WO2019245050A1

WO2019245050A1 - Hollow fiber cell culture device, cell culture method, and method for producing culture supernatant

Info

Publication number: WO2019245050A1
Application number: PCT/JP2019/024872
Authority: WO
Inventors: 漆畑　直樹; 勝幸隠岐
Original assignee: 株式会社バイオミメティクスシンパシーズ
Priority date: 2018-06-22
Filing date: 2019-06-24
Publication date: 2019-12-26
Also published as: JP2019216694A; JP6469287B1; CN111448305A; CN111448305B

Abstract

[Problem] To provide a culture device. [Solution] A hollow fiber cell culture device for producing a culture supernatant, said culture device comprising: a filter housing 1; hollow fibers 3 existing in the filter housing and having a pore size of from 5 nm to 1 μm inclusive, into which cells are introduced via a cell introduction part 5; a first end cap 6a for sealing a first end 4a of the hollow fibers 3 and the filter housing 1; a culture liquid introduction part 7 for introducing a culture liquid into the filter housing; and a culture liquid discharge part 9 for discharging the culture liquid having passed through the inside of the filter housing.

Description

Hollow fiber cell culture device, cell culture method, method for producing culture supernatant

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 Patent Application Laid-Open Publication No. 2017-176043 describes a cell culture method using hollow fibers. In this application, a mesenchymal stem cell is seeded in a hollow fiber, cells and a culture solution are circulated in the hollow fiber, and a useful component having a physiological activity secreted from the cell is added to the hollow fiber. Keep the concentration, and collect the culture supernatant from the hollow fiber. In this document, it is intended to restrict as much as possible the secretion from cells in the hollow fiber into the culture flowing outside the hollow fiber.

JP 2017-176043 A

(4) An object of the present invention is to provide a technique that has high robustness assuming that a culture supernatant is used as a drug, and that can be used for mass production with efficiency and reduced production cost.

Basically, the solution described in the present specification is capable of culturing cells stably by culturing the cells inside the hollow fiber and circulating the culture solution outside the hollow fiber. Based on the knowledge that high-quality cells and culture supernatants can be obtained inexpensively and in large quantities.

The apparatus disclosed in the present specification is a hollow fiber cell culture apparatus for producing a culture supernatant. And this device is
Filter housing 1,
The hollow fiber 3 present inside the filter housing, having a pore size of 5 nm or more and 1 μm or less, and the first and second ends 4 a and 4 b of the hollow fiber 3 are the first and second ends of the filter housing 1. 1a and 1b, the hollow fiber 3 having a cell introduced therein by a cell introduction unit 5;
A first end 4a of the hollow fiber 3, a first end cap 6a for sealing the filter housing 1,
A culture solution introduction unit 7 connected to the filter housing 1 for introducing a culture solution into the filter housing, and connected to a culture solution supply passage 8 for supplying a culture solution into the filter housing; When,
A culture medium discharge section 9 connected to the filter housing 1 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 comprising:

This device comprises a culture solution supply channel (8),
The device is preferably provided in the culture solution supply passage (8) and further having a liquid sending device (11) for sending the culture solution into the filter housing (1) through the culture solution introduction section (7). .

装置 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 inner lumen is made circular.

This device further has a cell introduction part (5),
The cell introduction part (5) is connected to a site corresponding to the hollow fiber (3) connecting 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 space volume provided at a part corresponding to the hollow fiber filter connecting the first end cap 6a, and one end of the cylindrical volume part has: A pusher with a gasket is inserted, and the other end of the cylindrical volume is connected to a hollow fiber filter. By pushing the pusher in the direction of the hollow fiber filter, cells are inserted into the lumen of the hollow fiber filter. Preferably, the device is introduced.

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

Using this apparatus, a culture supernatant can be produced. This manufacturing method
A cell introduction step of introducing cells into the hollow fiber 3 via the cell introduction part 5;
A culture solution supply step of supplying a culture solution into the filter housing via the culture solution introduction unit 7;
A culture solution discharging step of discharging the culture solution inside the filter housing from the culture solution discharge section 9, and a step of culturing cells inside the hollow fiber 3;
A culture supernatant collecting step of collecting a culture supernatant from the discharged liquid obtained in the culture liquid discharging step;
And a method for producing a culture supernatant.

According to the present invention, it is possible to provide a technique which has high robustness assuming that a culture supernatant is used as a drug, and which can be used for mass production with an efficient and reduced production cost.

The present invention is characterized in that cells are sealed in the lumen of the hollow fiber, and the movement of the culture solution in the lumen is mainly caused by diffusion to the outside of the hollow fiber through the hollow fiber filter. It has a great effect on cells where each other cells are tightly aggregated, such as leaf stem cells. Considering the long-term culture of mesenchymal stem cells by the conventional hollow fiber culture method, the mesenchymal stem cells seeded in the hollow fiber lumen easily form aggregates and proliferate there. If the culture conditions are provided, the circulating culture solution is blocked while closing the hollow fiber lumen. Even if the whole of the multiple hollow fibers is not occluded by cells, it is difficult to achieve the same culture conditions, cell properties, and quality between the plugged and non-embedded hollow fibers. The risk is expected to be adopted as a robust and robust manufacturing method for drug development. In addition, when a hollow fiber is used that is so thick that the inner cavity of the hollow fiber is not shielded by cells, cell death may occur due to nutrient depletion in the deep part of the cell aggregate after cell proliferation.

In addition, the only reason in the industry that cell culture using a hollow fiber filter has so far been to circulate the culture medium on the side where cells are present is due to the efficiency of products from cells in the lumen of the hollow fiber. In order to recover the cells, it is necessary to collect the cells through the filter not as the culture medium in the opposite space but as the culture medium in the space where the cells exist. 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 lumen of a hollow fiber having an appropriate inner diameter is closed with cells and a culture medium is not actively sent in the lumen of the hollow fiber, based on a flexible idea that is not restricted to the conventional method. . Instead, by appropriately exchanging and circulating the culture medium outside the hollow fiber, the cells inside the hollow fiber can be maintained healthy for a long time, and the culture supernatant having sufficient biological activity can be obtained. , It was found that it can be recovered outside the hollow fiber for the duration.

