Method for producing culture supernatant liquid

Abstract

[Problem] To provide a method for producing a culture supernatant liquid, the method making it possible to produce a large amount of a culture supernatant liquid containing an active ingredient at a high concentration. [Solution] The method for producing a culture supernatant liquid comprises: (a) a step for supplying a culture solution containing a carrier, mesenchymal stem cells, and serum (hereinafter referred to as "FBS-DMEM") to a culture tank; (b) a step for adhering the mesenchymal stem cells to the carrier; (c) a step for culturing the mesenchymal stem cells using the FBS-DMEM; (d) a step for removing the FBS-DMEM while leaving the carrier-adhered mesenchymal stem cells in a filter; (e) a step for washing the culture tank, the carrier-adhered mesenchymal stem cells, and the filter; (f) a step for supplying a serum-free culture solution for a supernatant liquid (hereinafter, referred to as "CM-DMEM") to the culture tank; (g) a step for culturing the mesenchymal stem cells using the CM-DMEM; and (h) a step for recovering the CM-DMEM while leaving the carrier-adhered mesenchymal stem cells in the filter.

Description

Method for producing culture supernatant

[0001] The present invention relates to a method for producing a stem cell culture supernatant.

[0002] In medical treatments using mesenchymal stem cells, it is known that not only do the stem cells themselves bring about therapeutic effects, but also various physiologically active substances secreted by the stem cells, such as cytokines and exosomes, contribute significantly to the therapeutic effect.When mesenchymal stem cells are artificially cultured, these cytokines and other substances are released from the cells into the culture medium, and the culture medium from which the mesenchymal stem cells have been removed can be collected and effectively utilized as culture supernatant.

[0003] Patent Document 1 discloses a method for producing a culture supernatant, which includes the steps of supplying a culture medium to mesenchymal stem cells seeded on the inner surface of a permeable membrane hollow fiber, bringing the culture medium into contact with the mesenchymal stem cells to culture the mesenchymal stem cells, and recovering the culture medium containing components secreted by the mesenchymal stem cells. The invention of Patent Document 1 enables the easy production of a culture supernatant.

[0004] Patent No. 6958350

[0005] However, the invention of Patent Document 1 has a problem in that the amount of culture supernatant produced is small. Therefore, an object of the present invention is to provide a method for producing a culture supernatant containing a high concentration of an active ingredient in large quantities using mesenchymal stem cells.

[0006] This embodiment is a method for producing a culture supernatant obtained by removing mesenchymal stem cells from a culture medium in which mesenchymal stem cells have been cultured. The production method includes the steps of (a) supplying a culture medium containing a carrier, mesenchymal stem cells, and serum (hereinafter referred to as FBS-DMEM) to a culture vessel, (b) adhering the mesenchymal stem cells to the carrier, (c) culturing the mesenchymal stem cells using the FBS-DMEM, (d) leaving the mesenchymal stem cells adhered to the carrier in a filter and removing the FBS-DMEM, (e) washing the culture vessel, the mesenchymal stem cells adhered to the carrier, and the filter, (f) supplying a serum-free culture medium for the supernatant (hereinafter referred to as CM-DMEM) to the culture vessel, (g) culturing the mesenchymal stem cells using CM-DMEM, and (h) leaving the mesenchymal stem cells adhered to the carrier in the filter and recovering the CM-DMEM.

[0007] The step (b) may include a first period in which the FBS-DMEM is stirred so that the carriers are suspended in the FBS-DMEM, and a second period in which stirring is stopped. The step (c) may include stirring the FBS-DMEM so that the carriers are suspended in the FBS-DMEM. The step (g) may include stirring the CM-DMEM so that the carriers are suspended in the CM-DMEM.

[0008] The mesh size of the filter is preferably 70 to 400 mesh. After steps (a) to (e), steps (f) to (h) are preferably repeated consecutively. Steps (f) to (h) may also be repeated two to four times consecutively.

