture medium discharge path 10 are connected to the culture medium introduction section 7 and the culture medium discharge section 9, respectively. These components are referred to as components 1 here. It is preferable to sterilize by the sterilization method of the method which the raw material of the component 1 accept | permits.

  In addition, the cell introduction part 5 for introducing cells into the hollow fiber described above is not a sterilized product, and if sterilization is necessary, the sterilization process is performed according to the sterilization principle of the method allowed by the material. It is preferable to apply.

  However, the present invention is not limited to the representative examples shown here, and by using an existing bioprocess technology, it is possible to select an optimal apparatus connection mode according to the purpose of the practitioner. For example, the connection of tubes, etc., that form the flow path of the culture solution, when using existing aseptic connection technology frequently used in the bio industry, or when incorporating a stainless steel container as a culture solution storage container The connection style of the device can be taken according to In addition, it is used in the manufacture of general biotechnology-applied pharmaceuticals, etc. according to the requirements of manufacturing scale, facility requirements, overall plan for aseptic operation, containers used, liquid feeding lines, etc. The technology etc. may be designed by adopters.

<Introduction of cells>
Next, remove “component 1” in the aseptic operation area ISO class 5 or in the management area where substantially aseptic operation is possible, including the end cap 6a gasket and sealing material, and place the cells in it. The introduction part 5 is connected. Thereafter, the cell suspension prepared for seeding is placed in the cell introduction part. The cell suspension is preferably a culture solution, but is not particularly limited as long as it is an acceptable solution, a thickener, or a gelling agent as long as it does not adversely affect the culture. If the culture is a small scale and the cell introduction part 5 is a syringe, remove the plunger and put the cell suspension in the outer cylinder, then attach the plunger and push the plunger to remove the cell suspension. Introduce into the hollow fiber. At this time, the filter housing end 1b and the hollow fiber end 4b on the opposite side of the hollow fiber are sealed in some cases including an end cap 6b gasket and a sealing material. The solution portion is discharged from the hollow fiber lumen into the filter housing 1 through the membrane of the hollow fiber 3. Since the hollow fiber membrane has a pore size that does not allow cells to pass therethrough, the cells are completely retained in the hollow fiber lumen without being discharged into the culture medium circulation channel. In order to avoid the occurrence of uneven cell distribution in the hollow fiber lumen, it is desirable to avoid injecting gas into the hollow fiber as much as possible. Furthermore, in order to suppress the formation of cell aggregates, the cell suspension contains cellulose-based water-soluble polymers such as methylcellulose MC and carboxymethylcellulose CMC, other cellulose derivatives, or other water-soluble polymers such as polyethylene glycol PEG and polyvinylpyrrolidone. A method of increasing the viscosity of the culture solution by adding PVP, sodium alginate or the like or a gelling agent thereof may be used.

As a typical example of the range of the number of cells introduced into the hollow fiber lumen, the case of mesenchymal stem cells is illustrated as 1 × 10 ⁵ per 1 cm ³ of the total lumen volume of the hollow fibers constituting the hollow fiber filter module. The range can be selected from ⁴ to 5 × 10 ⁷ , more preferably 1 × 10 ⁵ to 5 × 10 ⁶ , more preferably 5 × 10 ⁵ to 1 × 10 ⁶ Range is selected.

  Next, the cell introduction part 5 is removed, and the inside of the hollow fiber 3 and the inside of the filter housing 1 are sealed by attaching a case where an end cap 6a gasket or a sealing material may be included in the filter housing end 1a and the hollow fiber end 4a. This is designated as component 2. After sealing, the hollow fiber filter module should be kept horizontal to avoid cell bias inside the hollow fiber.

<Start of culture>
The culture solution supply path 8 is connected to a culture solution storage container into which the culture solution has been injected aseptically. When the culture solution is a circulation type, the culture solution discharge path 10 is also connected to the same culture solution storage container. If the circulation type is not used, the culture solution discharge channel 10 is connected to a culture solution storage container different from the culture solution storage container to which the culture solution supply path 8 is connected. This is referred to as a component 3.