As a result, a method for producing a culture supernatant that has high robustness and can reduce production costs has been completed, and the production method 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 device. FIG. 2 is a conceptual diagram illustrating an example of a ferrule. FIG. 3 is a conceptual diagram illustrating an example of a cell introduction unit. FIG. 4 is a photograph instead of a drawing showing 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 using 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 result of quantitative analysis of glucose. FIG. 8 is a conceptual diagram showing the appearance of the end of the filter housing.

The first aspect disclosed herein relates to a hollow fiber cell culture device for producing a culture supernatant. Basically, this device cultivates cells (for example, stem cells) in a hollow fiber and circulates a culture solution outside the hollow fiber, so that cells can be cultured stably and the culture supernatant is easily removed. It can be collected. This device is also called a hollow fiber filter module.

(1) FIG. 1 is a conceptual diagram showing a configuration example of a culture device. As shown in FIG. 1, the apparatus comprises: 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, and a culture solution discharge unit 9. Having.

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

The hollow fiber (3) is a hollow fiber (a filter having a void inside) existing 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, preferably 0.1 μm or more and 0.65 μm or less. The hollow fiber 3 has, for example, an example of a diameter of 0.1 mm or more and 1.6 mm or less when the inner cavity is circular. With respect to the hollow fiber constituting the hollow fiber filter module, an example of 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 or more and 1.6 mm or less, or 0.1 mm or more and 0 mm or less. 0.5 mm or less. Hollow fiber materials include materials with low hydrophilicity and low protein adsorption, such as polyethersulfone (PES), cellulose acetate (CE), polysulfone (PS), polyvinylpyrrolidone (PVDF) and polyacrylonitrile (PAN). It is preferable to select it, but it has the characteristics of a filter and is resistant in any sterilization process such as gamma sterilization, autoclave sterilization, ethylene oxide gas sterilization, etc. without impairing the substantial performance of the filter. , The material is not limited. An example of the nominal pore size of the hollow fiber membrane is an ultrafiltration membrane or microfiltration membrane of 5 nm (corresponding to 3 kDa) to 1 μm, preferably a microfiltration membrane 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 and tertiary structure of the target recovered supernatant in the culture supernatant, hydrophobicity, ionic interaction, and the like. Further, the length of the tube on the long diameter side of the hollow fiber is preferably 20 cm to 100 cm, which is easy to handle and suitable, but is not limited as long as it is allowable in designing the hollow fiber cell culture apparatus constituting the present invention. Absent.

The first and second ends 4a, 4b of the hollow fiber 3 are preferably connected to the first and second ends 1a, 1b of the filter housing 1. However, after closing the first and second ends 4a and 4b of the hollow fiber 3 and the first and second ends 1a and 1b of the filter housing 1 with the end caps 6a and 6b, respectively, It is preferable that the inner space of the hollow fiber 3 and the internal space of the filter housing 1 are configured to have no spatial connection. FIG. 8 is a conceptual diagram showing an example of the end of the filter housing after housing the hollow fiber. In the example shown in FIG. 8, a hollow fiber is accommodated in the housing 21 of the filter housing, and the openings 23 leading to the hollow fiber lumen are aligned at the end of the filter housing. In this state, when the end cap is attached to the filter housing, the opening 23 is closed by the end cap, the lumen of the hollow fiber forms a closed system, and the lumen of the hollow fiber 3 and the internal space of the filter housing 1 are separated. , To have no spatial connection. However, since there are a plurality of micro holes in the hollow fiber main body itself, it is possible to exchange substances capable of passing through the micro holes. As in the example shown in FIG. 8, the terminal region of the filter housing may be sealed by an assembly 25 other than the holes of the hollow fibers. In this way, after the stem cells have been introduced into the hollow fiber 1 by the cell introduction unit 5 and the first end cap 6a to be attached to the front empty fiber filter module is attached, the stem cells and the culture solution Is prevented from occurring through the opening at the first end 4a of the hollow fiber 3. That is, the state is such that the culture solution does not flow from the first end 4a of the hollow fiber 3. Further, when the second end cap 6b is attached, the second end 4b of the hollow fiber 3 is sealed so that the movement of the stem cells and the culture solution does not occur through the opening of the second end 4b of the hollow fiber 3. Is done. Then, the culture solution does not flow from the second end 4 b of the hollow fiber 3.

The hollow fiber 3 is a cell into which a cell is 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. It is a cell type that secretes or produces a target component outside the cell, and may be used for the purpose of recovering the target component. Particularly preferred cells that can enjoy the effects of the present invention are adherent cells, and more preferably, mesenchymal stem cells are selected. Mesenchymal stem cells used are those isolated and cultured from humans, dogs, horses, cats, pigs, mice, rats, and the like. Tissues from which mesenchymal stem cells are separated include umbilical cord, cord blood, bone marrow, amniotic membrane, placenta, dental pulp, fat, cartilage, menstrual blood, breast milk, etc., and the source tissues and animal species are particularly limited Not something. In addition, mesenchymal stem cells must have been transduced or modified to establish immortalized cell lines, to provide cell characteristics that can withstand long-term culture, or to improve the effectiveness of culture supernatants. May be used.

