WO2016148230A1 - 幹細胞培養上清の製造方法 - Google Patents
幹細胞培養上清の製造方法 Download PDFInfo
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- WO2016148230A1 WO2016148230A1 PCT/JP2016/058462 JP2016058462W WO2016148230A1 WO 2016148230 A1 WO2016148230 A1 WO 2016148230A1 JP 2016058462 W JP2016058462 W JP 2016058462W WO 2016148230 A1 WO2016148230 A1 WO 2016148230A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/10—Hollow fibers or tubes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
Definitions
- the present invention relates to a method for producing a culture supernatant of stem cells.
- a mesenchymal stem cell that is a stem cell is a kind of somatic stem cell, and has a differentiation ability into a mesenchymal cell, that is, a bone cell, a cardiac muscle cell, a chondrocyte, an adipocyte, etc.
- a mesenchymal stem cell that is, a bone cell, a cardiac muscle cell, a chondrocyte, an adipocyte, etc.
- mesenchymal stem cells also have anti-inflammatory and immunoregulatory effects, it is known that they are already widely used for various diseases such as liver diseases, graft-versus-host diseases, and autoimmune diseases. It has been.
- Non-patent Document 1 Ueda et al. Also reported that the culture supernatant of mesenchymal stem cells has a wound healing effect and a periodontal tissue regeneration promoting effect (Non-patent Documents 2 and 3).
- exosomes secreted from cells contain various proteins and RNA, and this may be a substance responsible for communication between cells. From the above, in cell culture, not only the cells obtained thereby but also the culture supernatant are very valuable, and it is extremely important to produce them efficiently.
- the object of the present invention relates to a method for producing a culture supernatant of stem cells having excellent tissue regeneration ability, excellent disease healing action, or high physiological activity.
- a culture solution in which stem cells such as mesenchymal stem cells are cultured contains physiologically active substances having various actions, which contribute to tissue regeneration and disease healing effects. .
- Such a cell culture supernatant is usually obtained by implanting cells in a flask or petri dish, culturing the cells for a certain period, and then collecting the culture solution used for culturing at this time.
- the amount of the physiologically active substance released into the culture solution increases in proportion to the number of cells. For this reason, as long as the number of cells is good, for example, adherent cells such as mesenchymal stem cells spread further on the bottom of a petri dish or flask and proliferate, so as many physiologically active substances released from the cells as possible, In order to recover the culture supernatant contained in a high concentration, this culture method is by no means efficient.
- the outline of the present invention is as follows. 1.
- a method for producing a stem cell culture supernatant comprising the following steps [a] to [c].
- the cell culture container is a module storing a permeable membrane hollow fiber, and has two openings communicating with the lumen of the hollow fiber, and two communicating with the inner space of the module and the outer lumen of the hollow fiber. 2.
- the method according to 1 or 2 wherein the stem cells are cultured using a cell culture apparatus including the following configurations [d] to [g]. [D] the cell culture container [e] at least one culture medium storage container [f] a channel network connecting the cell culture container and the culture medium storage container [g] provided in the channel network, 3. means for controlling the supply of the culture solution to the culture vessel and / or the discharge of the culture solution from the cell culture vessel 4.
- the number of cells growing on the bottom of the petri dish reaches about 30 to 40,000 cells / cm 2 .
- a petri dish having a diameter of about 10 cm cells grow to about 2,000,000 to 3,000,000 cells, and about 10 ml of culture solution is used for this.
- 10 ml of the culture supernatant is recovered from a diameter of 10 cm ⁇ height of 1.5 cm, that is, a container volume of 120 cm 3 .
- culture is performed using a module of a permeable membrane hollow fiber, and the culture solution is passed through the hollow fiber module at a very low speed, so that the culture is recovered as compared with the number of cells.
- the supernatant is significantly less than in the conventional method.
- a hollow fiber module having a length of about 1 cm, which is 10 cm long and contains several hundred permeable membrane hollow fibers the number of cells reaches 10,000,000 or more, but the culture flows directly in contact with the cells.
- the total volume of the liquid is less than 10 ml, the ratio of the number of cells to the conventional method is about 1/5, and the concentration of the substance secreted into the culture supernatant is about 5 times.
- the container volume is about 8 cm 3 , the recovery efficiency per volume is also improved. That is, according to the present invention, it has become possible to easily and efficiently produce a culture supernatant containing a high concentration of a physiologically active substance secreted from mesenchymal stem cells to be cultured.
- the structural example of the hollow fiber module used for implementation implementation of the stem cell culture supernatant of this invention is shown.
- the example of the cell culture form used for implementation of stem cell culture supernatant manufacture of this invention is shown with a schematic diagram.
- the schedule of culture supernatant preparation in Example 1 of this invention is shown. It shows the VEGF concentration in the culture supernatant in Example 1 of the present invention. It shows the IGF concentration in the culture supernatant in Example 1 of the present invention. 3 shows the TNF- ⁇ production inhibitory effect of the culture supernatant in Example 1 of the present invention.