[0009] If the amount of at least one of cytokines and exosomes in the recovered CM-DMEM is less than a predetermined value, steps (c) and (e) may be performed only once. If the amount of at least one of cytokines and exosomes in the recovered CM-DMEM is greater than a predetermined value, steps (f) to (h) may be repeated consecutively.

[0010] The mesenchymal stem cells are preferably immortalized stem cells, and more preferably, the immortalized stem cells are deciduous tooth pulp stem cells.

[0011] This embodiment is a method for producing a culture supernatant obtained by removing mesenchymal stem cells from a culture medium in which mesenchymal stem cells have been cultured. The method for producing the culture supernatant includes the steps of: (p) culturing mesenchymal stem cells adhered to a carrier in a culture vessel using a serum-containing culture medium (hereinafter referred to as FBS-DMEM), then leaving the mesenchymal stem cells adhered to the carrier in a filter placed in the culture vessel, and removing the FBS-DMEM; (q) after step (p), washing the culture vessel, the mesenchymal stem cells adhered to the carrier, and the filter; and (r) after step (q), supplying a serum-free culture medium for the supernatant (hereinafter referred to as CM-DMEM) to the culture vessel, culturing the mesenchymal stem cells using CM-DMEM, then leaving the mesenchymal stem cells adhered to the carrier in the filter, and recovering the CM-DMEM.

[0012] Steps (p) and (q) may be performed only once, and then step (r) may be repeated two to four times in succession, and again steps (p) and (q) may be performed only once, and then step (r) may be repeated two to four times in succession.

[0013] The capacity of the culture vessel is preferably 5 L to 20 L, and the mesh size of the filter is preferably 70 mesh to 400 mesh. Furthermore, the mesenchymal stem cells are preferably immortalized stem cells.

[0014] According to the method for producing a culture supernatant of the present invention, a large amount of culture supernatant containing an active ingredient at a high concentration can be continuously provided.

[0015] 1 is a schematic cross-sectional view showing an example of a culture vessel 100 without a filter 30 attached. (A) is a perspective view of the filter 30, and (B) is a perspective view of the carrier 40. FIG. 2 is a flow chart showing an example of a method for producing a culture supernatant. (A) is a conceptual diagram showing a state in which the culture vessel 100 is filled with culture medium, and (B) is a conceptual diagram showing a state in which most of the culture medium has been discharged from the culture vessel 100.

[0016] <Stem Cells to be Cultured> The stem cells of this embodiment are not particularly limited, but bone marrow mesenchymal stem cells or adipose tissue-derived mesenchymal stem cells are suitable. The animal species is also not particularly limited, and stem cells derived from any animal, such as humans, mice, or rats, can be used. The type of cell is also not particularly limited, and may be, for example, fibroblasts, endothelial cells, epithelial cells, nerve cells, stem cells, leukocytes, bone cells, muscle cells, or adipocytes, with fibroblasts or endothelial cells being preferred. More preferred stem cells are human-derived dental pulp stem cells, particularly deciduous tooth dental pulp stem cells.

[0017] <Immortalization of Mesenchymal Stem Cells> A method for immortalizing mesenchymal stem cells is disclosed, for example, in U.S. Patent 10,494,606 B2. This disclosed method allows for the immortalization of mesenchymal stem cells by introducing four types of genes, hTERT, bmi-1, E6, and E7, into primary cultured cells obtained by initial culturing of mesenchymal stem cells. Another method for immortalizing mesenchymal stem cells is disclosed in U.S. Patent 6,146,888. This disclosed method allows for the immortalization of mesenchymal stem cells by introducing the SV40 gene using a viral vector. Another method for immortalizing mesenchymal stem cells includes introducing the telomerase reverse convertase (TERT) gene into mesenchymal stem cells, which also allows for the immortalization of mesenchymal stem cells.

[0018] <Mesenchymal stem cell culture supernatant> In this specification, the supernatant obtained by culturing mesenchymal stem cells and from which the mesenchymal stem cells have been removed is referred to as the culture supernatant. In order to enhance safety, it is preferable that the culture supernatant does not contain animal serum. The culture supernatant can also be freed of animal serum by dialysis, solvent substitution, or the like.