The composition and type of the culture are not particularly limited. A culture solution similar to that used in the conventional adhesion culture method using a flask or the like may be used. When mesenchymal stem cells are taken as an example, they are generally grown in an αMEM medium containing 10% serum. In order to further improve cell growth and shorten the process, a medium sold by each company or a medium with a unique formulation may be used. Also, considering that the industrial value of the culture supernatant is an agent used in living organisms such as therapeutic applications and cosmetic raw materials, the composition of the culture solution may not use human or animal-derived components. Desirable (eg, cell culture kit Procul キット AD®; Rohto Pharmaceutical). In addition, a technique has been reported to enhance the action of mesenchymal stem cells by various stimuli. In order to prepare cells having a more effective therapeutic effect and a culture supernatant, a culture solution utilizing these techniques has been reported. Can also be used. As described above, there are various options for a medium for growing mesenchymal stem cells, and a medium having a performance suitable for the origin of the tissue and the species of the organism is selected. Any culture solution that can be used to prepare the sera may be used.

方法 In addition, depending on the method of producing the culture supernatant, there has been reported a method of maintaining a culture solution close to the basal medium, which is not intended for cell growth, at the stage of collecting the culture supernatant. This is for the purpose of eliminating as much serum as possible as possible 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, add the cellulosic water-soluble polymer methylcellulose (MC) or carboxymethylcellulose (CMC) to the culture medium. ), Other cellulose derivatives, or other water-soluble polymers such as polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), or sodium alginate and their gelling agents, using a technique to increase the viscosity of the culture solution. May be. In such a case, the concentration of the water-soluble polymer can be set with reference to the previous report. As a result, it is possible to form spheroids of uniform size in the cell culture space in the hollow fiber and to arrange them uniformly in space.

The first end cap 6a is for sealing the first end 4a of the hollow fiber 3 and the end 1a of the filter housing. End caps are used to seal both ends of the hollow fiber filter module. The ends of the hollow fibers 4a and b are preferably sealed so that movement of cells and culture medium does not occur through the openings at both ends of the hollow fiber. For this purpose, a disc-shaped silicon gasket is most suitable as an end cap that comes into contact with the ends 4a and 4b of the hollow fibers, and a resin cap for fixing the hollow fiber filter module to 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 mounted to seal the cell introduction side after the cells are introduced. The end cap to be attached after the cells are introduced may be replaced with the cell introduction unit 5 configured to also have a function as the end cap 4a. It is preferable that the end caps on either side are not 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 is connected to the lumen of the hollow fiber filter through the ferrule connection port. It is introduced to. Another example of the cell introduction part 5 is a cylindrical volume part having a spatial volume provided at a position corresponding to the filter housing end 1a and the hollow fiber end 4a to which the first end cap 6a is connected, A pusher having a gasket is inserted into one end of the cylindrical volume, and the other end of the cylindrical volume is connected to the hollow fiber 3 and pushes the pusher in the direction of the hollow fiber 3. Thus, the device is preferably one in which 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 depending on the size of the hollow fiber filter module used and the amount of the cell suspension to be introduced.

For example, in the industrialization stage of pharmaceutical production, etc., it is assumed that a cell suspension of several liters or more is introduced per hollow fiber filter module. In this case, a tubing having 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 other side, and a peristatic pump is connected. The cells can be sent to the ferrule connection port side by the driving liquid sending. FIG. 2 is a conceptual diagram illustrating an example of a ferrule. FIG. 2 is quoted from the website of Nitto Metal Industry Co., Ltd. The cell introduction part 5, the filter housing end 1a and the hollow fiber end 4a are connected via a ferrule, and cells can be aseptically introduced into the hollow fiber filter. After the cells are introduced into the hollow fiber, the hollow fiber filter can be sealed. Ferrule connections are also known as sanitary piping and sanitary fittings. Ferrule connections can be achieved, for example, using ferrules, gaskets, and clamp bands. The ferrule has a groove in a connection portion, and is fixed by clamping with a clamp band with a cross-shaped (or O-ring shaped) gasket interposed therebetween.

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

Furthermore, it is also possible to select a part that combines the purpose of the cell introduction part and the end cap described above. 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 corresponding to the connection type of the filter housing ends 1a and 1b, and more preferably a ferrule connection port is selected for both. Depending on another method, the hollow fiber filter module and the cell introduction part 5 may be connected in advance at the time of their manufacture. In the example of FIG. 3, a cell suspension is sealed inside the cell introduction unit 5. Then, it is connected to the filter housing end 1a using a ferrule connection or the like. Then, by pressing the piston pusher, the cell suspension is injected into the hollow fiber 3. 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. The cell introduction section 5 is at least composed of an outer cylinder, a gasket, a pusher, and a lock mechanism. When this is used, when the cells are pushed into the hollow fiber lumen by the gasket, the end 4a of the hollow fiber 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 the cell introduction, thereby reducing the risk of contamination. Further, a dedicated end cap 6a may be used for closing the filter housing end 1a and the hollow fiber end 4a on the cell introduction side.

The culture solution introduction unit 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 solution introduction unit 7 is an element for introducing a culture solution into the inside of the filter housing 1. On the other hand, the culture solution introducing section 7 is not connected to the inside of the hollow fiber 3 but is for introducing the culture solution into a region inside the filter housing existing around the hollow fiber. Then, the culture solution introduction unit 7 is connected to a culture solution supply passage 8 for supplying a culture solution into the filter housing. The culture solution supply path 8 exists outside the filter housing 1. As a result, the culture solution is introduced from outside the filter housing 1 into the filter housing. The culture solution supply path 8 is connected to, for example, a culture solution tank containing a culture solution, and is supplied with the culture solution.

That is, the apparatus includes a flow channel culture solution supply passage 8 for introducing a culture solution into the filter housing 1 and a flow channel culture solution discharge passage 10 for discharging the culture solution outside the hollow fiber filter housing. Is preferred. These are connected to the culture solution introduction unit 7 and the culture solution discharge unit 9 of the hollow fiber filter module. As a material for the tube, a silicon-based material, a thermoplastic elastomer-based material, a polyvinyl chloride-based material, or the like is selected as a suitable material. However, the present invention is not necessarily limited to these, and does not adversely affect the results of cell culture, can sterilize materials, does not denature or unintentionally adsorb target substances and components, and removes cells from these eluates. If there is no toxicity, irritation, carcinogenicity, pyrogenicity, etc., it can be arbitrarily selected.