  In order to grow or maintain cells, an apparatus for controlling the hollow fiber filter module in the range of 30 ° C. to 42 ° C. is required depending on the type of cell and the purpose, and more preferably, in many cases, 37 C is selected. In addition, when the pH of the culture solution is controlled using sodium bicarbonate and carbon dioxide, it is also necessary to control the carbon dioxide concentration. As a typical example, the component 3 after injecting the culture solution is placed in a carbon dioxide incubator maintained at a temperature and carbon dioxide concentration optimum for the cells to be used. In this case, carbon dioxide permeates into the culture fluid from the culture fluid channels 8 and 10 made of silicon, and the pH is controlled. However, if the hollow fiber filter module part in which the cells are present is controlled to the target temperature and the target pH, the control mode is not limited. Specifically, the entire apparatus of the present invention may be installed in a chamber of a constant temperature apparatus, or may be controlled by a jacket type heating apparatus for heating. Alternatively, heating is performed in the vicinity of the culture solution supply path 8 and the culture solution introduction section 7 on the side of supplying the medium to the hollow fiber filter module, and the temperature is set to the optimum temperature immediately before the culture solution is supplied to the hollow fiber filter module. In addition to controlling to reach the hollow fiber filter module, the hollow fiber filter module also has a mechanism for maintaining the optimum temperature, and further, the culture liquid discharge section 9 and the culture liquid discharge path 10 on the side from which the culture liquid is discharged from the hollow fiber filter module May be cooled to 4 to 10 ° C, and the culture medium storage container may also be cooled to 4 to 10 ° C to take measures to suppress the denaturation and decomposition of medium components and cell secretion components in long-term culture.

    Thereafter, a peristaltic pump or the like is attached to one or both of the culture fluid channels 8 and 10, and the circulation of the culture fluid is started by driving it. However, as described above, the circulation mode of the culture solution is not particularly limited by selecting an existing technique. Moreover, the condition for feeding the culture solution may be intermittent or continuous. Moreover, when intermittent, the interval between the on and off intervals may be arbitrarily changed during a series of culture periods, or may be constant. In addition, the amount of liquid delivered per unit time depends on various variables such as the cells used, the culture solution, the type of hollow fiber filter, the scale of the hollow fiber filter module, and the target component to be recovered in the culture supernatant. The optimum liquid feeding speed can be determined.

Taking mesenchymal stem cells as an example, the amount of fluid delivered per hour per total lumen volume of 1 cm ^{3 of} the hollow fibers constituting the hollow fiber filter module can be selected from 1 mL to 10,000 mL. More preferably, the range of 10 mL to 1,000 mL is selected, and more preferably, the range of 50 mL to 500 mL is selected. The volume of the culture solution filled in the culture device can be selected from 0.01 mL to 300 mL per 1 cm ³ of the total lumen volume of the hollow fiber, taking mesenchymal stem cells as an example. Preferably a range of 0.2 mL to 30 mL is selected, more preferably a range of 1 mL to 5 mL is selected. The amount of the culture solution can be reduced when the number of cells at the initial stage of culture is small, and the amount of the culture solution can be increased when the number of cells after growth is large.

<Medium exchange>
The introduction of cells into the hollow fiber lumen and the medium exchange in which the cells are in the growth phase will be determined depending on the type of cells used and the culture medium. In the case of mesenchymal stem cells, for example, it is desirable to base culture on a total medium exchange once every 4 days. However, in carrying out the present invention, the present invention is not limited to this, and is within a range that can be accommodated by half replacement of the culture solution storage container until the number of cells increases. In addition, the present invention can be satisfactorily performed by using a fed-batch culture or continuous culture technique as a control of the culture solution. The practitioner may set an arbitrary range within which the cells can be maintained without being killed.