[0019] The culture supernatant may be frozen or lyophilized, or the lyophilized culture supernatant may be dissolved in an appropriate solvent. Lyophilization provides good storage stability. Lyophilization of the cell culture supernatant can be performed using methods commonly used for lyophilizing protein-containing liquids.

[0020] The mesenchymal stem cell culture supernatant of this embodiment is preferably a culture supernatant of deciduous dental pulp stem cells, which have high proliferation capacity and contain large amounts of cytokines, exosomes, and the like.

[0021] The culture medium used in the method for producing a culture supernatant of this embodiment is not particularly limited. For example, the culture medium may be a basal culture medium such as DMEM, αMEM, IMDM, Ham's F-12, RPMI-1640, or a mixture thereof, to which serum such as fetal bovine serum (FBS) or a serum substitute such as KSR (KnockOut™ Serum Replacement), glucose, amino acids, vitamins, antibiotics, or the like is appropriately added.

[0022] In this embodiment, two types of culture medium are preferably prepared: a serum-containing culture medium (hereinafter referred to as FBS-DMEM) and a serum-free culture medium for collecting supernatant (hereinafter referred to as CM-DMEM). The FBS-DMEM preferably contains DMEM, 5-20% by volume of FBS, antibiotics, etc. The CM-DMEM preferably contains DMEM and antibiotics, etc., but contains little or no serum, in order to obtain a supernatant.

[0023] <Culture Tank and Cultivation Device> Fig. 1 is a schematic cross-sectional view showing an example of a culture vessel 100 used in this embodiment. Fig. 1 is a view showing a state before a filter 30, which will be described later, is attached.

[0024] The culture vessel 100 has a culture tank 10 with a capacity of 5 L to 20 L. The culture tank 10 is a component that holds a culture solution therein, and its shape, capacity, and material are appropriately selected according to the purpose of the culture. In this embodiment, the culture tank 10 has a circular bottom and cylindrical sides. The material of the culture tank 10 is not particularly limited, but in this embodiment, the culture tank 10 is preferably made of glass, polycarbonate, stainless steel, or the like, and is preferably made of a transparent material, particularly so that the culture status or stirring status can be visually confirmed. A heater 25 that adjusts the temperature inside the culture tank 10 is wrapped around the bottom of the culture tank 10.

[0025] The culture vessel 100 has a top plate 22. The top plate 22 is a member that covers the upper opening of the culture tank 10, and multiple O-rings OR are arranged between the top plate 22 and the culture tank 10 to increase airtightness. The top plate 22 can be made of, for example, polycarbonate or stainless steel. Multiple openings are formed in the top plate 22, allowing for the attachment of various components. In this embodiment, the top plate 22 is equipped with an agitation shaft 13, various sensors 18, a gas supply tube 19, a culture medium discharge nozzle 24, a culture medium supply nozzle (not shown), a gas discharge tube (not shown), and the like.

[0026] A stainless steel stirring paddle 12 is attached to the tip of a stainless steel stirring shaft 13, and a rotary motor 16 is attached to the rear end of the stirring shaft 13. The stirring shaft 13 is rotatably held by a bearing 15. For example, the stirring shaft 13 can be rotated at 5-100 rpm by the rotary motor 16 to stir the culture solution. Instead of providing the stirring paddle 12, rotary motor 16, and stirring shaft 13 in the culture vessel 100, a Teflon (registered trademark)-coated stirrer (magnetic bar) may be placed in the culture vessel 10, and the stirrer may be rotated by a magnetic stirrer (agitator) to stir the culture solution.

