Method for producing culture supernatant

Abstract

To provide a culture supernatant fluid production method for producing a large amount of culture supernatant fluid containing high concentration of active ingredients.SOLUTION: A method for producing culture supernatant fluid includes the following steps: (a) supplying a culture medium containing a carrier, mesenchymal stem cells, and serum (hereinafter referred to as FBS-DMEM) to a culture vessel; (b) causing the mesenchymal stem cells to adhere to the carrier; (c) culturing the mesenchymal stem cells using the FBS-DMEM; (d) removing the FBS-DMEM while leaving the mesenchymal stem cells adhering to the carrier in a filter; (e) cleaning the culture vessel, the mesenchymal stem cells adhering to the carrier, and the filter; (f) supplying a culture medium for supernatant fluid free of serum (hereinafter referred to as CM-DMEM) to the culture vessel, (g) culturing the mesenchymal stem cells using the CM-DMEM, and (h) collecting the CM-DMEM while leaving the mesenchymal stem cells adhering to the carrier in the filter.SELECTED DRAWING: Figure 3

Description

[Technical Field]

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

[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 mesenchymal stem cells into contact with the culture medium 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 makes it possible to easily produce a culture supernatant. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 6958350 Summary of the Invention [Problem to be solved by the invention]

[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. [Means for solving the problem]

[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: (a) a culture medium containing a carrier, mesenchymal stem cells, and serum; Culture solution (b) a step of adhering the mesenchymal stem cells to a carrier; and (c) Serum-containing culture medium (d) leaving the mesenchymal stem cells adhered to the carrier in the filter; Serum-containing culture medium (e) washing the culture vessel, the mesenchymal stem cells attached to the carrier, and the filter; and (f) washing the supernatant liquid containing no serum. Culture solution (g) supplying the above-mentioned solution to a culture tank; Serum-free culture medium for supernatant fluid (h) leaving the mesenchymal stem cells attached to the carrier in the filter; Serum-free culture medium for supernatant fluid and recovering the

[0007] In step (b), the carrier Serum-containing culture medium As if floating inside Serum-containing culture medium and a second period during which the stirring is stopped. In addition, in the step (c), the carrier Serum-containing culture medium As if floating inside Serum-containing culture medium may be stirred. In addition, in the step (g), the carrier Serum-free culture medium for supernatant fluid As if floating inside Serum-free culture medium for supernatant fluid may be stirred.

[0008] The mesh size of the filter is preferably between 70 mesh and 400 mesh. It is preferable that steps (a) to (e) are followed by steps (f) to (h) being repeated consecutively. Furthermore, steps (f) to (h) may be repeated two to four times in succession.

[0009] Recovered Serum-free culture medium for supernatant fluid When at least one of cytokines and exosomes is less than a predetermined value, Supplying a serum-containing culture solution to the culture tank; (c) process; (d) Process and (e) step is performed only once; Recovered Serum-free culture medium for supernatant fluidFurthermore, if at least one of cytokines and exosomes is greater than a predetermined value, steps (f) to (h) may be repeated continuously.

[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 further comprises: (p) a culture medium containing serum; Culture solution The mesenchymal stem cells adhered to the carrier are cultured in a culture tank using the method, and then the mesenchymal stem cells adhered to the carrier are left in a filter placed in the culture tank, Serum-containing culture medium (q) after step (p), a step of washing the culture vessel, the mesenchymal stem cells adhered to the carrier, and the filter; and (r) after step (q), a step of removing the serum-free supernatant. Culture solution is supplied to the culture tank, Serum-free culture medium for supernatant fluid After culturing mesenchymal stem cells using the filter, the mesenchymal stem cells attached to the carrier were left in the filter. Serum-free culture medium for supernatant fluid and recovering the

[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. [Effects of the Invention]

[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. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 is a schematic cross-sectional view showing an example of a culture vessel 100 in a state where a filter 30 is not attached. [Figure 2] 1A is a perspective view of the filter 30, and FIG. 1B is a perspective view of the carrier 40. FIG. [Figure 3] 1 is a flowchart showing an example of a method for producing a culture supernatant. [Figure 4] 1A is a conceptual diagram showing a state in which the culture vessel 100 is filled with culture medium, and FIG. 1B is a conceptual diagram showing a state in which most of the culture medium has been discharged from the culture vessel 100. FIG. BEST MODE FOR CARRYING OUT THE INVENTION

