JP6958350B2 - Method for producing stem cell culture supernatant - Google Patents

Description

The present invention relates to a method for producing a culture supernatant of stem cells.

Mesenchymal stem cells, which are stem cells, are a type of somatic stem cells and have the ability to differentiate into mesenchymal cells, that is, bone cells, myocardial cells, chondrocytes, adipocytes, etc. It is expected to be applied to regenerative medicine such as myocardial reconstruction. In addition, since mesenchymal stem cells also have anti-inflammatory and immunomodulatory effects, it is known that they are already widely used for various diseases such as liver disease, graft-versus-host disease, and autoimmune disease. Has been done.

Under these circumstances, it is clear that physiologically active substances such as various cytokines secreted from mesenchymal cells greatly contribute to the function of these mesenchymal stem cells transplanted in vivo in cell transplantation therapy. It has been.

When mesenchymal stem cells are cultured in vitro, these bioactive substances are released into the culture medium. Therefore, it has been reported that the culture medium used for culturing mesenchymal stem cells was recovered, and the tissue was successfully regenerated by using this culture medium containing a large amount of substances released from the cells. In an experiment using rats, Ueda et al. Showed that the culture supernatant of bone marrow mesenchymal stem cells has the ability to regenerate bone (Non-Patent Document 1). Among these, the culture supernatant of bone marrow mesenchymal stem cells contains a large amount of insulin-like growth factor (IGF) and vascular endothelial growth factor (VEGF), and these factors are used for tissue regeneration and the like. It has been suggested that it is involved (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).

In addition, Arimura et al. Reported that the culture supernatant of bone marrow mesenchymal stem cells has an anti-inflammatory effect and exhibits a preventive / therapeutic effect on enteritis (Patent Document 1). Furthermore, Su et al. Reported the antiallergic effect of the culture supernatant of mesenchymal stem cells (Non-Patent Document 4).

More recently, it has been pointed out that vesicles called exosomes, which are secreted from cells, contain various proteins and RNA, which may be substances responsible for cell-cell transmission.
From the above, in cell culture, not only the cells obtained thereby but also the culture supernatant are extremely valuable, and it is extremely important to efficiently produce them.

Japanese Unexamined Patent Publication No. 2013-018756

Tissue Engineering Part A. 2012; 18: 1479-1489 Cytotherapy, 2012; 14 (10): 1171-1181 Cytotherapy, 2015; 17 (4): 369-381 The Journal of allergy and clinical immunology, 2015; 136 (2): 423-432

An object of the present invention relates to a method for preparing a culture supernatant of stem cells having excellent tissue regeneration ability, excellent disease healing effect, or high physiological activity.

As described above, the culture medium in which stem cells such as mesenchymal stem cells are cultured contains physiologically active substances having various actions, which contribute to the effects of tissue regeneration and disease healing. .. The culture supernatant of such cells is usually obtained by implanting the cells in a flask or a petri dish, culturing the cells for a certain period of time, and then collecting the culture solution used for the culture at this time.

In this case, the amount of the physiologically active substance released into the culture medium increases in proportion to the number of cells. For this reason, it is sufficient if the number of cells is large, but since adherent cells such as mesenchymal stem cells spread further to the bottom of the petri dish or flask and proliferate, as much bioactive substance as possible is released from the cells. In order to recover the culture supernatant contained in a high concentration, this culture method cannot be said to be efficient.

That is, in the conventional cell culture method, since the volume of the culture solution to be contacted is large with respect to the number of cells, the effective component secreted in the culture supernatant is diluted and its content concentration becomes thin. Therefore, when the obtained culture supernatant is used for tissue regeneration or the like, it is necessary to concentrate the culture supernatant or freeze-dry it.

Against this background, there is a demand for a culture form and method for efficiently producing a culture supernatant containing a physiologically active substance secreted from cultured cells at a concentration as high as possible.