[0027] Various sensors 18 are attached to the openings in the top plate 22 and measure the oxygen concentration, carbon dioxide concentration, pH, temperature, etc. in the culture solution. A gas supply tube 19 is attached to the openings in the top plate 22 and supplies gases such as oxygen into the culture solution. The gas supply tube 19 is made of a chemical-resistant material such as fluororesin and supplies gas through porous through-holes. A gas exhaust tube (not shown) is also attached to the openings in the top plate 22 and exhausts unnecessary gases such as carbon dioxide. A culture solution supply nozzle (not shown) is attached to the openings in the top plate 22 and supplies the culture solution to the culture tank 10. A culture solution discharge nozzle 24 is attached to the openings in the top plate 22 and is made of stainless steel or the like and discharges the culture solution or supernatant from the culture tank 10. Note that instead of the openings in the top plate 22, an opening may be provided in the culture tank 10 to which the various sensors 18 or the culture solution discharge nozzle 24, etc., are attached.

[0028] A filter holding frame 28 is attached to the top plate 22 or the culture tank 10. The filter holding frame 28 is provided to hold a filter 30 (described later) in a predetermined shape, for example, a cylindrical shape. The filter holding frame 28 is made by combining stainless steel wires lengthwise and crosswise to form a cylindrical shape.

[0029] A control unit (not shown) supplies the culture solution to the culture tank 10 through a culture solution supply nozzle (not shown) and stirs the culture solution with a stirring paddle 12 according to a control program. Based on measurement results from various sensors 18, a gas supply tube 19 supplies oxygen from the gas supply tube 19, exhausts carbon dioxide and the like through a gas exhaust tube (not shown), and raises the temperature of the culture solution with a heater 25. The control program also discharges the culture solution from the culture solution exhaust nozzle 24 to the outside of the culture tank 10 at a scheduled time. The control program of this embodiment can automate the supply of culture solution, management of the temperature, pH, oxygen concentration, etc. of the culture solution, collection of the culture solution, and cleaning of the inside of the culture tank.

[0030] <Filter> FIG. 2(A) is a perspective view showing an example of a filter 30 used in this embodiment. The filter 30 has an overall cylindrical shape and includes a cylindrical portion 32 and a bottom portion 33 formed at the bottom of the cylindrical portion 32. The bottom portion 33 is circular and bowl-shaped. An opening 31 is formed at the top of the cylindrical portion 32. An attachment string 35 for attaching the filter 30 to the filter holding frame 28 or the top plate 22 is provided around the opening 31. The size of the filter 30 depends on the size of the culture tank 10, but for example, the length LL is 150 mm to 500 mm and the diameter ΦDM is Φ100 mm to 300 mm. The filter 30 is preferably made of polypropylene, polyester, or a combination of polypropylene and polyethylene, due to its light weight, excellent chemical resistance, and excellent thermal adhesiveness.

[0031] As will be described later, mesenchymal stem cells adhere to the carrier 40 and float. For this reason, the filter 30 preferably has a mesh size that allows the carrier 40 to penetrate but not the non-adhered mesenchymal stem cells to penetrate. Live mesenchymal stem cells remain adhered to the carrier 40, but dead stem cells detach from the carrier 40, or mesenchymal stem cells that detach from the carrier 40 die. Since the carrier 40 is generally 200 μm or larger in size and dead mesenchymal stem cells are around 20 μm in size, for example, the mesh size of the filter 30 is preferably 70 mesh (mesh size of approximately 185 μm) to 400 mesh (mesh size of approximately 35 μm).

[0032] <Carrier (Carrier)> Figure 2(B) is a perspective view showing an example of a carrier 40 used in this embodiment. This embodiment employs a culture method in which stem cells adhered to a carrier are grown suspended in a culture medium. To ensure a large culture area and culture volume and perform mass culture, carriers are preferably used for suspension culture. The carrier may be porous and spherical, polygonal, or disc-shaped. The size of porous spherical or other carriers is generally 140 μm to 280 μm. In this embodiment, for mass culture, a disc-shaped nonwoven fabric carrier 40 with a thickness LT of 200 μm to 500 μm and a diameter ΦDU of 3 mm to 9 mm is used. It is preferable that the nonwoven fabric be porous. The fibers constituting the nonwoven fabric are preferably small in diameter, with an average fiber diameter of preferably 10 to 100 μm, and particularly preferably 15 to 50 μm.