- One embodiment of the present invention is a method for producing a stem cell culture supernatant (conditioned medium, conditioned medium) comprising the following steps [a] to [c]. [A] supplying a culture solution to the stem cells seeded on the surface of the permeable membrane hollow fiber in the cell culture container [b] culturing the stem cells by bringing the culture solution into contact with the stem cells [c] Recovering the culture solution containing the secreted components
- the cell culture supernatant is obtained by separating a culture solution that has been in direct or indirect contact with a cell when the cell is cultured for a certain period (several hours to several days). To tell. Consent with culture medium conditioned medium, conditioned medium, etc.
- Stem cells that are one of the objects according to the present invention are not particularly limited, but bone marrow mesenchymal stem cells or adipose tissue-derived mesenchymal stem cells are suitable. Not only primary cells but also mesenchymal stem cells established by genetic modification or the like can be used.
- the animal species is not particularly limited, and any animal species such as human, mouse and rat can be used. Moreover, it can be suitably used for various somatic stem cells having adhesiveness other than mesenchymal stem cells.
- composition of the culture solution used in the method for producing the stem cell culture supernatant of the present invention is not particularly limited.
- those prepared by using DMEM, ⁇ MEM, RPMI1640, or the like as a basal medium and appropriately adding animal blood, cell growth factor, hormone or the like thereto can be used.
- the culture medium used in the present invention does not contain animal serum. This is because animal serum contains abundant physiologically active substances such as cell growth factors, and sometimes the presence of these physiologically active substances interferes with the purpose of using the culture supernatant or acts negatively. This is because there is a possibility of doing so.
- the permeable membrane hollow fiber used in the present invention is not particularly limited as long as it can hold a cell in the hollow fiber lumen and can take a structure that allows a solution or a low-molecular substance to permeate. Absent.
- a permeable membrane hollow fiber in the present invention is that the number of cell growth per culture area is larger than that of a plastic material such as a petri dish. This is because cells that adhere and grow on membrane hollow fibers do not expand as much as on plastic, so the number of cells adhering per unit area is several times higher on membrane hollow fibers than on plastic. Because. Furthermore, the culture area per unit volume can be remarkably increased by tightly bundling the membrane hollow fibers.
- the material of the permeable membrane hollow fiber used in the present invention is not particularly limited.
- cellulose-based materials such as cellulose acetate, cellulose triacetate and regenerated cellulose
- polysulfone-based materials such as polysulfone and polyethersulfone
- polyethylene polypropylene
- An insoluble carrier having a skeleton structure of a thermoplastic polymer such as polystyrene, polyvinyl alcohol, polymethyl methacrylate, polyacrylonitrile, fluorine resin, polyamide, polyvinylidene fluoride (PVDF) can be suitably used.
- PVDF polyvinylidene fluoride
- the permeable membrane hollow fiber used in the present invention may be one obtained by chemically modifying the above material.
- the permeable membrane hollow fiber used in the present invention is preferably hydrophilized.
- hydrophilizing the permeable membrane hollow fiber it becomes easy to supply a liquid component such as a culture solution to the cultured cells.
- the method of hydrophilizing the permeable membrane hollow fiber include a method of treating the permeable membrane hollow fiber with a hydrophilic polymer such as ethylene-vinyl alcohol copolymer, glycerin, and ethanol.
- a coating agent such as collagen or fibronectin may be used in order to improve adhesion to the hollow fiber depending on the cells to be used.
- the size of the permeable membrane hollow fiber used in the present invention is not particularly limited, but a preferable lower limit of the inner diameter of the hollow fiber is 10 ⁇ m, more preferably 20 ⁇ m, and further preferably 50 ⁇ m. On the other hand, the preferable upper limit of the inner diameter of the hollow fiber is 1000 ⁇ m, more preferably 500 ⁇ m.
- the film thickness is not particularly limited, but the hollow fiber may have an appropriate strength and the permeability of the substance may be set within a favorable range, and is preferably about 10 to 200 ⁇ m, for example.
- the pore diameter of the permeable membrane hollow fiber used in the present invention is not particularly limited as long as it is a pore that does not allow cells to pass but allows water, salts, and nutrients of relatively low molecular weight to pass through.
- the substance exchange has a somewhat efficient pore size and does not permeate useful components such as physiologically active substances produced by the cells. . Therefore, the pore diameter (pore radius) of the permeable membrane hollow fiber is preferably 1 to 500 mm, more preferably 5 to 300 mm, and further preferably 10 to 150 mm.
- the pore diameter of the permeable membrane hollow fiber is closely related to the permeability of the permeable membrane hollow fiber.
- the permeability of the permeable membrane hollow fiber is indicated, for example, by the amount of liquid passing per unit time / unit area.
- the permeability is not particularly limited. For example, 1 to 1000 ml / m 2 / hr / mmHg, more preferably 10 to 500 ml / m 2 / hr / mmHg. Is preferred.