It is preferable that the liquid supply device 11 is further provided. 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 sending device 11 is an element for sending a 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 sending device 11 is a pump. As a method for supplying or circulating the culture solution into the filter housing 1, a method using a peristatic pump is suitable as a method that can be used in any production scale. However, when the culture scale is large, a single-use pump head is required. A floating magnet pump (eg, PuraLev: LEVITRONIX) composed of a pump driver can also be adopted as an ideal liquid sending method. However, the driving method of the liquid sending can be selected without being limited to these.

The culture solution storage container is connected to, for example, a culture solution supply passage 8 for introducing a 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 bag shape, or a 3D process bag shape. Alternatively, in the case of a large-scale culture scale, a stainless steel container may be used. In the case where the culture solution is refluxed through the filter housing 1, a culture solution for discharging the culture solution in the filter housing 1 is used. Preferably, the discharge path 10 is also connected. This apparatus preferably has a culture solution sampling port for monitoring the state of culture, and preferably has various centers for 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 for storing a culture solution to be introduced into the filter housing 1, a culture solution for discharging the culture solution from the filter housing 1 is also provided. Those having another culture solution storage container are preferred.

(4) The culture solution discharge section 9 is connected to the filter housing 1. The culture solution discharge section 9 may be formed integrally with the filter housing 1. The culture solution discharge section 9 is an element for discharging the culture solution that has passed through the inside of the filter housing. The culture solution discharge section 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 section 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.

要素 The components such as the filter housing, housing terminal connection, hollow fiber fixing material, sealant, and flow path forming port can be designed with reference to existing technologies and products. For example, as for the housing terminal connection portion, a design corresponding to a highly versatile ferrule sanitary connection is preferable. The shape and design of these can be freely selected. In addition, regarding these materials, it is preferable to select from general materials that are conventionally used in the biotechnology-related field, but it is not necessarily limited to these, and adversely affect the results of cell culture. It is possible to sterilize the material without imparting it, and if there is no denaturation or unintended adsorption of the target substance or component, and if the eluate is toxic such as cytotoxicity, irritation, carcinogenicity, pyrogenicity, etc. Can be selected. For example, as a hollow fiber fixing material for fixing and fixing the hollow fibers at the end of the filter housing, urethane resin or epoxy resin is acceptable. Further, a silicon material is suitable for a sealing material used for joining each component to form a complete seal. It is recommended that the filter housing be made of polysulfone, which is tough, rigid, transparent, and resistant to various chemicals. The channel forming port can be similarly selected from polysulfone, polypropylene, and the like.

装置 In order to proliferate 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 chosen. As long as the hollow fiber filter module in which the cells are present is controlled to the target temperature, the mode of temperature control does not matter. 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 sent to the filter module 1 and the flow path forming port, and control is performed so that the optimum temperature is reached immediately before the culture solution is sent to the filter module 1. In addition, the filter module 1 also has a mechanism for maintaining the optimum temperature, and the flow path forming port and the flow path on the side where the culture solution is discharged from the filter module 1 are cooled to 4 to 10 ° C. to store the culture solution. The liquid container may also be cooled to 4 to 10 ° C. to take measures to suppress denaturation and decomposition of the medium components and cell secretion components in long-term culture.

Using this apparatus, a culture supernatant can be produced. This manufacturing method
A cell introduction step of introducing cells into the hollow fiber 3 via the cell introduction part 5;
A culture solution supply step of supplying a culture solution to the inside of the filter housing 1 through the culture solution introduction unit 7;
A culture solution discharging step of discharging the culture solution inside the filter housing 1 from the culture solution discharge section 9;
Culturing cells inside the hollow fiber 3;
A culture supernatant collecting step of collecting a culture supernatant from the discharged liquid obtained in the culture liquid discharging step;
And 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-described culture solution and cells are used. The cells to be seeded are preferably cells prepared by ordinary adherent plate culture and easily dispersible with a peeling agent. Alternatively, cells prepared by microcarrier culture or spheroid culture may be used. It may be used, as long as it is large enough to fit into the inner hole of the hollow fiber filter, and may contain microcarriers that are not adhered to the cells but may be attached, or cells may form aggregates .

<Assembly of device>
A hollow fiber filter module including at least a filter housing 1, a hollow fiber 3 disposed inside the filter housing 3, filter housing ends 4a and 4b, a culture solution introduction unit 7, and a culture solution discharge unit 9, is provided with end caps 6a and 6b. A gasket or a sealing material is attached to the ends 4a and 4b of the hollow fiber filter housing, and the culture solution supply passage 8 and the culture solution discharge passage 10 are connected to the culture solution introduction unit 7 and the culture solution discharge unit 9, respectively. Here, these components are referred to as component 1. It is preferable to perform a sterilization process by a sterilization method that is allowed by the material of the component 1.

Also, when the cell introduction section 5 for introducing cells into the hollow fiber is not a sterilized product and needs to be sterilized, sterilization is performed according to the sterilization principle of a method permitted by the material. Preferably, it is applied.

However, the present invention is not limited to the representative examples shown here, and by using the existing bioprocess technology, the most appropriate connection mode of the device can be selected according to the purpose of the practitioner. For example, when connecting tubes and the like that form the culture medium flow path, when using existing aseptic connection technology often used in the bioindustry, or when incorporating a stainless steel container as a culture medium storage container, such a connection is required. The connection mode of the device according to the above can be taken. In addition, depending on the production scale, facility requirements, overall plan for aseptic operation, requirements for containers and liquid transfer lines used, etc., this technology is used in the production of general biotechnology-based drugs, etc. The technology may be designed by adopters.