[0033] The carrier material may be organic, inorganic, or a composite material thereof. Examples of organic materials include synthetic polymers such as polystyrene, polyester, polyurethane, polyethylene, polypropylene, acrylic polymers, acrylamide polymers, polyvinyl alcohol, silicone polymers, and epoxy resins; natural polymers such as collagen and gelatin; polygalacturonic acids such as pectin and pectate; and polysaccharides such as alginate, cellulose, cross-linked agarose, dextran, and chitosan. Examples of inorganic materials include glass and ceramics.

[0034] The amount of carriers placed in the culture vessel 10 is preferably 5 g to 40 g (dry state) per 1000 ml of culture solution. If the amount of carriers is less than 5 g, the number of mesenchymal stem cells cultured will be reduced, and the amount of kite sign and other proteins in the recovered supernatant will be reduced. On the other hand, if the amount of carriers is more than 40 g, it will be difficult to control the culture conditions, and the mesenchymal stem cells will be more likely to die.

[0035] <Cultivation method> Figure 3 is a flowchart showing a production method for producing a supernatant. A filter 30 is attached using an attachment string 35 in the culture tank 10 of the culture vessel 100 shown in Figure 1. Figures 4(A) and (B) are conceptual diagrams showing the filter 30 attached to a filter holding frame 28. A culture solution discharge nozzle 24 is arranged on the outside of the filter 30. Note that, to make the carrier 30 easier to see, the various sensors 18, gas supply tube 19, etc. shown in Figure 1 are not shown.

[0036] Mesenchymal stem cells, carriers 40 washed with EDTA solution or the like, and FBS-DMEM warmed to 36-38°C are placed in the culture tank 10 (step S31). The mesenchymal stem cells are stem cells that have been cultured by culturing frozen mesenchymal stem cells in a flask until the amount required for the culture tank 10 is reached. Various sensors 18 measure the temperature, pH, and oxygen concentration as appropriate, and the temperature, pH, and oxygen concentration of the FBS-DMEM are adjusted by a control program.

[0037] Next, the mesenchymal stem cells are allowed to adhere to the carriers 40 (step S32). To allow the mesenchymal stem cells to adhere to the carriers 40, the stirring paddle 12 stirs the FBS-DMEM at a predetermined rotation speed until the carriers 40 are suspended in the FBS-DMEM. Furthermore, to ensure that the mesenchymal stem cells adhere to the carriers 40, the rotation of the stirring paddle 12 is stopped and the FBS-DMEM in the culture vessel 10 is allowed to settle. The first period during which the FBS-DMEM is stirred is, for example, 1 to 59 minutes per hour, while the second period during which stirring is stopped is 59 to 1 minute. This stirring and halting of stirring is continued for 48 to 72 hours. The rotation of the stirring paddle 12 may be constant or variable.

[0038] Next, mesenchymal stem cells are cultured (step S33). The stirring paddle 12 stirs the FBS-DMEM at a predetermined rotation speed, causing the carriers 40 to float in the FBS-DMEM. This allows the mesenchymal stem cells to proliferate three-dimensionally on the carriers 40. As the mesenchymal stem cells proliferate and the weight of the carriers 40 increases, the carriers 40 will no longer float in the FBS-DMEM unless the rotation speed of the stirring paddle 12 is increased. For this reason, it is preferable to gradually increase the rotation speed of the stirring paddle 12. When the mesenchymal stem cells occupy the entire area of ​​the carriers 40, they enter a plateau phase (stationary phase). Therefore, the carriers 40 remain suspended in the FBS-DMEM even when the rotation speed of the stirring paddle 12 is maintained constant. The culture is complete when the plateau phase is reached, and this culture period lasts for 72 to 96 hours. Figure 4(A) shows the carriers 40 suspended in the FBS-DMEM.

[0039] Next, the culture medium discharge nozzle 24 removes the FBS-DMEM (step S34). At this time, the stirring paddle 12 is stopped. As the culture medium discharge nozzle 24 discharges the FBS-DMEM, the carriers 40 become caught on the filter 30. Figure 4(B) is a diagram showing the state in which the FBS-DMEM has been almost completely removed and the carriers 40 are caught in the filter 30.