[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 preferred. 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 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 No. 10,494,606 B2. This disclosed method makes it possible to immortalize mesenchymal stem cells by introducing four types of genes, namely hTERT, bmi-1, E6, and E7, into primary cultured cells obtained by initial culture of mesenchymal stem cells. Another method for immortalizing mesenchymal stem cells is disclosed in U.S. Patent 6,146,888. This disclosed method involves introducing the SV40 gene using a viral vector to immortalize mesenchymal stem cells. Another method for immortalizing mesenchymal stem cells involves introducing the telomerase reverse transcriptase (TERT) gene into mesenchymal stem cells, which can also be used to immortalize 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 a culture supernatant. To enhance safety, the culture supernatant preferably 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] <Culture solution and its preparation> The culture medium used in the method for producing a culture supernatant of this embodiment is not particularly limited, and may be, for example, 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] The culture medium of this embodiment contains serum. Culture solutionand serum-free supernatant recovery Culture solution It is preferable to prepare two types. Serum-containing culture medium The medium preferably contains DMEM, 5-20% by volume of FBS, an antibiotic, and the like. Serum-free culture medium for supernatant fluid In order to obtain a supernatant, it is preferable that the medium contains DMEM and antibiotics, etc., but contains little or no serum.

[0023] <Culture tank and culture equipment> 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 inside, 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 which the culture tank 10 is made 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 a plurality of 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. The top plate 22 has a plurality of openings formed therein, allowing various components to be attached. 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, 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 the filter 30, which will be described later, in a predetermined shape, for example, a cylindrical shape. The filter holding frame 28 is made by combining wires, such as stainless steel, 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 agitates the culture solution with an agitation paddle 12 according to a control program. Based on measurement results from various sensors 18, a gas supply tube 19 supplies oxygen, a gas exhaust tube (not shown) exhausts carbon dioxide and the like, and a heater 25 raises the temperature of the culture solution. 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 does not allow the carrier 40 to penetrate but allows 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> FIG. 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 solution. To ensure a large culture area and culture capacity 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 carriers is generally 140 μm to 280 μm. For mass culture, this embodiment employs 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. The nonwoven fabric is preferably porous. The fibers constituting the nonwoven fabric preferably have a small fiber 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 also 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] <Culture method> Fig. 3 is a flowchart showing a method for producing a supernatant. A filter 30 is attached to the culture tank 10 of the culture vessel 100 shown in Fig. 1 using an attachment string 35. Figs. 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 Fig. 1 are not shown.

[0036] Mesenchymal stem cells, carriers washed with EDTA solution, etc., and heated to 36-38°C Serum-containing culture medium are introduced into 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 they reach the required amount in the culture tank 10. Various sensors 18 measure the temperature, pH, and oxygen concentration as appropriate. Serum-containing culture medium The temperature, pH and oxygen concentration are adjusted by a control program.

[0037] Next, the mesenchymal stem cells are allowed to adhere to the carriers 40 (step S32). Serum-containing culture medium The mixture is stirred at a predetermined rotation speed, and the carrier 40 Serum-containing culture medium Furthermore, to ensure that the mesenchymal stem cells adhere to the carriers 40, the rotation of the stirring paddle 12 is stopped and the inside of the culture vessel 10 is cooled. Serum-containing culture medium Calm down. Serum-containing culture mediumThe first period during which the mixture is stirred is, for example, 1 to 59 minutes per hour, and the second period during which stirring is stopped is, for example, 59 minutes to 1 minute per hour. This cycle of stirring and stopping stirring continues for 48 to 72 hours. The rotation of the stirring paddle 12 may be constant or variable.

[0038] Next, the mesenchymal stem cells are cultured (step S33). Serum-containing culture medium The mixture is stirred at a predetermined rotation speed, and the carrier 40 Serum-containing culture medium As a result, the mesenchymal stem cells grow three-dimensionally on the carriers 40. If the mesenchymal stem cells grow and the weight of the carriers 40 increases, the rotation speed of the stirring paddle 12 must be increased in order to prevent the carriers 40 from becoming too heavy. Serum-containing culture medium Therefore, 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 carrier 40, the carrier 40 enters a plateau phase (stationary phase). Therefore, even if the rotation speed of the stirring paddle 12 is kept constant, the carrier 40 Serum-containing culture medium The culture is completed when the plateau phase is reached, and the culture time is 72 to 96 hours. Serum-containing culture medium This is a diagram showing the state in which it is floating in the air.

[0039] Next, the culture solution discharge nozzle 24 Serum-containing culture medium At this time, the stirring paddle 12 is stopped. Serum-containing culture medium As the water is discharged, the carrier 40 is caught by the filter 30. Serum-containing culture medium 10 is a diagram showing a state in which the carrier 40 is caught in the filter 30 after the removal of the carrier 40.

[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). Then, the culture solution discharge nozzle 24 recovers the phosphate buffer, which is then removed.

[0041] It was then warmed to 36-38°C. Serum-free culture medium for supernatant fluid is introduced into the culture tank 10 (step S36). The various sensors 18 measure the temperature, pH, and oxygen concentration as appropriate. Serum-free culture medium for supernatant fluid The temperature, pH and oxygen concentration are adjusted by a control program.