<Monitoring of culture environment>
As a method for nondestructively evaluating the degree of cell proliferation, it can be carried out by measuring the culture supernatant collected from a sterile sampling port provided in the culture medium circulation channel of the culture apparatus. As an example of the measurement item, the production amount per unit time, the glucose consumption amount, the pH, the dissolved oxygen concentration, the dissolved carbon dioxide concentration, or the lactic acid concentration of the target product substance in the culture supernatant is an index. The degree of cell proliferation in the hollow fiber lumen can be evaluated in advance by evaluating the correlation between the degree of cell proliferation in the hollow fiber lumen and these values. Depending on the measurement item, continuous measurement may be performed with a non-contact sensor installed in the culture supernatant flow path.

As a method for monitoring the degree of cell death, it can be evaluated by quantifying the double-stranded DNA (dsDNA) concentration, lactate dehydrogenase (LDH) concentration, etc. in the culture supernatant. By evaluating the degree of cell death in the hollow fiber lumen and the correlation between these values in advance, the degree of cell proliferation in the hollow fiber lumen can be evaluated.
By these methods, it is possible to know the start time of the recovery of the culture supernatant containing the target product at a high concentration and the period until the recovery is stopped that limits the soundness and homogeneity of the cells.

<Preparation and collection of culture supernatant>
Begin preparation and collection of culture supernatants when the cells grow and the secretion of the desired amount of the desired component can be confirmed. For example, when the culture medium is circulating, the timing for collecting the culture supernatant circulating in the hollow fiber housing is taken as an example of mesenchymal stem cells. When batch culture is used to prepare the culture supernatant, The collection after 1 to 10 days from the medium exchange just before the collection of the culture supernatant is preferred, more preferably after 2 to 8 days, and most suitably from the 3rd to 5th days. In addition, it is emphasized as a great effect of the present invention that the culture supernatant can be collected for a very long time after the introduction of the cells into the hollow fiber lumen. It is sufficient to collect the necessary amount of culture supernatant. In the case of mesenchymal stem cells, in flat adherent culture using flasks, after reaching confluence, the maintenance of cells in a healthy state is limited to several days. It can be kept healthy for more than 50 days.

  In addition, since the cells exist only in the hollow fiber lumen, the technical common sense in the field prior to the present invention is as a practical method for recovering the culture supernatant containing cell secretions. In this method, the culture medium on the side where the cells are present is circulated separated by a hollow fiber membrane, and the culture medium on the same side is recovered as a culture supernatant (Patent Document 1). On the other hand, in the present invention, the culture supernatant is recovered from the outside of the hollow fiber by diffusion to the outside of the hollow fiber through the hollow fiber membrane from the hollow fiber lumen where the cells are sealed and without active circulation. In addition to batch culture, the culture supernatant may be obtained by continuous culture such as fed-batch culture or perfusion culture.

[Example 1]
1. Hollow fiber culture of adipose tissue-derived mesenchymal stem cells (AD-MSC) and preparation of culture supernatant 1.1. Cell preparation method (1) Primary culture (P0)
Provision of subcutaneous fat after obtaining consent for research use from surplus tissue after separation of subcutaneous adipose tissue, which is a raw material necessary for preparation of cells for administration, from patients undergoing regenerative medicine using AD-MSC And subjected to primary culture. The subcutaneous adipose tissue was subjected to centrifugation (400 × g for 5 minutes) and separated into three layers from the top in the order of lipid fraction, adipose tissue fraction, and aqueous fraction. The upper and lower layers were discarded, leaving the middle adipose tissue fraction. To the remaining adipose tissue fraction, 4 times the amount of 0.15% collagenase enzyme solution per tissue weight was added and permeated at 37 ° C. for 1 hour for enzyme treatment. After the adipose tissue is dispersed, it is subjected to centrifugation (400 × g for 5 minutes), and the precipitated fraction is suspended in 30 mL of PBS (−) solution as a stromal vascular cell fraction containing mesenchymal stem cells. did. Thereafter, the suspension was passed through a cell strainer (70 μm diameter), and the passed fraction was subjected to centrifugation again (400 × g for 5 minutes), and the tissue residue captured by the cell strainer was discarded. The precipitate fraction is suspended in 6 mL of serum-free medium (Procul AD; Rohto Pharmaceutical Co., Ltd.), and the whole volume is seeded in a T-25 flask (CellBIND (registered trademark); Corning) ₂ ) It left still and the primary culture was started.