[0040] Next, phosphate-buffered saline (PBS) is supplied to the culture vessel 10, and the stirring paddle 12 is rotated at high speed for several minutes to approximately 10 minutes. This cleans the entire culture vessel 10, including the filter 30, carrier 40, various sensors 18, gas supply tube 19, etc. (Step S35). The culture solution discharge nozzle 24 then recovers the phosphate buffer, which is then removed.

[0041] Next, CM-DMEM warmed to 36-38°C is introduced into the culture tank 10 (step S36). The various sensors 18 measure the temperature, pH, and oxygen concentration as appropriate, and the temperature, pH, and oxygen concentration of the CM-DMEM are adjusted by a control program.

[0042] Next, the mesenchymal stem cells are cultured (step S37). The stirring paddle 12 stirs the CM-DMEM at a predetermined rotation speed so that the carriers 40 are suspended in the CM-DMEM. As a result, the mesenchymal stem cells are cultured on the carriers 40. The culture time is 40 to 60 hours.

[0043] Next, the CM-DMEM is collected by the culture solution discharge nozzle 24 (step S38), while the stirring paddle 12 is stopped. The culture solution discharge nozzle 24 collects the CM-DMEM, which is then stored as a culture supernatant of mesenchymal stem cells.

[0044] The recovered CM-DMEM (culture supernatant) is then subjected to a determination of whether the mesenchymal stem cells are in a good state (step S39). The determination of whether the mesenchymal stem cells are in a good state may be based on, for example, whether the measured levels of at least one of cytokines and exosomes are greater than a predetermined threshold. If the levels of at least one of secreted cytokines and exosomes are greater than a predetermined value, this indicates that the mesenchymal stem cells are in a good state.

[0045] If the mesenchymal stem cells are in good condition, steps S36-S38 are continuously repeated. If the mesenchymal stem cells are in bad condition, the process proceeds to step S40, in which FBS-DMEM heated to 36-38°C is added to the culture tank 10. The process then proceeds to steps S33-S34. In other words, nutrients in the form of serum are provided to the mesenchymal stem cells, causing them to proliferate or return to a good condition.

[0046] In step S39, whether the condition of the mesenchymal stem cells is good or not is determined by whether cytokines, etc., are greater than a predetermined threshold, but it is not necessary to measure every time. If it is confirmed that the secreted cytokines, etc., are greater than a predetermined threshold by alternately repeating culture in FBS-DMEM and culture in CM-DMEM, it is also possible to alternately repeat culture in FBS-DMEM and culture in CM-DMEM without measuring every time. Even if culture in CM-DMEM is performed four times in succession and then culture in FBS-DMEM once, it is also possible to perform a cycle of four consecutive cultures in CM-DMEM and one culture in FBS-DMEM, as long as it is confirmed that the secreted cytokines, etc., are greater than a predetermined threshold.

[0047] <Culturing of immortalized human dental pulp stem cells> Human deciduous dental pulp stem cells (SHED) prepared from the dental pulp tissue of human deciduous teeth were used as human dental pulp stem cells. These SHED were immortalized by introducing the SV40 gene using a viral vector. These immortalized SHED (hereinafter referred to as IM-SHED) were stored in a -80°C freezer, thawed to room temperature, and cultured in flasks in FBS-DMEM at 37°C. After repeated passage, 32 flasks were placed in a culture vessel 10 when they reached full confluence.

[0048] <Culturing IM-SHED in FBS-DMEM> IM-SHED, 60 g (dry weight) of a disk-shaped nonwoven fabric carrier 40, and 4000 ml of FBS-DMEM were placed in a 10 L (liter) culture vessel 10. The mesh size of the filter 30 used was 180 mesh (mesh size: approximately 84 μm). The FBS-DMEM was prepared at pH 7.0, dissolved oxygen 2.0, and a temperature of 37°C.