[0042] Next, the mesenchymal stem cells are cultured (step S37). Serum-free culture medium for supernatant fluid The mixture is stirred at a predetermined rotation speed, and the carrier 40 Serum-free culture medium for supernatant fluid The mesenchymal stem cells are then allowed to float in the carrier 40. This allows the mesenchymal stem cells to be cultured on the carrier 40. The culture time is 40 to 60 hours.

[0043] Next, the culture medium is discharged through the culture medium discharge nozzle 24. Serum-free culture medium for supernatant fluid The culture medium is collected (step S38). At this time, the stirring paddle 12 is stopped. Serum-free culture medium for supernatant fluid The supernatant is then collected and stored as a mesenchymal stem cell culture supernatant.

[0044] Recovered Serum-free culture medium for supernatant fluid The (culture supernatant) is then judged to determine whether the state of the mesenchymal stem cells is good (step S39). Whether the state of the mesenchymal stem cells is good may be judged, for example, by whether at least one of the measured cytokines and exosomes is greater than a predetermined threshold. If at least one of the secreted cytokines and exosomes is greater than a predetermined value, this indicates that the state of the mesenchymal stem cells is good.

[0045] If the condition of the mesenchymal stem cells is good, steps S36-S38 are repeated continuously. If the condition of the mesenchymal stem cells is poor, proceed to step S40 and heat the mesenchymal stem cells to 36-38°C. Serum-containing culture medium is introduced into the culture tank 10. Then, the process proceeds to steps S33-S34. That is, nutrients, which are serum, are provided to the mesenchymal stem cells, and the mesenchymal stem cells proliferate or return to a healthy state.

[0046] In step S39, it is determined whether the state of mesenchymal stem cells is good based on whether cytokines and the like are more than a predetermined threshold, but it is not always necessary to measure every time. Serum-containing culture medium cultivation by Serum-free culture medium for supernatant fluid If the cultivation by Serum-containing culture medium and the cultivation by Serum-free culture medium for supernatant fluid are alternately repeated, and it is known that the secreted cytokines and the like are more than a predetermined threshold, then without measuring every time, Serum-free culture medium for supernatant fluid the cultivation by Serum-containing culture medium can be alternately repeated with the cultivation by Serum-free culture medium for supernatant fluid It may also be a cycle of the cultivation by Serum-containing culture medium and the cultivation by

Example

[0047] <Culture of immortalized human dental pulp stem cells> As human dental pulp stem cells, human deciduous dental pulp stem cells (SHED) prepared from the dental pulp tissue of human deciduous teeth were used. In addition, this SHED was immortalized by introducing the SV40 gene using a viral vector. This immortalized SHED (hereinafter referred to as IM-SHED) was stored in a freezer at -80 °C, thawed at room temperature, and cultured in a flask with FBS-DMEM at 37 °C. After repeating subculturing, when 32 flasks became fully confluent, they were placed in the culture tank 10.

[0048] <Culture of IM-SHED with FBS-DMEM> IM-SHED, the carrier 40 which is a 60 g (dry weight) disk-shaped non-woven fabric, and 4000 ml of FBS-DMEM were put into the culture tank 10 with a capacity of 10 l (liters). The mesh size of the filter 30 adopted was 180 mesh (mesh size of about 84 μm). FBS-DMEM was prepared at pH 7.0, dissolved oxygen 2.0, and temperature 37 °C.

[0049] The IM-SHED, nonwoven fabric carrier 40, and FBS-DMEM were stirred with 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, an additional 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 had been 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. For this reason, 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, carrier 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, carrier 40, etc., was washed.

[0052] Next, 7500 ml of CM-DMEM warmed to 37°C was poured into the culture tank 10, and 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 IM-SHED was 48 hours.

[0053] Next, the CM-DMEM was collected using the culture medium 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 cytokines, such as 3711.5 pg / ml for VEGF and 2566.7 pg / ml for HGF. For example, the threshold values ​​for VEGF and HGF were set to 3400 pg / ml and 2300 pg / ml, respectively. Therefore, CM-DMEM was added to the culture vessel 10 without adding FBS-DMEM, and the culture supernatant was continuously collected. In other words, steps S36 to S38 in FIG. 3 were continuously repeated. [Table 1]

[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. To ensure that the threshold was exceeded, in this example, the culture supernatant was automatically collected after two consecutive additions of CM-DMEM and one addition of FBS-DMEM. As a result, even after 40 cycles (80 additions of CM-DMEM and 40 additions of FBS-DMEM) 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. [Explanation of symbols]

[0058] 10 ... culture tank, 12 ... stirring paddle, 13 ... stirring shaft 15 ... bearing, 16 ... rotation motor, 18 ... various sensors 19 ... Gas supply tube, 22 ... Top plate, 24 ... Culture medium discharge nozzle 25 ... Heater, 28 ... Filter holder frame 30 ... filter, 31 ... opening, 33 ... bottom 40 ... carrier, 100 ... culture vessel