(2) Subculture (P0 → P1)
The whole medium was changed once every three days, the supernatant was discarded, and the cells that grew on the bottom of the flask were selectively grown. On day 11 after seeding, 2 mL of the enzyme solution (TrypLE Select (registered trademark); Thermo Fisher Scientific) was added to the cells grown to semi-confluent in the T-25 flask and detached (37 ° C., 5 minutes). Still). Cells were diluted with PBS (−) and subjected to centrifugation (400 × g for 5 minutes). As a result of suspending the precipitated cells in the culture medium and counting the number of cells by trypan blue staining, 1.2 × 10 ⁶ viable cells were recovered, so the total amount was T-150 flask (CellBIND (registered trademark); Corning was inoculated with 24 mL of serum-free medium (4,000 cells / cm ² ), and left in an incubator (37 ° C., 5% CO ₂ ) for subculture.

The whole medium was changed once every three days, the supernatant was discarded, and the cells that grew on the bottom of the flask were selectively grown. 6 mL of an enzyme solution (TrypLE Select (registered trademark); Thermo Fisher Scientific) was added to the cells grown to semi-confluent on the 4th day after seeding, and detached (37 ° C., left for 5 minutes). Cells were diluted with PBS (−) and subjected to centrifugation (400 × g for 5 minutes). As a result of suspending the precipitated cells in serum-free medium and measuring the number of cells by trypan blue staining, it was confirmed that the survival rate was 98.7% and the number of viable cells was recovered 9.8 × 10 ^6. This was stored at 4 ° C. until the start of culture.

1.2. Hollow fiber culture (P2)
(1) Device assembly
Filter housing ends 1a and 1b are sanitary connection port shapes, and a hollow fiber filter module (PES MidiKros TC module / 0.2 μm fraction size, filter membrane area) with a luer lock connect in the culture medium introduction part and culture medium discharge part 290 cm ² , total length 44 cm; T04-P20U-05-N; Spectrum Laboratories) was used. An end cap 6b having a ferrule sanitary connection port and containing a silicon gasket and a silicone sealant was sealed by tightening a clamp so as to close the filter housing end 1b and the hollow fiber end 4b.

In addition, a 3 cm silicon tube is attached to the hose port of a part of the cell introduction part (Pro-Connex 3/4 ”sanitary fitting; ACPX-SM4-06N; Spectrum Laboratories) having a ferrule sanitary connection and hose port, A luer lock port was attached to the opposite side of the hose port, and these were fixed with a tie band, and a sealing plug for luer lock connect was attached to the luer lock to form the cell introduction part (5).
Next, the above-mentioned cell introduction part 5 was attached to the filter housing end 1a by tightening a clamp with a ferrule sanitary connection port equipped with a silicon gasket.

  Furthermore, the culture medium introduction section 8 and the culture medium discharge path 10 of the 50 cm silicon tube were respectively attached to the culture medium introduction section 7 and the culture medium discharge section 9 of the hollow fiber filter module via a luer lock connect. A female CPC coupling and its cap (male type) were attached and sealed to the open ends of the culture medium introduction part 8 and the culture medium discharge path 10. These were sterilized by high-temperature pressurized steam sterilization (121 ° C./20 minutes) to prepare Component 1.