[0049] The IM-SHED, nonwoven fabric carrier 40, and FBS-DMEM were stirred by the stirring paddle 12. The stirring paddle 12 was programmed to rotate at 20-40 rpm for a predetermined time, then stop rotating and rotate again for a predetermined time so that the IM-SHED would adhere to the carrier 40. After 24 hours, 1000 ml of FBS-DMEM was added to the culture vessel 10.

[0050] After the process of adhering the IM-SHED to the carriers 40 was carried out for 48 to 72 hours, the stirring paddle 12 was used to stir the FBS-DMEM at a rotation speed of 40 rpm so that the carriers 40 were suspended in the FBS-DMEM. As the IM-SHED attached to the carriers 40 proliferated over time, the carriers 40 became less likely to float in the FBS-DMEM. Therefore, the rotation speed of the stirring paddle 12 was gradually increased from 40 rpm to 70 rpm to stir the FBS-DMEM.

[0051] 216 hours after the introduction of the IM-SHED, carriers 40, and FBS-DMEM, the cultivation of the IM-SHED was completed, and the FBS-DMEM was collected through the culture solution discharge nozzle 24. Then, 5000 ml of phosphate buffer solution was supplied to the culture vessel 10, and the entire culture vessel 10 including the filter 30, carriers 40, etc. was washed.

[0052] Next, 7500 ml of CM-DMEM warmed to 37°C was poured into the culture vessel 10, and the IM-SHED was cultured. The stirring paddle 12 stirred the CM-DMEM at a rotation speed of 50 to 60 rpm, so that the carriers 40 were suspended in the CM-DMEM. The culture time for the IM-SHED was 48 hours.

[0053] Next, the CM-DMEM was collected using the culture solution discharge nozzle 24 and stored in a refrigerator or freezer as the culture supernatant of IM-SHED.

[0054] As shown in Table 1, the recovered culture supernatant contained high concentrations of secreted cytokines, such as 3711.5 pg / ml for VEGF and 2566.7 pg / ml for HGF. For example, the threshold values ​​for VEGF were set to 3400 pg / ml and 2300 pg / ml for HGF. For this reason, CM-DMEM was added to the culture vessel 10 without adding FBS-DMEM, and the culture supernatant was continuously recovered. In other words, steps S36 to S38 in FIG. 3 were continuously repeated.

[0055] In Table 1, IM-SHED-AUTO (automated culture of immortalized human dental pulp stem cells) is the concentration of cytokines in this example, IM-SHED (immortalized human dental pulp stem cells) is the concentration when an operator uses a flask, and SHED (non-immortalized human dental pulp stem cells) is the concentration when an operator uses a flask.

[0056] It was confirmed that when CM-DMEM was added to the culture vessel 10 five consecutive times without adding FBS-DMEM, the VEGF concentration fell below the threshold value. To ensure that the threshold value was exceeded, in this example, the culture supernatant was automatically collected in a cycle in which CM-DMEM was added two consecutive times and FBS-DMEM was added once. As a result, even after 40 cycles (80 additions of CM-DMEM and 40 additions of FBS-DMEM) were repeated over a nine-month period, a culture supernatant containing a high concentration of cytokines as shown in Table 1 was obtained.

[0057] In this example, not only can high concentrations of cytokines be obtained, but large quantities of culture supernatant can also be obtained. For example, when IM-SHED is cultured using flasks, 30 ml of culture supernatant can be obtained from one flask. The maximum amount that two workers can culture using flasks is approximately 100, and the culture supernatant that can be obtained per month is 24 L. In contrast, in this example, 80 L of culture supernatant could be obtained automatically per month with almost no operator intervention. If the capacity of the culture tank 10 is 20 L, it is possible to obtain 160 L of culture supernatant.