Claims

1. A method for producing a culture supernatant obtained by removing immortalized mesenchymal stem cells from a culture medium in which the mesenchymal stem cells have been cultured, comprising: (a) supplying a culture solution containing a carrier, the mesenchymal stem cells, and serum to a culture vessel; (b) after the step (a), adhering the mesenchymal stem cells to the carrier; (c) culturing the mesenchymal stem cells by stirring the serum-containing culture solution; (d) leaving the mesenchymal stem cells adhered to the carrier in the filter, and removing the serum-containing culture medium from the culture vessel; (e) after the step (d), washing the culture vessel, the mesenchymal stem cells adhered to the carrier, and the filter; (f) after the step (e), supplying a serum-free culture medium for a supernatant to the culture tank; (g) culturing the mesenchymal stem cells by stirring the serum-free supernatant culture medium; (h) leaving the mesenchymal stem cells adhered to the carrier in the filter, and recovering the serum-free culture medium for supernatant; Equipped with A method for producing a culture supernatant, wherein after steps (a) to (e), steps (f), (g) and (h) are successively repeated.

2. The method for producing a culture supernatant according to claim 1 , wherein the steps (f), (g), and (h) are repeated two to four times consecutively.

3. the step (b) includes a first period during which the serum-containing culture medium is stirred so that the carrier is suspended in the serum-containing culture medium, and a second period during which stirring is stopped; In the step (c), the serum-containing culture medium is stirred so that the carrier is suspended in the serum-containing culture medium; 2. The method for producing a culture supernatant according to claim 1, wherein in the step (g), the serum-free culture medium for the supernatant is stirred so that the carrier is suspended in the serum-free culture medium for the supernatant.

4. 2. The method for producing a culture supernatant according to claim 1, wherein the carrier is made of a nonwoven fabric composed of fibers, and the average fiber diameter of the fibers of the nonwoven fabric is 10 to 100 μm.

5. The method for producing a culture supernatant according to claim 4, wherein the carrier made of the nonwoven fabric is a disk-shaped carrier having a diameter of 3 mm to 9 mm.

6. 5. The method for producing a culture supernatant according to claim 4, wherein the carrier made of the nonwoven fabric is supplied in an amount of 5 g to 40 g per 1000 ml of the serum-containing culture medium or the serum-free culture medium for the supernatant.

7. The method for producing a culture supernatant according to any one of claims 1 to 6, wherein the mesh size of the filter is from 70 mesh to 400 mesh.

8. 7. The method for producing a culture supernatant according to claim 1, wherein when at least one of cytokines and exosomes in the collected culture medium for the serum-free supernatant is less than a predetermined value, a serum-containing culture medium is supplied to the culture tank, and steps (c), (d), and (e) are performed only once; and when at least one of cytokines and exosomes in the collected culture medium for the serum-free supernatant is more than a predetermined value, steps (f), (g), and (h) are continuously repeated.

9. The method for producing a culture supernatant according to claim 1 , wherein the immortalized stem cells are deciduous dental pulp stem cells.

10. A method for producing a culture supernatant obtained by removing immortalized mesenchymal stem cells from a culture medium in which the mesenchymal stem cells have been cultured, comprising: (p) agitating the serum-containing culture solution to culture the mesenchymal stem cells adhered to the carrier in a culture vessel, and then leaving the mesenchymal stem cells adhered to the carrier in a filter placed in the culture vessel, and removing the serum-containing culture solution from the culture vessel; (q) after the step (p), washing the culture vessel, the mesenchymal stem cells adhered to the carrier, and the filter; (r) after step (q), supplying a serum-free supernatant culture medium to the culture vessel, stirring the serum-free supernatant culture medium to culture the mesenchymal stem cells, and then leaving the mesenchymal stem cells adhered to the carrier in the filter and recovering the serum-free supernatant culture medium; Equipped with A method for producing a culture supernatant, wherein the step (r) is continuously repeated after the steps (p) to (q).

11. 11. The method for producing a culture supernatant according to claim 10, wherein steps (p) and (q) are performed only once, then step (r) is repeated two to four times in succession, then steps (p) and (q) are performed again only once, then step (r) is repeated two to four times in succession.

12. 12. The method for producing a culture supernatant according to claim 10 or 11, 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.

13. The carrier is made of a nonwoven fabric composed of fibers, and the average fiber diameter of the fibers of the nonwoven fabric is 10 to 100 μm; The method for producing a culture supernatant according to claim 10 or 11, wherein the carrier made of the nonwoven fabric is supplied in an amount of 5 g to 40 g per 1000 ml of the serum-containing culture medium or the serum-free culture medium for the supernatant.

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