(2) Introduction of culture medium and cells
Inside the safety cabinet, fill 2D process bag (FLEXBOY BAG 500 mL MPC MALE COUPLERS) with 350 mL culture solution (Procul AD; Rohto Pharmaceutical Co., Ltd.) and connect it to two CPC connection ports of the process bag. The CPC coupling of the culture fluid supply path 8 and the culture fluid discharge path 10 of the above-described component 1 subjected to sterilization was connected to form a closed circulation path for the hollow fiber filter module and the culture fluid storage container.

Next, the plug of the luer lock connect of the cell introduction part 5 is removed, and the 20 mL capacity syringe barrel (Nipro syringe lock type, medical device notification number 27B1X00045000033: Nipro) with the plunger removed is connected to the end of the filter housing ( The whole cell suspension prepared in 15 mL was connected to 1a) and introduced into a syringe (9.0 × 10 ⁶ cells). Place the plunger on the syringe, and place the cell suspension side up to fill the hollow fiber lumen by pushing the plunger for about 10 seconds with the cell introduction side facing up and the hollow fiber filter module vertically. Completed. At this time, it was operated so that air could not enter the hollow fiber lumen. Since the total volume of the hollow fiber filter lumen derived by calculation is about 15 cm ³ , the cell seeding density was about 6.0 × 10 ⁵ cells / cm ³ . Next, the cell introduction device is removed by removing the clamp, and the end cap 6a having a ferrule sanitary connection port and including a silicone gasket and a silicone sealant is attached to the exposed filter housing end 1a, and the filter housing end 1a. The clamp was tightened to seal the hollow fiber end 4a. Immediately, the long axis direction of the hollow fiber filter module was placed horizontally, thereby completing the introduction and encapsulation of the cells into the lumen of the hollow fiber 3 and completing the component 2.

(3) Circulation start of culture solution
Component 2 was placed in a carbon dioxide incubator (37 ° C., 5% carbon dioxide concentration). Since the culture solution (Procul AD; Rohto Pharmaceutical Co., Ltd.) is a bicarbonate buffer system, gas exchange was performed through the silicon tube of the culture solution supply channel and the culture solution discharge channel. A peristatic pump was attached to one place of the culture solution supply path 8 and was driven to start circulation of the culture solution in the flow path leading to the hollow fiber filter housing. The liquid feeding speed was about 30 mL / hour, and the pump was driven continuously. This was set to be 2 mL / hour per 1 cm ³ volume of the hollow fiber lumen.

(4) Culture medium exchange and culture supernatant
From the start of hollow fiber culture to the 12th day, the culture medium was semi-exchanged once every 3 to 4 days. Thereafter, all exchanges were made once every 3 to 4 days. In addition, for the evaluation of the culture supernatant, the culture start date (Day 0), the culture start date (Day 4), the culture start date (Day 7), the culture start date (Day 11), the culture start date (Day 11), and the culture start date (14 days) (Day 14), 18 days after start of culture (Day 18), 21 days after start of culture (Day 21), 25 days after start of culture (Day 25), 28 days after start of culture (Day 28), 32 days after start of culture (Day 32), culture 35 days after start (Day 35), 39 days after start of culture (Day 39), 42 days after start of culture (Day 42), 46 days after start of culture (Day 46), 50 days after start of culture (Day 50), 3 mL each was collected from the sampling port in the process bag containing the culture solution using a syringe, and the culture was terminated at Day 50. The collected culture supernatant is filtered with a 0.2 μm PES syringe filter (25 mm GD / X syringe filter (sterically sterilized with PES 0.2 μm); 6896-2502; GE Healthcare Japan) until it is used for analysis. Refrigerated storage at -28 ° C.

(5) Confirmation of cells
After completion of the culture, the apparatus was disassembled, and the cell detachment enzyme solution TrypLE Select (Thermo Fisher Scientific) was filled inside the hollow fiber filter module housing. The enzyme treatment was performed at 37 ° C. for 30 minutes to loosen the adhesion between the hollow fiber membrane and the cells, and then the cell mass in the hollow fiber lumen was taken out, and the cell mass was observed with a phase contrast microscope. FIG. 4 is a photograph replacing a drawing showing the cells taken out from the hollow fiber lumen.