As a result of diligent studies on the above problems, the present inventors have found that the above problems can be solved, and have completed the present invention. That is, the outline of the present invention is as follows.
1. 1. A method for producing a stem cell culture supernatant, which comprises the following steps [a] to [c].
[A] The culture solution is supplied to the stem cells seeded on the inner surface of the permeable membrane hollow thread in the cell culture vessel at a linear velocity of 0.01 to 1 mm / min, and the culture solution is 0 on the side where the cells are not seeded. A step of culturing the stem cells by supplying the cells at a linear velocity of .02 to 5 mm / min [b] A step of proliferating the stem cells using a culture solution in which serum is added to the culture solution when culturing the stem cells [b] c] Step of contacting the proliferated stem cells with a serum-free culture medium or a low-serum culture medium and collecting the obtained culture supernatant. The cell culture vessel is a module that stores a permeable membrane hollow fiber, and has two openings that communicate with the inner cavity of the hollow fiber and two openings that communicate with the inside of the module and the outer cavity of the hollow fiber. The method according to 1, wherein the method has an opening.
3. 3. The method according to 1 or 2, wherein the stem cells are cultured using a cell culture apparatus containing the following configurations [d] to [g].
[D] The cell culture container [e] At least one culture solution storage container [f] A flow path network connecting the cell culture container and the culture solution storage container [g] The cell provided in the flow path network. 4. Means for controlling the supply of the culture solution to the culture container and / or the discharge of the culture solution from the cell culture container. The method according to any one of 1 to 3, wherein the pore radius of the permeable membrane hollow fiber is 1 to 500 Å.
5. The method according to any one of 1 to 4 , wherein the stem cell is a mesenchymal stem cell.

For example, in cell culture using dish a conventional method, cells that grows in a Petri dish bottom, the cell number reached approximately 30,000 to 40,000 pieces / cm ^2. In a petri dish with a diameter of about 10 cm, about 2,000,000 to 3,000,000 cells grow, and about 10 ml of a culture solution is used. 10 ml of the culture supernatant is collected from a diameter of 10 cm × a height of 1.5 cm, that is, a container volume of 120 cm ^3.

On the other hand, in the present invention, the culture using the permeable membrane hollow fiber module is performed, and the culture solution is passed through the hollow fiber module at an extremely low speed, so that the culture is collected in comparison with the number of cells. The amount of supernatant is significantly smaller than that of the conventional method. For example, in a 10 cm long hollow fiber module having a thickness of about 1 cm and containing hundreds of permeable membrane hollow fibers, the number of cells reaches 10,000,000 or more, but the culture flows in direct contact with the cells. The total volume of the liquid is less than 10 ml, the cell number ratio is about 1/5 of the conventional method, and the concentration of the substance secreted in the culture supernatant is conversely about 5 times. Moreover, since 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 prepare a culture supernatant containing a high concentration of a physiologically active substance secreted from mesenchymal stem cells to be cultured.

The configuration example of the hollow fiber module used for carrying out the production of the stem cell culture supernatant of the present invention is shown. An example of the cell culture form used for carrying out the production of the stem cell culture supernatant of the present invention is shown in a schematic diagram. It shows the schedule of the culture supernatant preparation in Example 1 of this invention. 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. It shows the effect of suppressing TNF-α production by the culture supernatant in Example 1 of the present invention.

One of the embodiments of the present invention is a method for producing a stem cell culture supernatant (conditioned medium, conditioned medium), which comprises the following steps [a] to [c].
[A] A step of supplying a culture solution to stem cells seeded on the surface of a permeable membrane hollow thread in a cell culture vessel [b] A step of bringing the culture solution into contact with the stem cells to culture the stem cells [c] The stem cells Step of collecting the culture solution containing the secreted components

(Cell culture supernatant)
In the present invention, the cell culture supernatant is obtained by separating a culture solution that has been in direct or indirect contact with a cell from the cell when the cell is cultured for a certain period of time (several hours to several days). To tell. Consent with culture medium, Conditioned medium, etc.