<Introduction of cells>
Next, in the aseptic operation area ISO class 5 or the control area where practically aseptic operation is possible, remove “Component 1” from the case where the end cap 6a includes a gasket or a sealing material and remove the cells. Connect the introduction unit 5. Thereafter, the cell suspension prepared for seeding is put into the cell introduction part. The cell suspension is preferably a culture solution, but is not particularly limited as long as it is a solution, a thickener, or a gelling agent that is acceptable within a range that 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 end 1b of the filter housing and the end 4b of the hollow fiber on the opposite side of the hollow fiber are sealed with an end cap 6b, which may include a gasket or 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 through, the cells are completely retained in the hollow fiber lumen without being discharged into the culture solution circulation channel. In addition, it is desirable to avoid injecting gas into the hollow fiber as much as possible in order to avoid the uneven distribution of cells in the hollow fiber lumen. Furthermore, in order to suppress the formation of cell aggregates, a cell-based water-soluble polymer such as methylcellulose MC and carboxymethylcellulose CMC, other cellulose derivatives, or other water-soluble polymers such as polyethylene glycol PEG and polyvinylpyrrolidone are used to suppress cell aggregate formation. A method of increasing the viscosity of the culture solution by adding PVP or sodium alginate or a gelling agent thereof may be used.

Also, for the range of cell number to be introduced into the hollow fiber lumen, To illustrate the case of mesenchymal stem cells as a representative example, the total in luminal body volume 1 cm ³ per hollow fiber constituting the hollow fiber filter module, 1 × 10 It can be selected from the range of ⁴ to 5 × 10 ⁷ , more preferably the range of 1 × 10 ⁵ to 5 × 10 ⁶ , even more preferably the range of 5 × 10 ⁵ to 1 × 10 ⁶ 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 hermetically sealed by attaching an end cap 6a to the end 1a of the filter housing and the end 4a of the hollow fiber if there is a gasket or a sealing material. This is designated as component 2. After sealing, the hollow fiber filter module should be kept horizontal to avoid bias of cells 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 aseptically injected. When the culture solution is circulated, the culture solution discharge passage 10 is also connected to the same culture solution storage container. When not using the circulation type, the culture solution discharge passage 10 is connected to a culture solution storage container different from the culture solution storage container to which the culture solution supply passage 8 is connected. This is designated as component 3.

In order to grow or maintain cells, a device for controlling the hollow fiber filter module in the range of 30 ° C. to 42 ° C. is required depending on the type and purpose of the cells. ° C is selected. In addition, when the pH of the culture solution is controlled using sodium hydrogen carbonate and carbon dioxide, it is necessary to control the carbon dioxide concentration. As a typical example, the component 3 after the injection of the culture solution is placed in a carbon dioxide incubator maintained at an optimum temperature and carbon dioxide concentration for the cells to be used. In this case, carbon dioxide permeates the culture solution from the culture solution channels 8 and 10 made of silicon material, and the pH is controlled. However, as long as the hollow fiber filter module 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 unit 7 on the side where the culture medium is sent to the hollow fiber filter module, and the temperature is adjusted to the optimum temperature immediately before the culture solution is sent to the hollow fiber filter module. In addition to controlling the medium to reach the temperature, the hollow fiber filter module also has a mechanism for maintaining the optimum temperature, and further includes a culture medium discharge section 9 and a culture medium discharge path 10 on the side where the culture medium is discharged from the hollow fiber filter module. May be cooled to 4 to 10 ° C., and the culture solution storage container may be cooled to 4 to 10 ° C. to prevent denaturation and degradation of the medium components and cell secretion components in long-term culture.

Thereafter, a peristatic pump or the like is attached to one or both of the culture solution flow paths 8 and 10, and the culture medium circulation is started by driving it. However, as described above, the circulation type of the culture solution is not particularly limited by selecting an existing technique. The conditions for feeding the culture solution may be intermittent or continuous. In the case of intermittent operation, 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 cell used, the culture solution, the type of hollow fiber filter, the scale of the hollow fiber filter module, and the target component collected in the culture supernatant. It is possible to determine the optimal liquid sending speed.

When the mesenchymal stem cells as an example, a total of the luminal body volume 1 cm ³ per hollow fiber constituting the hollow fiber filter module, feed volume per hour is selectable in the range from 1 mL of 10,000 mL, More preferably, a range of 10 mL to 1,000 mL is selected, and even more preferably, a range of 50 mL to 500 mL is selected. The liquid volume of the culture liquid to be filled in the culture apparatus, when the mesenchymal stem cells as an example, a total of the luminal body volume 1 cm ³ per hollow fiber, may be selected in the range of 0.01 mL of 300 mL, and more Preferably, a range of 0.2 mL to 30 mL is selected, and more preferably, a range of 1 mL to 5 mL is selected. It is also possible to reduce the amount of the culture solution at a stage where the number of cells is small in the initial stage of the culture, and to increase the amount of the culture solution at a stage where the number of cells after the proliferation is large.

<Medium exchange>
The cells are introduced into the lumen of the hollow fiber, and the medium is exchanged when the cells are in the growth phase, which is determined depending on the type of the cells and the culture solution to be used. In the case of mesenchymal stem cells, for example, it is preferable that the culture be performed on a batch basis with a total medium exchange about once every four days. However, the present invention is not limited to this, and it is within a range that can be dealt with by half replacement of the culture solution storage container until the number of cells increases. The present invention can also be satisfactorily carried out by using a fed-batch culture or a continuous culture technique as a control of the culture solution. The practitioner may set the value within an arbitrary range in which cells can be maintained without dying.

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

In addition, as a method of monitoring the degree of cell death, it can be evaluated by quantifying the concentration of double-stranded DNA (dsDNA), lactate dehydrogenase (LDH), and the like in the culture supernatant. The degree of cell proliferation in the hollow fiber lumen can be evaluated by previously evaluating the degree of cell death in the hollow fiber lumen and the correlation between these values.
By these methods, it is possible to know the start time of the collection of the culture supernatant containing the target product at a high concentration and the period until the stop of the collection at which the soundness and homogeneity of the cells are limited.