[Example 2]
1. 1. Flat adhesion culture of adipose tissue-derived mesenchymal stem cells (AD-MSC) and preparation of culture supernatant 1.1. Cell preparation method (1) Primary culture (P0)
About the remainder of AD-MSC (P1 passage cell) prepared in Example 1 and seeded on the hollow fiber filter, 8 × 10 ⁵ cells were seeded on one T-150 flask (CellBIND; Corning International). At this time, the volume of the culture solution (Procul AD; Rohto Pharmaceutical) was 24 mL. Four days after seeding, the culture supernatant was collected when it reached semi-confluence. Thereafter, the culture medium is completely exchanged once every 3 to 4 days, 7 days after starting culture (Day 7), 11 days after starting culture (Day 11), and 14 days after starting culture. On (Day 14), the culture supernatant was collected before the medium exchange. These culture supernatants are filtered through a 0.2 μm PES syringe filter (25 mm GD / X syringe filter (PES 0.2 μm sterilized); 6896-2502; GE Healthcare Japan) until use for analysis -28 Refrigerated storage at ℃.

[Example 3]
1. Analysis of culture supernatant 1.1. Quantification by ELISA (TIMP2)
The culture supernatant derived from mesenchymal stem cells contains various proteins, and at least some of them are considered to exert pharmacological action. Among them, the Tissue Inhibitor of Metalloproteinase (TIMP) family, which is an endogenous inhibitor of Metalloproteinase (MMP), and Hepatocyte Growth Factor (HGF), which is a growth factor, are contained in high concentrations in the culture supernatant of mesenchymal stem cells. This is a known factor.

Although the presence of analgesic activity has been reported in the culture supernatant of mesenchymal stem cells (Sci. Rep. 2017 Aug 29; 7 (1): 9904. Therapeutic effect of human adipose-derived stem cells and their secretome in experimental diabetic pain). TIMP2 has also been reported to have the same therapeutic effect (Nat. Med. 2008 Mar; 14 (3): 331-6. Distinct roles of matrix metalloproteases in the early- and late-phase development of neuropathic pain.) . The mesenchymal stem cell-derived culture supernatant has been reported to have a therapeutic effect on multiple sclerosis, and HGF has been identified as an efficacy factor (Nat. Neurosci. 2012 Jun; 15 ( 6): 862-70. Hepatocyte growth factor mediates mesenchymal stem cell induced recovery in multiple sclerosis models.). Therefore, TIMP2 and HGF were selected as representative secretory factors for the culture supernatant efficacy index, and their contents were evaluated by using commercially available ELISA kits (Human TIMP2 ELISA Kit; ab188395; abcam, and Human HGF). ELISA Kit; ab100534) was used to carry out quantitative tests of TIMP2 and HGF in the supernatants prepared in Example 1 and Example 2 by the method described in the protocol.

Tables 1 and 2 and FIG. 5 show the quantification results of the TIMP2 protein and the HGF protein contained in the culture supernatant of the conventional method prepared by planar adhesion culture and the culture supernatant prepared by the present invention. FIG. 5 is a graph instead of a drawing showing the results of quantitative analysis of TIMP2 protein (FIG. 5 (a) and HGF protein (FIG. 5 (b)) measured by ELISA. Day indicates the number of days of culture.