- the method for producing a stem cell culture supernatant of the present invention includes the following steps. [A] Step of supplying a culture solution to stem cells existing in the permeable membrane hollow fiber lumen [b] The culture solution is moved (passed) through the hollow fiber lumen continuously or intermittently to the stem cells. The step of culturing the stem cells in contact with the culture solution [c] The step of recovering the culture solution obtained by the step may be performed in the order of [a] [b] [c] Two or more processes may overlap in the middle. However, the process start of [b] does not precede the process start of [a], and the process start of [c] does not precede the process start of [b].
- the method for producing a stem cell culture supernatant of the present invention is not particularly limited, and for example, the hollow fiber can be used to culture stem cells on the hollow fiber lumen side.
- stem cells are seeded on the surface of the hollow fiber by injecting a stem cell suspension into the hollow fiber lumen. To supply.
- culture is performed on the lumen side.
- the supplied culture medium is moved (passed) through the hollow fiber lumen continuously or intermittently, thereby bringing the culture medium into contact with the stem cells and culturing the stem cells.
- intermittent movement (passing) refers to repeating the process of temporarily stopping or advancing the flow of the culture solution in the hollow fiber.
- the interval setting for stopping or advancing the flow is not particularly limited, and may be equal or irregular.
- the culture solution has a role of supplying nutrients and oxygen necessary for the cells and discharging waste products. During the culture period, it is preferable to continue to supply the medium on both the inner and outer lumen sides for gas exchange, nutrient supply to the cells, and removal of waste products. At that time, by using a peristaltic pump or the like, it can be supplied / discharged or circulated at an appropriate speed. Thus, if the culture solution after performing culture
- the permeable membrane hollow fiber used in the method for producing a stem cell culture supernatant of the present invention stores a hollow fiber bundle in which several tens to several tens of thousands of hollow fibers are bundled in a cylindrical container to form a module.
- a culture vessel may be used (such a module is hereinafter also referred to as a hollow fiber module).
- the hollow fiber module needs to have at least one opening that can supply a medium to the culture substrate and one or more openings that can discharge the medium.
- the structure of the module using such a permeable membrane hollow fiber is not particularly limited.
- the module case 1 having four openings (end conduit and side conduit) is permeable to the main body.
- a form in which the required number of membrane hollow fibers 2 are appropriately bundled and filled is mentioned.
- the two end conduits 3 are each provided with an appropriate sealing material (for example, a polyurethane potting agent) so as not to block the hollow portion of each hollow fiber at both ends of the hollow fiber bundle. ), And the liquid or the like introduced from one of the end conduits 3 is led out from one end conduit 3 that passes through the hollow fiber lumen (that is, flows in one direction).
- an appropriate sealing material for example, a polyurethane potting agent
- the spaces inside the remaining two side conduits 4 are the spaces inside the module case 1 and the outer cavities of the hollow fibers (hereinafter simply referred to as “external side”).
- the liquid introduced from one side of the side conduit 4 is led out from the other side conduit 4 that passes through the outer lumen side of the membrane hollow fiber (that is, one-way). To flow).
- the hollow fiber module is used as a culture vessel in the method for producing a stem cell culture supernatant of the present invention
- seeding is performed by injecting a stem cell suspension from the end conduit, and after the seeding is finished, What is necessary is just to supply by perfusing the hollow fiber lumen in which the stem cells exist.
- the culture medium is injected into the hollow fiber outer space side from the side conduit and perfused simultaneously with the perfusion of the culture solution to the hollow fiber lumen side.
- the hollow fiber or the hollow fiber module is preferably supplied after being sterilized by an appropriate method.
- the sterilization method is not particularly limited, and examples thereof include autoclaving sterilization, gamma ray sterilization, and ethylene oxide gas sterilization.
- the present invention can be implemented more efficiently.
- a fresh medium can be uniformly supplied to cells in the hollow fiber module lumen at a low speed, and the culture supernatant can be efficiently collected.
- culture may be performed using a cell culture system including the following configurations [d] to [g]. [D] the cell culture container [e] at least one culture medium storage container [f] a channel network connecting the cell culture container and the culture medium storage container [g] provided in the channel network, Means for controlling supply of culture solution to culture vessel and / or discharge of culture solution from said cell culture vessel
- the cell culture vessel used in the system is a module using a porous membrane hollow fiber, and has two openings connected to the lumen from both ends of the hollow fiber and two openings connected to the outer cavity. If it is a cell culture container which has this, it will not specifically limit.
- culture medium storage container Although the form and material of the culture solution storage container used in the system are not particularly limited, it is desirable to have gas permeability.
- a culture apparatus including the hollow fiber module may be configured, and the culture supernatant may be prepared by installing the culture apparatus in a CO 2 incubator. If the culture solution storage container having the above is used, the culture can be easily performed without installing a gas exchange tube having problems such as bubble generation. Examples of such culture medium storage containers include commercially available cell culture bags (available from Nipro, Toyo Seikan, etc.), plastic bottles, and the like.
- connection network In the system, several channels (channel networks) are connected to the cell culture container.
- the configuration (piping) is not particularly limited, but includes at least a connection portion between the cell culture container and the liquid medium storage container so that the medium can be supplied from the liquid medium storage container to the liquid medium storage container. It needs to be piped.