(Stem cells to be cultured)
The stem cell that is one of the subjects of the present invention is not particularly limited, but bone marrow mesenchymal stem cells or adipose tissue-derived mesenchymal stem cells are preferable. Not limited to primary cells, mesenchymal stem cells established by gene modification or the like can also be used. The animal species is not particularly limited, and any animal-derived animal such as human, mouse, or rat can be used. Further, it can be suitably used for various somatic stem cells having adhesiveness other than mesenchymal stem cells.

(Culture solution)
The composition of the culture solution used in the method for producing the stem cell culture supernatant of the present invention is not particularly limited. For example, DMEM, αMEM, RPMI1640 or the like is used as a basal medium, and those prepared by adding animal blood, cell growth factors, hormones and the like as appropriate can be used.

In some cases, the culture medium used in the present invention preferably does not contain animal serum. This is because animal serum is rich in bioactive substances such as cell growth factors, and sometimes the presence of these bioactive substances interferes with the purpose of using the culture supernatant or has a negative effect. This is because there is a possibility of doing so.

(Transparent membrane hollow fiber)
The permeable membrane hollow fiber used in the present invention is not particularly limited as long as it can hold cells in the hollow fiber lumen and can have a structure that allows a solution or a small molecule substance to permeate. No.

One of the reasons for using the permeable membrane hollow fiber in the present invention is that the number of cells growing per culture area is larger than that of a plastic material such as a petri dish. This is because the cells that adhere to and proliferate on the hollow fiber membrane do not stretch as much as on the plastic, so the number of cells that adhere to the hollow fiber membrane per unit area is several times higher on the hollow fiber membrane than on the plastic membrane. Because. Further, by tightly bundling the membrane hollow fibers, the culture area per unit volume can be remarkably increased.

The material of the permeable membrane hollow yarn used in the present invention is not particularly limited, and for example, cellulose-based materials such as cellulose acetate, cellulose triacetate and regenerated cellulose, polysulfone-based materials such as polysulfone and polyethersulfone, polyethylene, polypropylene, and the like. An insoluble carrier having a skeletal structure made of a thermoplastic polymer such as polystyrene, polyvinyl alcohol, polymethylmethacrylate, polyacrylonitrile, fluororesin, polyamide, and polyvinylidene fluoride (PVDF) can be preferably used. Further, these derivatives may be the main component.

The permeable membrane hollow fiber used in the present invention may be a material obtained by chemically modifying the above-mentioned material. For example, the permeable membrane hollow fiber used in the present invention is preferably hydrophilized. By hydrophilizing the permeable membrane hollow fiber, it becomes easy to supply a liquid component such as a culture solution to cultured cells. Examples of the method for hydrophilizing the permeable membrane hollow fiber include a method for treating the permeable membrane hollow fiber with a hydrophilic polymer such as an ethylene-vinyl alcohol copolymer, glycerin, or ethanol. Alternatively, depending on the cells used, a coating agent such as collagen or fibronectin may be used to improve the adhesion to the hollow fiber.

The size of the permeable membrane hollow fiber used in the present invention is also not particularly limited, but the lower limit of the inner diameter of the hollow fiber is preferably 10 μm, more preferably 20 μm, and further preferably 50 μm. On the other hand, the preferable upper limit of the inner diameter of the hollow fiber is 1000 μm, more preferably 500 μm. The film thickness is also not particularly limited, but the hollow fiber may be set within a range in which the hollow fiber maintains an appropriate strength and the permeability of the substance is also good, and is preferably about 10 to 200 μm, for example.

The pore size of the permeable membrane hollow thread used in the present invention is not particularly limited as long as it allows cells to pass through but allows passage of culture solution components such as water, salts, and nutrients having a relatively small molecular weight. However, considering cell culture and recovery of culture supernatant, it is necessary that substance exchange has a pore size that is efficient to some extent and that useful components such as physiologically active substances produced by cells do not permeate. .. Therefore, the pore diameter (hole radius) of the permeable membrane hollow fiber is preferably 1 to 500 Å, more preferably 5 to 300 Å, and even more preferably 10 to 150 Å.