In planar adhesion culture, the amount of TIMP2 secretion decreases due to cell deterioration due to overgrowth, with the upper limit being 159.5 ng / mL on Day 7, and in long-term culture exceeding 1 week, the quality of the recovered culture supernatant A decrease was suggested. On the other hand, in the novel type hollow fiber culture in which cells are sealed in the hollow fiber lumen according to the present invention and the culture medium flows outside the hollow fiber, the maximum secretion amount reaches 201.5 ng / mL on Day 32, 150 The period of recovery at concentrations above ng / mL was very long, from Day 21 to Day 50. Similarly, HGF protein, which was another index, showed a decrease in concentration up to 5,794 pg / mL on Day 7, suggesting a decrease in the quality of the collected culture supernatant in long-term culture over one week. . On the other hand, in the novel type hollow fiber culture in which cells are sealed in the hollow fiber lumen according to the present invention and the culture medium flows outside the hollow fiber, the maximum secretion amount reaches 7,391 pg / mL on Day 35, and reaches 5,000. The period of recovery at a concentration of pg / mL or more was very long, from Day 21 to Day 50.
Thus, according to the present invention, it has been realized for the first time that the production efficiency of the culture supernatant can be raised to the industrialization level by making it possible to continuously recover the culture supernatant containing the efficacy factor at a high concentration for a long period of time.

1.2. Double-stranded DNA (dsDNA) quantification
In order to evaluate the health and cytotoxicity of the cells in the long-term culture process, the concentration of dsDNA secreted into the extracellular medium due to cell death was quantified. A commercially available kit was used for dsDNA quantification according to the manual (QuantiFluor (registered trademark) dsDNA System; Promega). The results are shown in Table 3 and FIG.

  FIG. 6 is a graph instead of a drawing showing the results of quantitative analysis of dsDNA. In the flat adhesion culture of T-150 flasks, a significant increase in dsDNA concentration was observed after Day 7, suggesting that the cells would die due to overgrowth. On the other hand, in the culture supernatant of the hollow fiber culture of the present invention, no significant increase in the dsDNA concentration was observed during the culture period up to Day 50, so that the mesenchymal system proliferated as an aggregate in the sealed space in the hollow fiber. Stem cells were confirmed to maintain cell integrity even in long-term culture.

1.3. Glucose determination
Glucose, which is the main energy source of cells, was quantified in order to evaluate the concentration change in the culture medium during the long-term culture process. A commercially available kit was used for glucose quantification according to the manual (Glucose Colorimetric Assay Kit 2; BioVision). The results are shown in Table 4 and FIG.

  FIG. 7 is a graph instead of a drawing showing the results of quantitative analysis of glucose. In the culture supernatant of the hollow fiber culture of the present invention, glucose was not completely depleted during the culture period up to Day 50, but was maintained at a low concentration of 1 nmol / μL or less after Day 18.

The industrial application of the present invention is the production of culture supernatant as a pharmaceutical or cosmetic raw material. By utilizing the technology of the present invention, it is possible to achieve a reduction in production area floor area, a reduction in production personnel, an increase in production scale per production unit, and the like, compared with the production of culture supernatants by conventional adhesion culture methods. . As a result, the production cost of the culture supernatant can be reduced, the product price can be suppressed, and an optimum and effective method can be developed.

Claims (7)