<Preparation and recovery of culture supernatant>
Preparation and recovery of the culture supernatant is started when the cells can proliferate and the expected amount of the target component can be confirmed. For example, when the culture medium is circulating, the timing of collecting the culture supernatant circulating in the hollow fiber housing is, for example, mesenchymal stem cells. If batch culture is used to prepare the culture supernatant, The collection is preferably 1 to 10 days after the medium exchange immediately before the collection of the culture supernatant, more preferably 2 to 8 days, most preferably 3 to 5 days. Also, a point that is emphasized as an enormous effect of the present invention is that the collection of the culture supernatant can be continued for a very long time after the cells are introduced into the hollow fiber lumen. The required amount of culture supernatant may be collected. In the case of mesenchymal stem cells, in flat adherent culture using a flask, the maintenance of cells in a healthy state after reaching confluence is limited to several days. It can be kept healthy for more than 50 days.

In addition, cells exist only in the hollow fiber lumen, and the technical common knowledge in the field prior to the present invention is that a substantially feasible method for recovering a culture supernatant containing secretions of cells is as follows. In this method, a culture solution on the side where cells are present is circulated through a hollow fiber membrane to collect the culture solution on the same side as a culture supernatant (Patent Document 1). On the other hand, in the present invention, the culture supernatant is collected from the outside of the hollow fiber by diffusion from the hollow fiber lumen through which the cells are closed and there is no active circulation to the outside of the hollow fiber through the hollow fiber membrane. In addition to the 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 of surplus tissue from subcutaneous adipose tissue as 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 of a lipid fraction, an adipose tissue fraction, and an aqueous fraction in order from the top. The upper and lower layers were discarded, leaving the middle adipose tissue fraction. To the remaining adipose tissue fraction, a 0.15% collagenase enzyme solution in an amount of 4 times the tissue weight was added, and the mixture was permeated at 37 ° C. for 1 hour to perform enzyme treatment. After the adipose tissue is dispersed, centrifuge (400 xg for 5 minutes) and suspend as a stromal vascular cell fraction containing mesenchymal stem cells in 30 mL of PBS (-) solution. did. Thereafter, the suspension was passed through a cell strainer (70 μm diameter), and the flow-through fraction was subjected to centrifugation again (400 × g for 5 minutes), and the tissue residue and the like captured by the cell strainer were discarded. The precipitate fraction was suspended in 6 mL of a serum-free culture solution (Procul AD; Rohto Pharmaceutical), and the whole volume was inoculated into a T-25 flask (CellBIND (registered trademark); Corning), and then in an incubator (37 ° C, 5% CO 2). _The primary culture was started by standing still in ₂ ).

(2) Subculture (P0 → P1)
The medium was completely exchanged once every three days, the supernatant was discarded, and the cells growing on the bottom of the flask were selectively grown. On the eleventh day from the seeding, 2 mL of an enzyme solution (TrypLE Select (registered trademark); Thermo Fisher Scientific) was added to the cells that had grown to semi-confluence in a T-25 flask, and detached (37 ° C., 5 minutes) Stationary). The cells were diluted with PBS (-) and subjected to centrifugation (400 xg for 5 minutes). The precipitated cells were suspended in a culture solution, and the number of cells was measured by trypan blue staining. As a result, 1.2 × 10 ⁶ viable cells could be recovered, and the entire amount was collected in a T-150 flask (CellBIND (registered trademark); Corning) were seeded in total with 24 mL of serum-free culture solution (4,000 cells / cm ² ), and allowed to stand in an incubator (37 ° C., 5% CO ₂ ) for subculture.

The medium was completely exchanged once every three days, the supernatant was discarded, and the cells growing on the bottom of the flask were selectively grown. On the fourth day after seeding, 6 mL of an enzyme solution (TrypLE Select (registered trademark); Thermo Fisher Scientific) was added to the cells that had grown to semi-confluence, and the cells were detached (rest at 37 ° C. for 5 minutes). The cells were diluted with PBS (-) and subjected to centrifugation (400 xg for 5 minutes). The precipitated cells were suspended in a serum-free culture medium, and the number of cells was counted by trypan blue staining. As a result, it was confirmed that the viability was 98.7% and the number of viable cells recovered was 9.8 × 10 ^6. And stored at 4 ° C. until the start of culture.

1.2. Hollow fiber culture (P2)
(1) Assembly of equipment
The filter housing ends 1a and 1b have a sanitary connection port shape, and a hollow fiber filter module (PES MidiKros TC module / 0.2μm fractionation size, filter membrane area) with a Luer lock connect at the culture solution introduction and culture solution discharge portions. 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 including a silicon gasket and a silicone sealant was closed tightly by clamping so as to close the filter housing end 1b and the hollow fiber end 4b.

In addition, a 3 cm silicon tube was 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 a hose port. A luer lock port was attached to the opposite side of the hose port, these were fixed with a binding band, and a luer lock connect stopper was attached to the luer lock to provide a 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.

{Circle around (5)} The culture medium introduction part 8 and the culture medium discharge path 10 of 50 cm silicon tube were attached to the culture medium introduction part 7 and the culture medium discharge part 9 of the hollow fiber filter module, respectively, via a Luer lock connect. A female CPC coupling and its cap (male type) were attached to the open-side end of the culture solution introduction part 8 and the culture solution discharge passage 10 and sealed. These were sterilized by high-temperature and high-pressure steam sterilization (121 ° C./20 minutes) to prepare Component 1.

(2) Introduction of culture solution and cells
In a safety cabinet, a 2D process bag (FLEXBOY BAG 500 mL MPC MALE COUPLERS; Sartorius Tedim Japan) is filled with 350 mL of the culture solution (Procul AD; Rohto Pharmaceutical), and the two CPC connection ports of the process bag are filled. Then, the CPC coupling of the culture solution supply channel 8 and the culture solution discharge channel 10 of the component 1 which had been sterilized was connected to form a closed circulation path for the hollow fiber filter module and the culture solution storage container.