- a channel network at least (1) a connection portion between the cell culture container and the opening on the hollow fiber lumen side of the culture solution storage container, and (2) the cell culture container and the culture solution storage container A flow path network including a connection portion with the opening on the hollow fiber outer cavity side.
- connection may be either directly connected or connected via a flow path such as a pipe.
- the piping material used in the system is not particularly limited.
- silicon tube Master Flex (registered trademark), C-Flex (registered trademark), Tygon (registered trademark), Farmed (registered trademark) BPT, Pharma Pure (registered trademark), Norprene (registered trademark), Sanitec (registered) Trademark) or the like.
- a gas permeable channel tube for the channel tube of the portion connecting the liquid medium storage container and the cell culture container.
- a silicon tube can be suitably used.
- the system is provided with means for controlling the supply of the culture solution to the cell culture vessel and / or the discharge of the culture solution from the cell culture vessel, provided in the channel network.
- the form of the method for supplying the cell suspension or culture solution is not particularly limited, but if the peristalsis using a peristaltic pump or the culture solution storage container is a soft one such as a culture bag, a roller is attached to the container. A method of squeezing out the medium by applying is suitable.
- the installation location of the pump is not particularly limited, but it is preferable to install the pump on the discharge side in order to avoid the risk of contamination of liquid when the liquid passes through the pump portion.
- FIG. 2 shows an embodiment of the present invention in which a culture system composed of the hollow fiber module, culture medium storage container, peristaltic pump, etc. is installed in a CO 2 incubator.
- 5 and 6 are gas-permeable culture medium storage containers (cell culture bags, etc.), respectively.
- a flow path is connected to the end conduit 3 a of the hollow fiber module 7 so that the medium contained in the culture solution storage container 5 can be transferred to the hollow fiber lumen side.
- a flow path is connected to the side conduit 4a of the hollow fiber module 7, so that the medium contained in the culture solution storage container 6 can be transferred to the hollow fiber outer space side.
- the culture solution that has passed through the lumen side of the hollow fiber module 7 is discharged from the end conduit 3b, is connected to the culture supernatant collection container 11 through the peristaltic pump 8, and passes through the hollow fiber module lumen.
- the collected culture supernatant can be collected.
- the culture solution that has passed through the outer cavity side of the hollow fiber module 7 is discharged from the side conduit 4 b and is collected in the waste solution collection container 10 via the peristaltic pump 9.
- the method for producing a stem cell culture supernatant of the present invention is carried out using the system, for example, when the permeable membrane hollow fiber module is used, the medium is supplied to the hollow fiber on the lumen side and the outer cavity. Two routes on the side are required. At that time, when supplying the culture medium, separate culture solution storage containers may be used on the lumen side and the outer cavity side, or the medium is supplied from one container to both the lumen side and the outer lumen side. Also good.
- the culture fluid that has flowed through the hollow fiber lumen contains a large amount of substances released by the cells, whereas there are no cells on the hollow fiber outer lumen side. This is because the amount of substances released by cells is extremely small. That is, the culture fluid that has flowed into the lumen from the opening at one end of the hollow fiber flows out of the other opening as it is without being affected by outflow to the outer space or interference from the inflow from the outer lumen as much as possible. It is desirable to go.
- the culture medium used for the culture is not particularly limited as long as it is normally used as a culture liquid for mesenchymal stem cells, but when applying the culture supernatant of stem cells to a living body for the purpose of treatment, It is desirable that the animal serum in the culture solution is as little as possible or does not contain animal serum. For this reason, when culturing stem cells using a culture solution containing serum, when the cells are in a sufficiently increased state, it is replaced with a low serum culture solution or a serum-free culture solution, and finally the serum content is low. Alternatively, a culture supernatant containing no serum can be obtained. It is also possible to perform cell culture from the beginning using a culture solution in which components have been adjusted so that stem cells can be cultured without serum, thereby obtaining a culture supernatant containing no serum.
- the culture solution flowing through the hollow fiber lumen must be collected in a container separately from the culture solution flowing through the hollow fiber outer lumen. Is preferred.
- the method for examining the degree of cell proliferation is not particularly limited, but can be performed based on the measurement results such as the concentration of glucose and lactate in the culture solution.
- a preferable flow rate of the culture solution flowing to the side where cells are seeded is 0.01 to 1 mm / min.
- the preferred flow rate of the culture solution to be flowed to the side where cells are not seeded is 0.02 to 5 mm / min.
- the culture fluid flowing through the hollow fiber lumen is unidirectional.
- one direction means that the route from introduction of the culture medium to the hollow fiber module is always in the same direction.
- the culture fluid that flows on the outer lumen side of the hollow fiber may flow in one direction, or may be a so-called circulation type in which the medium derived from the cell culture vessel is once again introduced from the inlet. Good.
- the cell seeding density was about 5100 cells / cm 2 .
- the culture solution is DMEM GlutaMAX (Life Technologies) supplemented with 10% fetal bovine serum (Life Technologies) from the start of culture (cell seeding) to 96 hours later, and after 96 hours when the culture supernatant is collected, MF -Medium, mesenchymal stem cell growth medium (Toyobo) was used.