A hollow fiber filter module for a hollow fiber cell culture device for producing a culture supernatant,
A filter housing (1),
A hollow fiber (3) present inside the filter housing, having a pore size of 5 nm or more and 1 μm or less, and a diameter when the lumen is rounded is 0.1 mm or more and 1.6 mm or less. The first and second ends (4a, 4b) of the yarn (3) are connected to the first and second ends (1a, 1b) of the filter housing (1), and the hollow fibers ( 3) The stem cell is introduced inside by the cell introduction part (5),
A first end cap (6a) for mounting on the hollow fiber filter module after the stem cells are introduced into the hollow fiber (1) by the cell introduction part (5), the stem cell And the movement of the culture solution does not occur through the opening of the first end (4a) of the hollow fiber (3), the first end (4a) of the hollow fiber (3) and the filter housing (1 A first end cap (6a) for sealing
A culture medium introduction part (7) connected to the filter housing (1) for introducing a culture medium into the filter housing, wherein the culture medium is supplied to supply the culture medium into the filter housing Connected to the road (8),
A culture medium discharge section (9) connected to the filter housing (1) and configured to discharge a culture medium containing a culture supernatant that has passed through the inside of the filter housing, wherein the culture supernatant inside the filter housing is Connected to the culture drainage path (10) for draining the containing culture medium;
The second end (4b) of the hollow fiber (3) and the filter housing so that movement of the stem cells and culture medium does not occur through the opening of the second end (4b) of the hollow fiber (3). A second end cap (6b) sealed with (1) ;
The culture solution does not flow from the first end (4a) of the hollow fiber (3) and the second end (4b) of the hollow fiber (3) ,
The hollow fiber cell culturing apparatus closes the lumen of the hollow fiber (3) with the stem cells, exchanges the culture solution circulating outside the hollow fibers (3) with the inside of the hollow fibers (3), and the stem cells. The culture supernatant produced by the above is moved to the outside of the hollow fiber (3), whereby the culture solution containing the culture supernatant can be discharged from the culture solution discharge part (9).
Hollow fiber filter module . The hollow fiber filter module for a hollow fiber cell culture device according to claim 1, wherein the pore size is 0.1 µm or more and 0.65 µm or less . A hollow fiber filter module for a hollow fiber cell culture device according to claim 1,
The hollow fiber filter module , wherein the stem cells are mesenchymal stem cells . A hollow fiber filter module for a hollow fiber cell culture device according to claim 1,
The cell introduction part (5),
The cell introduction part (5) is a cylindrical volume part having a spatial volume provided at a site corresponding to the hollow fiber filter connecting the first end cap (6a), and one of the cylindrical volume parts. A pusher with a gasket is inserted at the end of the tube, and the other end of the cylindrical volume is connected to the hollow fiber filter. By pushing the pusher toward the hollow fiber filter, the cells Hollow fiber filter module introduced into the filter lumen. A hollow fiber cell culture device for producing a culture supernatant comprising a hollow fiber filter module for a hollow fiber cell culture device according to claim 1,
A culture solution supply path (8) connected to the culture solution introduction section (7) and for introducing the culture solution into the culture solution introduction section (7);
A culture drainage path (10) connected to the culture medium discharge section (9) and for collecting the culture medium containing the nutrient supernatant discharged from the culture medium discharge section (9);
  Including
The lumen of the hollow fiber (3) is closed with the stem cells, the culture fluid circulating outside the hollow fiber (3) is exchanged with the inside of the hollow fiber (3), and the culture supernatant produced by the stem cells is removed from the hollow cells. The thread (3) is moved to the outside, whereby the culture solution containing the culture supernatant is discharged from the culture solution discharge part (9), and the culture supernatant is collected.
Hollow fiber cell culture device. It is a hollow fiber cell culture apparatus of Claim 5, Comprising: It is provided in the said culture solution supply path (8), and sends a culture solution in the said filter housing (1) via the said culture solution introduction part (7) A device further comprising a liquid delivery device (11) for A method for producing a culture supernatant using the apparatus of claim 5 ,
A cell introduction step of introducing mesenchymal stem cells into the hollow fiber (3) via the cell introduction portion (5);
After the cell introduction step, a first end cap (6a) is provided so that no movement of the mesenchymal stem cells or culture solution occurs through the opening of the first end (4a) of the hollow fiber (3). Using the first end (4a) of the hollow fiber (3) and sealing the filter housing (1);
A culture solution supply step for supplying the culture solution into the filter housing via the culture solution introduction part (7);
A culture solution discharging step of discharging the culture solution inside the filter housing from the culture solution discharge unit (9);
The culture solution supply step and the culture solution discharge step are continued, the culture solution is supplied into the filter housing via the culture solution introduction part (7), and the culture solution in the filter housing is discharged. In the hollow fiber (3), the culture solution is not introduced from the first end (4a) of the hollow fiber (3) and the second end (4b) of the hollow fiber (3). Culturing the mesenchymal stem cells ;
A culture supernatant recovery step of recovering the culture supernatant from the effluent obtained by the culture solution discharge step;
A method for producing a culture supernatant of mesenchymal stem cells .

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