Next, the stopper of the Luer lock connect of the cell introduction part 5 was removed, and the 20 mL capacity syringe (Nipro syringe lock type, medical device notification number 27B1X00045000033: Nipro) from which the plunger was removed was attached to the end of the filter housing (Luer lock connect). The cell suspension was connected to 1a), and the whole amount of the cell suspension prepared to 15 mL was introduced into a syringe (9.0 × 10 ⁶ cells). Attach the plunger to the syringe, turn the hollow fiber filter module vertically with the cell introduction side upward, and push the plunger in about 10 seconds to introduce the cell suspension so as to fill the hollow fiber lumen. Completed. At this time, operation was performed so that air did not enter the hollow fiber lumen. Since the total volume of the hollow fiber filter lumen derived by calculation was about 15 cm ³ , the cell seeding density was about 6.0 × 10 ⁵ cells / cm ³ . Next, the clamp is removed and the cell introduction device is removed, and an end cap 6a having a ferrule sanitary connection port and including a silicon gasket and a silicone sealing material is attached to the exposed filter housing end 1a. The clamp was tightened and sealed so as to close 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 cells into the lumen of the hollow fiber 3, thereby completing the component 2.

(3) Start circulation 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.) was a bicarbonate buffer system, gas exchange was performed through the silicon tubes in the culture solution supply channel and the culture solution discharge channel. A peristatic pump was attached to one position of the culture solution supply path 8, and by driving it, the circulation of the culture solution in the flow path leading into the hollow fiber filter housing was started. The liquid sending speed was about 30 mL / hour, and the pump was driven continuously. This was set so that the volume of the hollow fiber lumen was 2 mL / hour per 1 cm ³ .

(4) Culture medium exchange and culture supernatant
From the start of the hollow fiber culture to the twelfth day, half-replacement of the culture solution was performed once every three to four days. Thereafter, the entire exchange was performed once every three to four days. In addition, in order to evaluate the culture supernatant, the culture start date (Day 0), the culture start 4 days (Day 4), the culture start 7 days (Day 7), the culture start 11 days (Day 11), the culture start 14 days (Day 14), 18 days after the start of culture (Day 18), 21 days after the start of culture (Day 21), 25 days after the start of culture (Day 25), 28 days after the start of culture (Day 28), 32 days after the start of culture (Day 32), culture 35 days after the start (Day 35), 39 days after the start of culture (Day 39), 42 days after the start of culture (Day 42), 46 days after the start of culture (Day 46), 50 days after the start of culture (Day 50), the culture supernatant at the time A 3 mL portion was collected using a syringe from the sampling port of the process bag into which the culture solution was placed, and the culture was terminated at the time of Day 50. The collected culture supernatant is 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) and used for analysis. Stored frozen at -28 ° C.

(5) Confirmation of cells
After completion of the culture, the device was disassembled, and the inside of the housing of the hollow fiber filter module was filled with a cell detachment enzyme solution TrypLE Select (Thermo Fisher Scientific). After performing an enzyme treatment at 37 ° C. for 30 minutes to loosen the adhesion between the hollow fiber membrane and the cells, 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 instead of a drawing showing cells taken out from the hollow fiber lumen.

[Example 2]
1. Planar 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)
Regarding the remaining AD-MSC (P1 passage cell) prepared in Example 1 and seeded on the hollow fiber filter, 8 × 10 ⁵ 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 the seeding, the culture supernatant was recovered when it reached semi-confluence. After that, the culture medium was completely exchanged once every 3 to 4 days, and the culture medium was exchanged at 7 days after the start of culture (Day 7), 11 days after the start of culture (Day 11), and 14 days after the start of culture. On (Day 14), the culture supernatant was collected before replacing the medium. These culture supernatants were 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 they were used for analysis. Stored frozen at ℃.

[Example 3]
1. Analysis of culture supernatant 1.1. ELISA method (TIMP2)
Culture supernatants derived from mesenchymal stem cells contain various proteins, and it is thought that at least some of them exert pharmacological actions. In particular, the Tissue Inhibitor of Metalloproteinase (TIMP) family, which is an endogenous inhibitor of Metalloproteinase (MMP), and the Hepatocyte Growth Factor (HGF), a growth factor, are contained at high concentrations in the culture supernatant of mesenchymal stem cells. Is a factor that is known to

The presence of an analgesic effect in the culture supernatant of mesenchymal stem cells has been reported (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 a similar therapeutic effect (Nat. Med. 2008 Mar; 14 (3): 331-6. Distinct roles of matrices metalloproteases in the the early-and-late-phase development of of neuropathic pain). . In addition, culture supernatants derived from mesenchymal stem cells have been reported to have a therapeutic effect on multiple sclerosis, but HGF has been identified as an effective 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 typical secretion factors that serve as an index of the efficacy of the culture supernatant, and their contents were evaluated using commercially available ELISA kits (Human \ TIMP2 \ ELISA \ Kit; ab188395; abcam and Human \ HGF Using ELISA Kit; ab100534), the supernatants prepared in Examples 1 and 2 were subjected to TIMP2 and HGF quantitative tests by the method described in the protocol.