The pore size of the permeable membrane hollow fiber is closely related to the permeability of the permeable membrane hollow fiber. Permeability The permeability of the hollow fiber membrane is indicated by, for example, the amount of liquid passing per unit time and unit area. In the permeable membrane hollow fiber used in the present invention, the permeability is not particularly limited, but for example, 1 to 1000 ml / m ² / hr / mmHg, more preferably 10 to 500 ml / m ² / hr / mmHg. Is preferable.

(Manufacturing process of stem cell culture supernatant)
The method for producing a stem cell culture supernatant of the present invention includes the following steps.
[A] Step of supplying the culture solution to the stem cells existing in the permeable membrane hollow fiber cavity [b] The culture solution is continuously or intermittently moved (passed) through the hollow fiber cavity to the stem cells. Step of culturing the stem cells by contacting the culture broth [c] Step of collecting the culture broth obtained by the step The order may be, for example, [a] [b] [c]. Two or more steps may be overlapped in the middle of. However, the start of the process of [b] does not precede the start of the process of [a], and the start of the process of [c] does not precede the start of the process of [b].

The method for producing the stem cell culture supernatant of the present invention is not particularly limited, and for example, the stem cells may be cultured on the hollow fiber lumen side using the hollow fiber. For example, when culturing stem cells, the stem cells are seeded on the surface of the hollow fiber by injecting a stem cell turbid solution into the hollow fiber lumen, and after the seeding is completed, the culture solution is perfused into the hollow fiber lumen where the stem cells are present. Supply by letting. Next, culture is performed on the lumen side. For example, the supplied culture solution is continuously or intermittently moved (passed) through the hollow fiber lumen to bring the culture solution into contact with the stem cells to culture the stem cells. The intermittent movement (passage) refers to repeating the process of temporarily stopping or advancing the flow of the culture solution in the hollow fiber. Here, the interval setting for stopping or advancing the flow is not particularly limited, and may be evenly spaced or irregular. The culture solution has a role of supplying necessary nutrients and oxygen to cells, and conversely discharging waste products. During the culture period, it is preferable to continue to supply the medium on both the luminal side and the outer luminal side for gas exchange, nutrient supply to cells, and removal of waste products. At that time, by using a perista pump or the like, it is possible to supply / discharge or circulate at an appropriate speed. By collecting the culture broth after culturing for a certain period of time in this way, the stem cell culture supernatant of the present invention can be obtained.

(Hollow fiber module)
The permeable membrane hollow fiber used in the method for producing the stem cell culture supernatant of the present invention is modularized by storing a hollow fiber bundle in which dozens to tens of thousands of hollow fibers are bundled in a tubular container to form cells. It may be used as a culture container (hereinafter, such a module is also referred to as a hollow fiber module). The hollow fiber module needs to have at least one opening capable of supplying the medium to the culture substrate and one opening capable of discharging the medium.

The configuration of the module using such a permeable membrane hollow fiber is not particularly limited, but as shown in FIG. 1, for example, it is permeable to the main body of the module case 1 having four openings (end conduit and side conduit). Examples thereof include a form in which a required number of hollow fiber membranes 2 are bundled and filled. In this embodiment, of the four openings, the two end conduits 3 are suitable sealing materials (for example, polyurethane-based potting agents) so as not to block the hollow portions of the hollow fibers 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 the other end conduit 3 (that is, flows in one direction) even though it passes through the hollow fiber lumen. Has been done. On the other hand, in the opening, the space inside the remaining two side ducts 4 is the space inside the module case 1 and the outer cavity of the hollow fiber (hereinafter, simply "outer cavity side"). It is connected to (also referred to as), and liquid or the like introduced from one of the side ducts 4 is led out from the other side duct 4 even though it passes through the outer cavity side of the membrane hollow fiber (that is, one direction). It is configured to flow to).

When the hollow thread module is used as the culture vessel in the method for producing the stem cell culture supernatant of the present invention, for example, the stem cell suspension is seeded by injecting it from the end conduit, and after the seeding is completed, the culture solution is used. It may be supplied by perfusing the hollow thread lumen in which the stem cells are present. Further, at the time of culturing, it is preferable that the culture medium is perfused to the hollow fiber lumen side at the same time as the medium is injected from the side duct to the hollow fiber outer lumen side.

The hollow fiber or hollow fiber module is preferably sterilized and supplied by an appropriate method. The sterilization method is not particularly limited, and examples thereof include pressure heat sterilization, gamma ray sterilization, and ethylene oxide gas sterilization.

By using the hollow fiber module in the culture vessel, the present invention can be carried out more efficiently. The fresh medium can be uniformly supplied to the cells in the lumen of the hollow fiber module at a low speed, and the culture supernatant can be efficiently collected.

(Cell culture system used in the present invention)
In the method for producing a stem cell culture supernatant of the present invention, culturing may be performed using a cell culture system containing the following configurations [d] to [g].
[D] The cell culture container [e] At least one culture solution storage container [f] A flow path network connecting the cell culture container and the culture solution storage container [g] The cell provided in the flow path network. Means for controlling the supply of the culture solution to the culture container and / or the discharge of the culture solution from the cell culture container.

The cell culture vessel used in the system is a module using a porous membrane hollow fiber, and has two openings connected to the inner cavity and two openings connected to the outer cavity from both ends of the hollow fiber. It is not particularly limited as long as it is a cell culture vessel having.

(Culture storage container)
The form and material of the culture solution storage container used in the system are not particularly limited, but it is desirable that the culture solution storage container has gas permeability. In one aspect of the system, a culture apparatus including the hollow thread module may be configured _{and placed in a CO 2} incubator to prepare a culture supernatant, in which case gas permeability is achieved. By using the culture solution storage container having the above, it is possible to easily carry out the culture without installing a gas exchange tube having a problem such as bubble generation. Examples of such a culture solution storage container include commercially available cell culture bags (manufactured by Nipro, Toyo Seikan, etc. are available), plastic bottles, and the like.

(Channel network)
In the system, several channels (channel networks) are connected to the cell culture vessel. The configuration (pipe) 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. Must be piped. As such a flow path network, at least (1) the connection portion between the cell culture container and the opening on the hollow thread 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 an opening on the outer cavity side of the hollow thread. In addition, in this specification, "connection" may be either the case where it is directly connected or the case where it is connected through a flow path such as a pipe.

The material of the piping used in the system is not particularly limited. For example, Silicon Tube, Masterflex (registered trademark), C-Flex (registered trademark), Tygon (registered trademark), Farmed (registered trademark) BPT, Pharmapure (registered trademark), Norprene (registered trademark), Sanitech (registered trademark). Trademark) and the like can be used. As the flow path tube of the portion connecting the liquid medium storage container and the cell culture container, it is preferable to use a flow path tube having gas permeability. For example, a silicon tube or the like can be preferably used.

(Means to control the supply and discharge of the culture solution)
The system is provided with means provided in the channel network to control the supply of the culture solution to the cell culture vessel and / or the discharge of the culture solution from the cell culture vessel. The form of the method for supplying the cell suspension or the culture medium is not particularly limited, but perfusion using a perista pump or when the culture medium storage container is a soft one such as a culture bag, a roller is placed in the container. A method of squeezing out the medium by hitting it is preferable. The 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 dust being mixed in when the liquid passes through the pump portion.

(One aspect of cell culture system)
FIG. 2 shows an example of an embodiment of the present invention in which a culture system composed of the above-mentioned hollow fiber module, culture solution storage container, perista pump, etc. _{is installed in a CO 2 incubator.} In FIG. 2, 5 and 6 are gas-permeable culture medium storage containers (cell culture bag, etc.), respectively. From the culture solution storage container 5, a flow path is connected to the end conduit 3a of the hollow thread module 7, so that the medium contained in the culture solution storage container 5 can be transferred to the hollow thread lumen side. On the other hand, from the culture solution storage container 6, 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 cavity side. There is. The culture solution that has passed through the lumen side of the hollow fiber module 7 is discharged from the end conduit 3b, and the flow path is connected to the culture supernatant collection container 11 via the perista pump 8 and passes through the hollow fiber module lumen. The culture supernatant can be collected. On the other hand, the culture solution that has passed through the outer cavity side of the hollow fiber module 7 is discharged from the side conduit 4b and collected in the waste liquid recovery container 10 via the perista pump 9.

(Culturing stem cells in the hollow fiber lumen)
When the method for producing the 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 luminal side and the outer cavity. Two routes on the side are required. At that time, when supplying the medium, separate culture medium storage containers may be used on the lumen side and the outer lumen side, or the medium is supplied from one container to both the lumen side and the outer lumen side. Is also good. Further, it is desirable to suppress the flow of the culture solution from the lumen to the lumen or from the lumen to the lumen as much as possible due to the water pressure difference or the osmotic pressure difference between the hollow fiber lumen side and the outer lumen side. This is because the culture medium flowing through the hollow thread lumen contains a large amount of substances released by cells, whereas the culture solution flowing through the hollow thread outer cavity side contains no cells. This is because the amount of substances released by cells is extremely small. That is, the culture solution that has flowed into the lumen from the opening at one end of the hollow fiber is not affected by the outflow to the outer cavity or the interference of the inflow from the outer cavity as much as possible, and flows out from the other opening as it is. It is desirable to go.

(Culture solution for preparing culture supernatant)
The culture medium used for culturing is not particularly limited as long as it is usually used as a culture medium for mesenchymal stem cells, but when the culture supernatant of stem cells is applied to a living body for the purpose of treatment or the like, it may be used. It is desirable that the culture medium contains as little animal serum as possible or does not contain animal serum. Therefore, when culturing stem cells using a culture medium containing serum, the cells are replaced with a low serum culture medium or a serum-free culture medium when the cells are sufficiently increased, and finally the serum content is low. Alternatively, a culture supernatant containing no serum can be obtained. It is also possible to obtain a serum-free culture supernatant by culturing cells from the beginning using a culture medium whose components have been adjusted so that stem cells can be cultured even without serum.

(Recovery of culture supernatant)
For the above reasons, in order to obtain the culture supernatant according to the present invention, the culture solution flowing through the hollow fiber lumen should be collected in a container separately from the culture solution flowing through the hollow fiber outer cavity. Is preferable.

(Speed at which the culture solution flows)
It is preferable to strictly control the flow velocity of the culture medium in cell culture, particularly the culture medium flowing in contact with the cells through the hollow thread lumen. If the flow velocity is too slow, the nutrient supply to the cells is insufficient and the cells are difficult to proliferate. On the contrary, even if the flow velocity is too fast, the environment around the cell changes drastically, the cell does not adapt to the surrounding environment, and the cell becomes difficult to proliferate. As described above, the flow velocity of the culture solution, particularly the culture solution flowing through the hollow fiber lumen, is preferably adjusted according to the degree of cell proliferation and the environment. The method for examining the cell proliferation degree is not particularly limited, but it can be performed based on the measurement results such as the concentration of glucose and lactate in the culture solution. The preferred flow rate of the culture medium flowing to the side where the cells are seeded is 0.01 to 1 mm / min. On the other hand, the preferable flow rate of the culture solution to be flowed to the side where the cells are not seeded is 0.02 to 5 mm / min.

In the present invention, the flow of the culture solution flowing through the hollow thread lumen, that is, the culture solution flowing from the culture solution storage container through the hollow thread lumen of the hollow thread module to the culture supernatant recovery container is unidirectional. It is preferable to have. Here, unidirectional means that the route from the introduction of the medium into the hollow fiber module to the derivation is always the same direction. Specifically, for example, there is a form in which there is one inlet and one outlet for the medium in the hollow fiber module, and the medium always flows from the inlet to the outlet. On the other hand, the culture solution flowing on the outer cavity side of the hollow fiber may flow in one direction, or may be a so-called circulation type in which the medium once drawn out from the cell culture vessel is introduced again from the introduction port. good.

(1) Culturing mesenchymal stem cells using a hollow fiber module and preparing a culture supernatant A hollow fiber module (hollow fiber is made of PES / PVP, inner diameter 200 μm, outer diameter) coated with collagen (Nitta Gelatin) in advance before cell seeding. Using 260 μm, a film thickness of 30 μm, a hole radius of 75 Å, and Toyo Boseki), the same components as those shown in FIG. 2 _{were installed in a CO 2} incubator, and this example was carried out. Seeded hollow fiber lumen human bone marrow mesenchymal stem cells (CELL APPLICATIONS Inc.) (seeding cell number, 5.0 × ¹⁰ 5 cells / modules) was. At this time, since the total culture area of the hollow fiber module (based on the inner diameter of the hollow fiber) was 98 cm ² , the cell seeding density was about 5100 cells / cm ² . As the culture medium, DMEM GlutaMAX (Life Technologies) supplemented with 10% fetal bovine serum (Life Technologies) was used from the start of culture (cell seeding) to 96 hours after the culture supernatant was collected, and after 96 hours, MF was used. -Medium, mesenchymal stem cell growth medium (Toyo Boseki) was used. MF-medium is a low serum culture medium containing only 1% serum.

FIG. 3 shows a schedule for preparing a culture supernatant by the method of the present invention. Culturing was carried out for 7 days (168 hours later) from cell seeding (start of culturing). During this period, the flow velocity (linear velocity) of the culture solution flowing through the hollow thread lumen was 0.066 mm / min on average from 96 hours after cell seeding, and 0. From 20 mm / min, 144 hours to 168 hours, the average was 0.33 mm / min. On the other hand, the velocity of the culture solution flowing through the outer cavity of the hollow fiber was 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 culture, and stored at 4 ° C. until it was used for later performance evaluation. The total amount of culture supernatant was 7.9 ml. The flow velocity was calculated based on the cross-sectional area of the hollow fiber by measuring the flow rate flowing out from each of the hollow fiber lumen and the outer cavity. Digested cells 168 hours after incubation with trypsin, peeling recovered, the results obtained by counting the number of cells, 1.2 × 10 ⁷ cells were recovered.

(2) Culturing mesenchymal stem cells using a petri dish and preparing a culture supernatant (conventional method)
^{Human bone marrow mesenchymal stem cells (CELL APPLICATIONS Inc.) were seeded on two} collagen-coated petri dishes (culture area 55 cm 2, Asahi Techno Glass) so that the cell seeding density was about 5100 cells / cm ² . As the culture medium, DMEM GlutaMAX (Life Technologies) supplemented with 10% fetal bovine serum (Life Technologies) was used from cell seeding to 96 hours after cell seeding, and after 96 hours, MF-media and mesenchymal were used. The medium was replaced with a line stem cell growth medium (Toyobo).
FIG. 3 shows the schedule for preparing the culture supernatant by the conventional method. Culture solution exchange was performed 48 hours and 96 hours after the start of culturing. Then, without exchanging the culture medium, the culture was terminated when 100% confluence was reached within 168 hours from the start of the culture. The culture broth obtained after culturing for 72 hours from the last medium exchange to the end of culturing was collected as a culture supernatant. The total amount of the culture supernatant was 10.0 ml. After 168 hours from culturing, the cells were digested with trypsin, exfoliated and collected, and the number of cells was counted. As a result, 2.6 × 10 ⁶ cells were collected.

(3) Evaluation of collected culture supernatant (measurement of VEGF and IGF concentrations)
VEGF (Vascular Endothelial Growth Factor) and IGF (Insulin) contained in the culture supernatants are included in the culture supernatants in order to evaluate the performance related to tissue regeneration for each of the culture supernatants collected by the above methods (1) and (2). The concentration of (like growth factor) was measured.
Human VEGF ELISA Kit (R & D Systems) was used for the quantification of VEGF.

The result of measuring the VEGF concentration in the culture supernatant is shown in FIG. As a result, the VEGF concentration in the culture supernatant recovered by the conventional method was 561.6 pg / ml, whereas it was 3597.1 pg / ml in the culture supernatant recovered by the present invention. It showed a clear high concentration. Further, from this concentration, 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) was used for the quantification of IGF.

The result of measuring the IGF concentration in the culture supernatant is shown in FIG. As a result, the IGF concentration in the culture supernatant recovered by the conventional method was 1864.6 pg / ml, whereas in the culture supernatant recovered by the present invention, it was 3798.0 pg / ml. It showed a clear high concentration. Further, from this concentration, the total amount of VEGF recovered during the culturing time of 72 hours was 18.6 ng in the conventional method, whereas it was 109.0 ng in the present invention.

(4) Evaluation of collected culture supernatant (measurement of effect on activated macrophages)
The anti-inflammatory effect was evaluated for each of the culture supernatants collected by the above methods (1) and (2). This is done by stimulating RAW264.7 cells (Dainippon Pharmaceutical Co., Ltd.), which is a macrophage-like cell line, with lipopolysaccharide (LPS) and measuring the production of inflammatory cytokines (TNF-α) produced by the stimulation. evaluated.
The measurement was carried out as follows. That is, RAW264.7 cells were suspended in RPMI1640 medium (Life Technologies) containing 10% fetal bovine serum (Life Technologies) and ^{seeded on a 24-well plate at 5 × 10 5} cells / well. After culturing for 24 hours, half of the culture broth was replaced with the culture supernatant of (1) or (2). After 1 hour, LPS was added to a final 100 ng / ml, and after further culturing for 3 hours, the culture broth was collected, and TNF-α in the culture broth was collected using Mouse TNF-αELISA Kit (R & D Systems). Was measured.
The effect of suppressing TNF-α production by the culture supernatant is shown in FIG. As a result, it was found that the culture supernatant of the mesenchymal stem cells prepared by the method of the present invention significantly suppressed the production of TNF-α as compared with the culture supernatant prepared by the conventional method.

From the results shown in (3) and (4) above, the culture supernatant of the stem cells produced by the present invention is richer in physiologically active substances and excellent in tissue regeneration and anti-inflammatory action as compared with the conventional ones. It can be said that.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to easily and efficiently produce a culture supernatant of stem cells having various actions such as tissue regeneration and anti-inflammatory.

1 Module case 2 Permeable membrane hollow fiber 3 End conduit 4 Side conduit 5, 6 Culture solution storage container 7 Cell culture container (hollow fiber module)
8, 9 Perista pump 10 Waste liquid recovery container 11 Culture supernatant recovery container

Claims (5)

A method for producing a stem cell culture supernatant, which comprises the following steps [a] to [c].
[A] The culture solution is supplied to the stem cells seeded on the inner surface of the permeable membrane hollow thread in the cell culture vessel at a linear velocity of 0.01 to 1 mm / min, and the culture solution is 0 on the side where the cells are not seeded. A step of culturing the stem cells by supplying the cells at a linear velocity of .02 to 5 mm / min [b] A step of proliferating the stem cells using a culture solution in which serum is added to the culture solution when culturing the stem cells [b] c] A step of contacting the proliferated stem cells with a serum-free culture medium or a low-serum culture medium and collecting the obtained culture supernatant. The cell culture vessel is a module that stores a permeable membrane hollow fiber, and has two openings that communicate with the inner cavity of the hollow fiber and two openings that communicate with the inside of the module and the outer cavity of the hollow fiber. The method according to claim 1, wherein the method has an opening. The method according to claim 1 or 2, wherein the stem cells are cultured using a cell culture apparatus containing the following configurations [d] to [g].
[D] The cell culture container [e] At least one culture solution storage container [f] A flow path network connecting the cell culture container and the culture solution storage container [g] The cell provided in the flow path network. Means for controlling the supply of the culture solution to the culture container and / or the discharge of the culture solution from the cell culture container. The method according to any one of claims 1 to 3, wherein the pore radius of the permeable membrane hollow fiber is 1 to 500 Å. The method according to any one of claims 1 to 4 , wherein the stem cell is a mesenchymal stem cell.

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