[0058] DESCRIPTION OF SYMBOLS 10: Culture tank, 12: Stirring paddle, 13: Stirring shaft, 15: Bearing, 16: Rotary motor, 18: Various sensors, 19: Gas supply tube, 22: Top plate, 24: Culture medium discharge nozzle, 25: Heater, 28: Filter holding frame, 30: Filter, 31: Opening, 33: Bottom, 40: Carrier, 100: Culture vessel

Claims

1. A method for producing a culture supernatant obtained by removing mesenchymal stem cells from a culture medium in which the mesenchymal stem cells have been cultured, comprising: (a) a step of supplying a culture medium containing a carrier, the mesenchymal stem cells, and serum (hereinafter referred to as FBS-DMEM) to a culture vessel; (b) after step (a), a step of adhering the mesenchymal stem cells to the carrier; (c) a step of culturing the mesenchymal stem cells using the FBS-DMEM; (d) a step of leaving the mesenchymal stem cells adhered to the carrier in a filter and removing the FBS-DMEM; (e) after step (c), a step of washing the culture vessel, the mesenchymal stem cells adhered to the carrier, and the filter; (f) after step (d), a step of supplying a serum-free culture medium for the supernatant (hereinafter referred to as CM-DMEM) to the culture vessel; (g) a step of culturing the mesenchymal stem cells using the CM-DMEM; and (h). leaving the mesenchymal stem cells adhered to the carrier in the filter and recovering the CM-DMEM.

2. The method for producing a culture supernatant according to claim 1, wherein step (b) comprises a first period during which the FBS-DMEM is stirred so that the carriers are suspended in the FBS-DMEM, and a second period during which stirring is stopped.

3. The method for producing a culture supernatant according to claim 1, wherein in step (c), the FBS-DMEM is stirred so that the carriers are suspended in the FBS-DMEM.

4. The method for producing a culture supernatant according to claim 1, wherein in step (g), the CM-DMEM is stirred so that the carriers are suspended in the CM-DMEM.

5. A method for producing a culture supernatant according to any one of claims 1 to 4, wherein the mesh size of the filter is from 70 mesh to 400 mesh.

6. A method for producing a culture supernatant according to any one of claims 1 to 4, wherein steps (a) to (e) are followed by successively repeating steps (f), (g) and (h).

7. The method for producing a culture supernatant according to claim 6, wherein steps (f), (g) and (h) are repeated two to four times consecutively.

8. A method for producing a culture supernatant according to any one of claims 1 to 4, wherein, when the amount of at least one of cytokines and exosomes in the recovered CM-DMEM is less than a predetermined value, steps (c) and (e) are carried out only once; and, when the amount of at least one of cytokines and exosomes in the recovered CM-DMEM is greater than a predetermined value, steps (f), (g), and (h) are successively repeated.

9. A method for producing a culture supernatant according to any one of claims 1 to 4, wherein the mesenchymal stem cells are immortalized stem cells.

10. The method for producing a culture supernatant according to claim 9, wherein the immortalized stem cells are deciduous dental pulp stem cells.

11. A method for producing a culture supernatant obtained by removing mesenchymal stem cells from a culture medium in which mesenchymal stem cells have been cultured, the method comprising the steps of: (p) culturing the mesenchymal stem cells adhered to a carrier in a culture vessel using a culture medium containing serum (hereinafter referred to as FBS-DMEM), then leaving the mesenchymal stem cells adhered to the carrier in a filter placed in the culture vessel, and removing the FBS-DMEM; (q) after step (p), washing the culture vessel, the mesenchymal stem cells adhered to the carrier, and the filter; and (r) after step (q), supplying a serum-free culture medium for the supernatant (hereinafter referred to as CM-DMEM) to the culture vessel, culturing the mesenchymal stem cells using the CM-DMEM, and then leaving the mesenchymal stem cells adhered to the carrier in the filter, and recovering the CM-DMEM.

12. The method for producing a culture supernatant according to claim 11, wherein steps (p) and (q) are carried out only once, then step (r) is repeated two to four times in succession, then steps (p) and (q) are carried out once again, then step (r) is repeated two to four times in succession.

13. The method for producing a culture supernatant according to claim 11 or 12, wherein the capacity of the culture tank is 5 L to 20 L, and the mesh size of the filter is 70 mesh to 400 mesh.

14. The method for producing a culture supernatant according to claim 11 or 12, wherein the mesenchymal stem cells are immortalized stem cells.

Images