- MF-medium is a low serum medium containing only 1% serum.
- FIG. 3 shows a schedule for preparing a culture supernatant according to the method of the present invention. Cultivation was carried out for 7 days (after 168 hours) after cell seeding (culture start). During this time, the flow rate (linear velocity) of the culture fluid flowing through the hollow fiber lumen averaged 0.066 mm / min from 96 hours after cell seeding, and averaged from 0.6 hours to 144 hours. From 20 mm / min, 144 hours to 168 hours, the average was 0.33 mm / min. On the other hand, the speed of the culture solution flowing through the hollow fiber outer space was set to 3.4 mm / min from the start to the end of the culture.
- the cell culture supernatant was collected for 72 hours from 96 hours to 168 hours after the start of the culture, and stored at 4 ° C. until used for subsequent performance evaluation.
- the total amount of the culture supernatant was 7.9 ml.
- the flow rate was calculated based on the cross-sectional area of the hollow fiber by measuring the flow rate flowing out of the hollow fiber lumen and the outer lumen. After 168 hours of culture, the cells were digested with trypsin, detached and recovered, and the number of cells was counted. As a result, 1.2 ⁇ 10 7 cells were recovered.
- FIG. 3 shows a schedule for preparing a culture supernatant according to the conventional method.
- Culture medium exchange was performed 48 hours after the start of culture and 96 hours after. Thereafter, the culture medium was not changed, and the culture was terminated when it reached 100% confluence in 168 hours from the start of the culture.
- the culture solution which was cultured for 72 hours from the last medium exchange to the end of the culture was collected as a culture supernatant.
- the total amount of the culture supernatant was 10.0 ml.
- the cells were digested with trypsin, detached and recovered, and the number of cells was counted. As a result, 2.6 ⁇ 10 6 cells were recovered.
- VEGF vascular endothelial growth factor
- IGF insulin
- the results of measuring the VEGF concentration in the culture supernatant are shown in FIG.
- the VEGF concentration in the culture supernatant recovered by the conventional method was 561.6 pg / ml, whereas in the culture supernatant recovered by the present invention, it was 3597.1 pg / ml.
- a clear high concentration was shown.
- the total amount of VEGF recovered during the culture time of 72 hours was 5616 pg in the conventional method, whereas it was 28417.1 pg in the present invention.
- human IGF-I ELISA Kit R & D Systems
- the results of measuring the IGF concentration in the culture supernatant are shown in FIG.
- the IGF concentration in the culture supernatant recovered by the conventional method was 1864.6 pg / ml, whereas in the culture supernatant recovered according to the present invention, it was 133798.0 pg / ml.
- a clear high concentration was shown.
- the total amount of VEGF recovered during the culture time of 72 hours was 18.6 ng in the conventional method, whereas it was 109.0 ng in the present invention.
- the culture supernatant of stem cells prepared according to the present invention is richer in physiologically active substances and superior in tissue regeneration and anti-inflammatory action than conventional ones. It can be said that.
- the present invention makes it possible to easily and efficiently produce a culture supernatant of stem cells having various actions such as tissue regeneration and anti-inflammation.
Abstract
Description
以上のことから、細胞培養においては、それによって得られる細胞だけでなく、培養上清もまた非常に貴重なものであり、それを効率よく作製することは極めて重要なことである。
1.幹細胞培養上清液の製造方法であって、以下の[a]から[c]の工程を含む、方法。
[a]細胞培養容器内の透過性膜中空糸の内表面に播種した幹細胞に培養液を供給する工程
[b]前記幹細胞に前記培養液を接触させて前記幹細胞を培養する工程
[c]前記幹細胞が分泌した成分を含む培養液を回収する工程
2.前記細胞培養容器は、透過性膜中空糸を格納したモジュールであって、前記中空糸の内腔と連通する2つの開口部と、前記モジュールの内側かつ前記中空糸の外腔と連通する2つの開口部を有するものである、1に記載の方法。
3.以下の[d]から[g]の構成を含む細胞培養装置を用いて前記幹細胞の培養を行う、1または2に記載の方法。
[d]前記細胞培養容器
[e]少なくとも1つの培養液貯留容器
[f]前記細胞培養容器と前記培養液貯留容器とを接続する流路網
[g]前記流路網に設けられ、前記細胞培養容器への培養液の供給および/または前記細胞培養容器からの培養液の排出を制御する手段
4.前記透過性膜中空糸の内腔を通過する培養液の線速度が、0.01~1mm/minである、1から3のいずれかに記載の方法。
5.前記透過性膜中空糸の孔半径が、1~500Åであることを特徴とする、1から4のいずれかに記載の方法。
6.前記培養液が動物血清を含まないことを特徴とする、1から5のいずれかに記載の方法。
7.前記幹細胞が間葉系幹細胞であることを特徴とする、1から6のいずれかに記載の方法。
即ち、本発明により、培養する間葉系幹細胞から分泌される生理活性物質を高濃度に含む培養上清液を、簡便に効率よく作製することが可能となった。
[a]細胞培養容器内の透過性膜中空糸表面に播種した幹細胞に培養液を供給する工程
[b]前記幹細胞に前記培養液を接触させて前記幹細胞を培養する工程
[c]前記幹細胞が分泌した成分を含む培養液を回収する工程
本発明において、細胞の培養上清とは、細胞を一定期間(数時間から数日)培養した際に、細胞に直接または間接に接触していた培養液を細胞と分離して得られるものを言う。培養液馴化培地、コンディションドメディウム(Conditioned medium)などと同意である。
本発明に係る対象の一つとなる幹細胞としては、特に限定されるものではないが、骨髄間葉系幹細胞、あるいは脂肪組織由来間葉系幹細胞が好適である。プライマリー細胞に限らず、遺伝子改変等によって株化された間葉系幹細胞も用いることが出来る。動物種も特に限定されず、ヒト、マウス、ラット等のいずれの動物由来のものも使用できる。また、間葉系幹細胞以外の接着性を有する種々の体性幹細胞にも好適に使用できる。
本発明の幹細胞培養上清液の製造方法に用いる培養液の組成等は、特に限定されない。例えば、DMEM、αMEM、RPMI1640などを基礎培地とし、これに適宜、動物血、細胞増殖因子、ホルモンなどを添加することにより調製されたものが使用できる。
本発明で使用する透過性膜中空糸は、細胞を中空糸内腔に保持でき、溶液や低分子の物質を透過させるような構造をとることができるものであれば、特に限定されるものではない。
本発明の幹細胞培養上清液の製造方法は、以下の工程を含む。
[a]透過性膜中空糸内腔に存在する幹細胞に培養液を供給する工程
[b]前記培養液を連続的または間欠的に前記中空糸内腔を移動(通過)させることにより前記幹細胞に前記培養液を接触させて前記幹細胞を培養する工程
[c]前記工程により得られた培養液を回収する工程
その順序は、例えば[a][b][c]の順に行えばよいが、操作の途中で2つ以上の工程が重複してもよい。ただし、[b]の工程開始が[a]の工程開始に先んじることはなく、[c]の工程開始が[b]の工程開始に先んじることはない。
本発明の幹細胞培養上清液の製造方法で使用する透過性膜中空糸は、筒状容器に数十本~数万本の中空糸が束ねられた中空糸束を格納してモジュール化し、細胞培養容器としてもよい(このようなモジュールを、以下、中空糸モジュールとも呼ぶ)。中空糸モジュールには、培養基材に培地を供給することができる開口部と、培地を排出することができる開口部とを、それぞれ1つ以上有している必要がある。
本発明の幹細胞培養上清液の製造方法においては、以下の[d]から[g]の構成を含む細胞培養システムを用いて培養を行えばよい。
[d]前記細胞培養容器
[e]少なくとも1つの培養液貯留容器
[f]前記細胞培養容器と前記培養液貯留容器とを接続する流路網
[g]前記流路網に設けられ、前記細胞培養容器への培養液の供給、および/または前記細胞培養容器からの培養液の排出を制御する手段
前記システムで用いる培養液貯留容器の形態や素材については特に限定されないが、ガス透過性を有することが望ましい。前記システムの一つの態様においては、前記中空糸モジュールを含む培養装置を構成し、それをCO2インキュベーター内に設置して培養上清の作製を実施してもよいが、その際、ガス透過性を有する培養液貯留容器を用いれば、気泡発生などの問題点を有するガス交換用チューブを設置することなく、簡便に培養を行うことができる。このような培養液貯留容器としては、例えば、市販の細胞培養バッグ(ニプロ社製、東洋製罐社製のものなどが入手できる。)、プラスチック製ボトルなどが挙げられる。
前記システムにおいて、前記細胞培養容器にはいくつかの流路(流路網)が接続している。その構成(配管)は特に限定されないが、少なくとも前記細胞培養容器と前記液体培地貯留容器との接続部分を含み、前記液体培地貯留容器から前記液体培地貯留容器への培地の供給が可能なように配管されている必要がある。このような流路網として、少なくとも(1)前記細胞培養容器と前記培養液貯留容器の中空糸内腔側の開口部との接続部分、および(2)前記細胞培養容器と前記培養液貯留容器の中空糸外腔側の開口部との接続部分を含む流路網、が挙げられる。なお、本明細書において「接続」とは、直接繋がっている場合、および、配管などの流路を介して繋がっている場合のいずれであってもよい。
前記システムには、前記流路網に設けられ、前記細胞培養容器への培養液の供給、および/または前記細胞培養容器からの培養液の排出を制御する手段が設けられている。細胞懸濁液や培養液の供給方法の形態は、特に限定されるものではないが、ペリスタポンプを用いた灌流や、培養液貯留容器が培養バッグなど軟質のものである場合は前記容器にローラーを当てて培地を絞り出す方法等が好適である。ポンプの設置場所は特に限定されないが、液体がポンプ部分を通過する際にゴミが混入する等のリスクを避けるため、排出側に設置することが好ましい。
上記の中空糸モジュールや培養液貯留容器、ペリスタポンプなどにより構成される培養システムをCO2インキュベーター内に設置し、本発明の実施の一つの態様例としたものを図2に示す。図2において、5および6はそれぞれ、ガス透過性を有する培養液貯留容器(細胞培養バッグなど)である。培養液貯留容器5からは、中空糸モジュール7の端部導管3aに、流路が接続され、培養液貯留容器5に入っている培地が中空糸内腔側に移送できるようになっている。他方、培養液貯留容器6からは、中空糸モジュール7の側部導管4aに、流路が接続され、培養液貯留容器6に入っている培地が中空糸外腔側に移送できるようになっている。中空糸モジュール7の内腔側を通過した培養液は、端部導管3bから排出され、ペリスタポンプ8を経由して、培養上清回収容器11まで流路が接続され、中空糸モジュール内腔を通過した培養上清が回収できるようになっている。一方、中空糸モジュール7の外腔側を通過した培養液は、側部導管4bから排出され、ペリスタポンプ9を経由して、廃液回収容器10に回収される。
前記システムを用いて本発明の幹細胞培養上清液の製造方法を実施する場合、例えば、前記の透過性膜中空糸モジュールを用いる場合、中空糸への培地の供給は、内腔側および外腔側の2ルートが必要である。その際、培地の供給にあたり、培養液貯留容器は内腔側および外腔側で別々のものを用いても良いし、1つの容器から内腔側および外腔側の両方に培地を供給しても良い。また、中空糸内腔側と外腔側の水圧差や浸透圧差により、内腔から外腔、あるいは外腔から内腔へと培養液が流れることは極力抑えることが望ましい。何故なら、中空糸内腔を流れてきた培養液には、細胞が放出した物質が多く含まれるのに対し、中空糸外腔側には細胞が存在しないため、外腔側を流れる培養液中には細胞が放出する物質は、極めて少量であるためである。即ち、中空糸の一端の開口部から内腔へと流入した培養液は、なるべく外腔への流出や外腔からの流入の干渉などを受けず、そのまま、もう一方の開口部から流出していくことが望ましい。
培養に用いられる培養液は特に限定されず、間葉系幹細胞の培養液として通常用いられるものであれば良いが、幹細胞の培養上清液を治療等の目的で生体に適用する場合には、培養液中の動物血清がなるべく少ない、あるいは動物血清を含まないことが望ましい。このため、血清を含む培養液を用いて幹細胞を培養する場合、充分に細胞が増えた状態になったところで低血清培養液や無血清培養液に交換し、最終的に血清含有量が少ない、または血清を含まない培養上清液を得ることが出来る。また、無血清でも幹細胞の培養を可能とするよう成分を調整された培養液を用いて最初から細胞培養を行い、血清を含まない培養上清液を得ることも可能である。
前記のような理由により、本発明により培養上清を得るには、中空糸内腔を流れてきた培養液は、中空糸外腔を流れてきた培養液とは区別して、容器に回収することが好ましい。
細胞培養における培養液、特に中空糸内腔を細胞に接して流れる培養液の流速については、流速を厳密に制御することが好ましい。流速が遅すぎると、細胞への栄養供給が十分になされず、細胞が増殖しにくくなる。逆に、流速が速すぎても、細胞周囲の環境変化が激しく、細胞が周りの環境に馴染めず、細胞が増殖しにくくなる。このように、培養液、特に中空糸内腔を流れる培養液の流速は、細胞増殖度合いや環境に応じて、調整することが好ましい。細胞増殖度合いを調べる方法は、特に限定されないが、培養液中のグルコースや乳酸塩の濃度等の測定結果をもとに行うことが出来る。細胞が播種された側に流す培養液の好ましい流速は、0.01~1mm/minである。一方、細胞が播種されていない側に流す培養液の好ましい流速は、0.02~5mm/minである。
細胞播種前に予めコラーゲン(新田ゼラチン)をコートした中空糸モジュール(中空糸はPES/PVP製、内径200μm、外径260μm、膜厚30μm、孔半径75Å、東洋紡)を用い、図2に示すものと同様の構成装置をCO2インキュベーター内に設置し、本実施例を行った。中空糸内腔にヒト骨髄間葉系幹細胞(CELL APPLICATIONS Inc.)を播種(播種細胞数は、5.0×105cells/モジュール)した。このとき、中空糸モジュール(中空糸内径基準)の総培養面積は98cm2であるため、細胞播種密度は、約5100cells/cm2であった。培養液は、培養開始(細胞播種)から96時間後までは、10%ウシ胎児血清(ライフテクノロジーズ)を添加したDMEM GlutaMAX(ライフテクノロジーズ)を用い、培養上清を採取する96時間以降は、MF-medium,間葉系幹細胞増殖培地(東洋紡)を用いた。MF-mediumは、血清を1%しか含まない低血清培養液である。
2枚のコラーゲンコートシャーレ(培養面積55cm2、旭テクノガラス)にヒト骨髄間葉系幹細胞(CELL APPLICATIONS Inc.)を細胞播種密度が約5100cells/cm2となるよう播種した。培養液は、実施例1と同様に、細胞播種から96時間までは、10%ウシ胎児血清(ライフテクノロジーズ)を添加したDMEM GlutaMAX(ライフテクノロジーズ)を用い、96時間以降はMF-medium,間葉系幹細胞増殖培地(東洋紡)に培地を交換した。
図3に、従来法による培養上清作製のスケジュールを示す。培養開始48時間後、および96時間後に、培養液交換を実施した。その後、培養液交換をせず、培養開始から168時間で100%コンフルエントに達したところで培養を終了した。この最後の培地交換から培養終了までの72時間の培養を行った培養液を培養上清として回収した。培養上清の量は、計10.0mlであった。培養から168時間後に細胞をトリプシンで消化、剥離回収し、細胞数をカウントした結果、2.6×106個の細胞が回収された。
上記、(1)および(2)の方法にて回収したそれぞれの培養上清について、組織再生に関わる性能を評価するため、培養上清に含まれるVEGF(血管内皮細胞増殖因子)およびIGF(インスリン様成長因子)の濃度を測定した。
VEGFの定量には、human VEGF ELISA Kit(R&D Systems)を用いた。
IGFの定量には、human IGF-I ELISA Kit(R&D Systems)を用いた。
上記、(1)および(2)の方法にて回収したそれぞれの培養上清について、抗炎症作用を評価した。これは、マクロファージ様細胞株であるRAW264.7細胞(大日本製薬)を、リポ多糖類(LPS)で刺激し、刺激により産生される炎症性サイトカイン(TNF-α)の産生を測定することにより評価した。
測定は以下のように実施した。即ち、RAW264.7細胞を、10%ウシ胎児血清(ライフテクノロジーズ)を含むRPMI1640培地(ライフテクノロジーズ)に懸濁し、24ウェルプレートに5×105個/ウェルで播種した。24時間培養後に、培養液の半分を前記(1)または(2)の培養上清に交換した。この1時間後、LPSを最終100ng/mlとなるよう添加し、更に3時間培養した後、培養液を回収し、Mouse TNF-αELISA Kit(R&D Systems)を用いて、培養液中のTNF-αを測定した。
培養上清によるTNF-α産生抑制効果を図6に示す。この結果、本発明の方法により作製した間葉系幹細胞の培養上清は、従来法で作製した培養上清に比べ、顕著にTNF-αの産生を抑制していることがわかった。
2 透過性膜中空糸
3 端部導管
4 側部導管
5、6 培養液貯留容器
7 細胞培養容器(中空糸モジュール)
8、9 ペリスタポンプ
10 廃液回収容器
11 培養上清回収容器
Claims (7)
- 幹細胞培養上清液の製造方法であって、以下の[a]から[c]の工程を含む、方法。
[a]細胞培養容器内の透過性膜中空糸の内表面に播種した幹細胞に培養液を供給する工程
[b]前記幹細胞に前記培養液を接触させて前記幹細胞を培養する工程
[c]前記幹細胞が分泌した成分を含む培養液を回収する工程 - 前記細胞培養容器は、透過性膜中空糸を格納したモジュールであって、前記中空糸の内腔と連通する2つの開口部と、前記モジュールの内側かつ前記中空糸の外腔と連通する2つの開口部を有するものである、請求項1に記載の方法。
- 以下の[d]から[g]の構成を含む細胞培養装置を用いて前記幹細胞の培養を行う、請求項1または2に記載の方法。
[d]前記細胞培養容器
[e]少なくとも1つの培養液貯留容器
[f]前記細胞培養容器と前記培養液貯留容器とを接続する流路網
[g]前記流路網に設けられ、前記細胞培養容器への培養液の供給および/または前記細胞培養容器からの培養液の排出を制御する手段 - 前記透過性膜中空糸の内腔を通過する培養液の線速度が、0.01~1mm/minである、請求項1から3のいずれかに記載の方法。
- 前記透過性膜中空糸の孔半径が、1~500Åであることを特徴とする、請求項1から4のいずれかに記載の方法。
- 前記培養液が動物血清を含まないことを特徴とする、請求項1から5のいずれかに記載の方法。
- 前記幹細胞が間葉系幹細胞であることを特徴とする、請求項1から6のいずれかに記載の方法。
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JP2019514402A (ja) * | 2016-05-05 | 2019-06-06 | テルモ ビーシーティー、インコーポレーテッド | 自動化された製造及び収集 |
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JP2019216694A (ja) * | 2018-06-22 | 2019-12-26 | 株式会社 バイオミメティクスシンパシーズ | 中空糸細胞培養装置,細胞培養方法,培養上清の製造方法 |
WO2019245050A1 (ja) * | 2018-06-22 | 2019-12-26 | 株式会社バイオミメティクスシンパシーズ | 中空糸細胞培養装置,細胞培養方法,培養上清の製造方法 |
CN109913410A (zh) * | 2019-04-19 | 2019-06-21 | 华子昂 | 干细胞的仿真培养方法 |
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JP6958350B2 (ja) | 2021-11-02 |
KR20170121205A (ko) | 2017-11-01 |
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