Tables 1 and 2 and FIG. 5 show the results of quantification of the TIMP2 protein and the HGF protein contained in the culture supernatant prepared by the conventional method and the culture supernatant prepared according to the present invention, which were prepared by the planar adhesion culture. 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 flat adherent culture, the amount of TIMP2 secretion decreases due to cell deterioration due to overgrowth up to 159.5 ng / mL on Day 7, and in long-term culture exceeding one week, the quality of the recovered culture supernatant A decrease was suggested. On the other hand, in the novel method of hollow fiber culture in which the cells are sealed in the hollow fiber lumen according to the present invention and the culture solution flows outside the hollow fiber, the maximum secretion amount reached 201.5 ng / mL on Day 32, The period of recovery at concentrations of ng / mL or more was very long, from Day 21 to Day 50. Similarly, the concentration of HGF protein, which was another index, decreased at the upper limit of 5,794 pg / mL on Day 7, suggesting that the quality of the recovered culture supernatant deteriorated in long-term culture exceeding 1 week. . On the other hand, in the novel method of hollow fiber culture in which cells are sealed in the hollow fiber lumen according to the present invention and the culture solution flows outside the hollow fiber, the maximum secreted amount on Day 35 reaches 7,391 pg / mL, and 5,000 The period of recovery at pg / mL or higher was very long, from Day 21 to Day 50.
As described above, according to the present invention, it has been realized for the first time that the production efficiency of a culture supernatant can be raised to an industrial level by making it possible to continuously recover a culture supernatant containing a high concentration of an effective factor for a long period of time.

1.2. Double-stranded DNA (dsDNA) quantification
To evaluate cell integrity and cytotoxicity during long-term culture, we measured the concentration of dsDNA secreted into the extracellular culture by cell death. 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 adherent culture of T-150 flasks, a remarkable 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 obtained by the hollow fiber culture of the present invention, a remarkable increase in the dsDNA concentration was not observed during the culture period up to Day 50, so that the mesenchymal system that grew as an aggregate in the closed space in the hollow fiber was not observed. It was confirmed that the stem cells maintained their soundness even in long-term culture.

1.3. Glucose determination
Glucose, the main energy source of the cells, was quantified to evaluate changes in the concentration of the medium during long-term culture. A commercially available kit was used for glucose determination 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.

An industrial application of the present invention is the production of culture supernatants as raw materials for pharmaceuticals and cosmetics. By utilizing the technology of the present invention, it is possible to achieve a reduction in the floor area of the manufacturing area, a reduction in the number of manufacturing personnel, an increase in the manufacturing scale per manufacturing unit, and the like, as compared with the production of the culture supernatant by the conventional adhesion culture method. . By these means, it is possible to reduce the production cost of the culture supernatant, suppress the product price, and provide an optimal and effective means of developing a production method.

Claims

A hollow fiber filter module for a hollow fiber cell culture device for producing a culture supernatant,
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 having a circular inner diameter of 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 are connected to the hollow fiber (1). 3) is that the stem cell is introduced inside by the cell introduction part (5),
A first end cap (6a) for being mounted on the hollow fiber filter module after the stem cells are introduced into the hollow fiber (1) by the cell introduction part (5), wherein the stem cells are The first end (4a) of the hollow fiber (3) and the filter housing (1) so that the transfer of the culture medium and the culture solution does not occur through the opening of the first end (4a) of the hollow fiber (3). A) a first end cap (6a) for sealing
A culture solution introduction unit (7) connected to the filter housing (1) for introducing a culture solution into the filter housing, and supplying a culture solution to the inside of the filter housing; Connected to road (8),
A culture solution discharge portion (9) connected to the filter housing (1) for discharging a culture solution containing a culture supernatant having passed through the inside of the filter housing; One connected to a culture discharge passage (10) for discharging the culture medium containing
The second end (4b) of the hollow fiber (3) and the filter housing so that the movement of the stem cells and the culture solution does not occur through the opening of the second end (4b) of the hollow fiber (3). A second end cap (6b) enclosing (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 culture device closes the lumen of the hollow fiber (3) with the stem cell, exchanges a culture solution circulating outside the hollow fiber (3) with the inside of the hollow fiber (3), and The culture supernatant generated 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 portion (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,
Further comprising 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 having a gasket is inserted into the end of the hollow fiber filter, and the other end of the cylindrical volume is connected to the hollow fiber filter. A hollow fiber filter module that is introduced into the lumen of the filter.
A hollow fiber cell culture device for producing a culture supernatant comprising the hollow fiber filter module for a hollow fiber cell culture device according to claim 1,
A culture solution supply passage (8) connected to the culture solution introduction unit (7) for introducing a culture solution into the culture solution introduction unit (7);
A culture discharge path (10) connected to the culture liquid discharge section (9) for collecting a culture liquid containing the nutrient supernatant discharged from the culture liquid discharge section (9);
Including
The lumen of the hollow fiber (3) is closed with the stem cells, the culture solution circulating outside the hollow fiber (3) is exchanged for the inside of the hollow fiber (3), and the culture supernatant generated by the stem cells is removed from the hollow fiber (3). The yarn (3) is moved outside, whereby the culture solution containing the culture supernatant is discharged from the culture solution discharge section (9), and the culture supernatant is collected.
Hollow fiber cell culture device.
The apparatus for culturing hollow fiber cells according to claim 5, wherein the culture solution is provided in the culture solution supply passage (8), and sends the culture solution into the filter housing (1) via the culture solution introduction section (7). Further comprising a liquid sending device (11).
A method for producing a culture supernatant using the apparatus according to claim 5,
A cell introduction step of introducing mesenchymal stem cells into the hollow fiber (3) via the cell introduction part (5);
After the cell introduction step, the first end cap (6a) is moved so that the mesenchymal stem cells and the culture solution do not move through the opening of the first end (4a) of the hollow fiber (3). Sealing the first end (4a) of the hollow fiber (3) and the filter housing (1) using
A culture solution supply step of supplying the culture solution into the filter housing through the culture solution introduction section (7);
A culture solution discharging step of discharging the culture solution inside the filter housing from the culture solution discharge section (9);
The culture solution supply step and the culture medium discharge step are continued to supply the culture medium inside the filter housing via the culture medium introduction section (7), and to discharge the culture medium inside the filter housing. , Inside the hollow fiber (3) in a state where 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). Culturing the mesenchymal stem cells;
A culture supernatant collecting step of collecting a culture supernatant from the discharged liquid obtained in the culture liquid discharging step,
A method for producing a culture supernatant of mesenchymal stem cells, comprising: