JP7800918B2 - Method for producing culture supernatant - Google Patents

Description

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

In medical treatments using mesenchymal stem cells, it is known that not only do the stem cells themselves bring about therapeutic effects, but various physiologically active substances secreted by the stem cells, such as cytokines and exosomes, also 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 recovered and effectively utilized as culture supernatant.

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.

Patent No. 6958350

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.

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, comprising the steps of (a) supplying a culture medium containing carriers, mesenchymal stem cells, and serum to a culture vessel, (b) adhering the mesenchymal stem cells to the carriers, (c) culturing the mesenchymal stem cells in the serum-containing culture medium , (d) leaving the mesenchymal stem cells adhered to the carriers in a filter and removing the serum-containing culture medium , (e) washing the culture vessel, the mesenchymal stem cells adhered to the carriers, and the filter, (f) supplying a serum-free culture medium for the supernatant to the culture vessel, (g) culturing the mesenchymal stem cells in the serum-free culture medium for the supernatant, and (h) leaving the mesenchymal stem cells adhered to the carriers in the filter and recovering the serum-free culture medium for the supernatant .

The step (b) may include a first period during which the serum-containing culture medium is stirred so that the carriers are suspended in the serum-containing culture medium , and a second period during which stirring is stopped.
In addition, in the step (c), the serum-containing culture medium may be stirred so that the carriers are suspended in the serum-containing culture medium.
In addition, in the step (g), the serum-free culture medium for the supernatant may be stirred so that the carriers are suspended in the serum-free culture medium for the supernatant .

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.

When at least one of cytokines and exosomes is less than a predetermined value in the collected serum-free culture medium for the supernatant, a serum-containing culture medium is supplied to the culture tank, and steps (c), (d) , and (e) are carried out only once;
If the collected serum-free culture medium contains more than a predetermined amount of cytokines and/or exosomes, steps (f) to (h) may be repeated continuously.

It is preferable that the mesenchymal stem cells are immortalized stem cells, and it is even more preferable that the immortalized stem cells are deciduous tooth pulp stem cells.

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 comprises the steps of: (p) culturing mesenchymal stem cells adhered to a carrier in a culture vessel using a serum-containing culture medium , then leaving the mesenchymal stem cells adhered to the carrier in a filter placed in the culture vessel, and removing the serum-containing culture medium ; (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 to the culture vessel and culturing the mesenchymal stem cells using the serum-free culture medium for the supernatant , then leaving the mesenchymal stem cells adhered to the carrier in the filter, and recovering the serum-free culture medium for the supernatant .

Steps (p) and (q) may be performed once, followed by repeating step (r) two to four times in succession, and then steps (p) and (q) may be performed once again, followed by repeating step (r) two to four times in succession.

It is preferable that the capacity of the culture vessel be 5 to 20 liters, and the mesh size of the filter be 70 to 400 mesh. It is also preferable that the mesenchymal stem cells are immortalized stem cells.

The method for producing culture supernatant of the present invention makes it possible to continuously provide large amounts of culture supernatant containing active ingredients at high concentrations.

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. 1A is a perspective view of the filter 30, and FIG. 1B is a perspective view of the carrier 40. FIG. 1 is a flowchart showing an example of a method for producing a culture supernatant. 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.

<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.

<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 culture 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 is to introduce the telomerase reverse transactivation (TERT) gene into mesenchymal stem cells, which can also be used to immortalize mesenchymal stem cells.

<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.

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 cell culture supernatant can be achieved using methods commonly used for lyophilizing protein-containing liquids.

In this embodiment, the mesenchymal stem cell culture supernatant is preferably a culture supernatant of deciduous dental pulp stem cells. Deciduous dental pulp stem cells have high proliferation capacity, and their culture supernatant contains large amounts of cytokines, exosomes, etc.

<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 culture medium containing 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.

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

<Culture tank and culture 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.

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 the culture solution inside, and its shape, capacity, and material are selected appropriately 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, etc., 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.

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 placed 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 multiple openings formed therein, allowing for the attachment of various components. In this embodiment, the top plate 22 is equipped with a stirring 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.

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 supported by bearings 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) can be placed in the culture tank 10, and the stirrer can be rotated by a magnetic stirrer (agitator) to stir the culture solution.

The 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. The gas supply tube 19 is attached to the openings in the top plate 22 and supplies gases such as oxygen to 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. The 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. The 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, openings may be provided in the culture tank 10 to which the various sensors 18 or the culture solution discharge nozzle 24 are attached.

A filter holding frame 28 is also attached to the top plate 22 or the culture tank 10. The filter holding frame 28 is provided to hold the filter 30 (described below) in a predetermined shape, such as a cylindrical shape. The filter holding frame 28 is made by combining wires, such as stainless steel, vertically and horizontally to form a cylindrical shape.

A control unit (not shown) supplies culture medium to the culture tank 10 through a culture medium supply nozzle (not shown) and stirs the culture medium with a stirring paddle 12 using a control program. Based on measurement results from various sensors 18, the gas supply tube 19 supplies oxygen, the gas exhaust tube (not shown) exhausts carbon dioxide and other gases, and the heater 25 raises the temperature of the culture medium. The control program also discharges the culture medium from the culture medium 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 medium, management of the temperature, pH, oxygen concentration, etc. of the culture medium, collection of the culture medium, and cleaning of the inside of the culture tank.

<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.

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 between 70 mesh (mesh size of approximately 185 μm) and 400 mesh (mesh size of approximately 35 μm).

<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 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 employed. It is preferable that the nonwoven fabric be porous. The fibers constituting the nonwoven fabric are desirably small in diameter, with an average fiber diameter of preferably 10 to 100 μm, and particularly preferably 15 to 50 μm.

The carrier material may be organic, inorganic, or a composite of these. 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.

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

<Culture method>
Figure 3 is a flowchart showing a 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.

Mesenchymal stem cells, carriers 40 washed with an EDTA solution or the like, and a culture solution containing serum heated to 36-38°C are introduced into the culture tank 10 (step S31). The mesenchymal stem cells are stem cells that have been frozen and cultured in a flask until they reach the amount required for the culture tank 10. Various sensors 18 measure the temperature, pH, and oxygen concentration as appropriate, and the temperature, pH, and oxygen concentration of the culture solution containing serum are adjusted by a control program.

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 serum-containing culture solution at a predetermined rotation speed, causing the carriers 40 to float in the serum-containing culture solution . Furthermore, to ensure that the mesenchymal stem cells adhere to the carriers 40, the rotation of the stirring paddle 12 is stopped and the serum-containing culture solution in the culture vessel 10 is allowed to settle. The first period during which the serum-containing culture solution 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.

Next, mesenchymal stem cells are cultured (step S33). The stirring paddle 12 stirs the serum-containing culture solution at a predetermined rotation speed, causing the carriers 40 to float in the serum-containing culture solution . 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 serum-containing culture solution 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, even if the rotation speed of the stirring paddle 12 is maintained constant, the carriers 40 will remain suspended in the serum-containing culture solution . 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 serum-containing culture solution .

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

Next, phosphate-buffered saline (PBS) is supplied to the culture vessel 10, and the stirring paddle 12 is rotated at high speed for several to 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 medium discharge nozzle 24 then collects the phosphate buffer, which is then removed.

Next, the serum-free culture medium for the supernatant, which has been 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 serum-free culture medium for the supernatant are adjusted by the control program.

Next, the mesenchymal stem cells are cultured (step S37). The stirring paddle 12 stirs the serum-free supernatant culture medium at a predetermined rotation speed, so that the carriers 40 are suspended in the serum-free supernatant culture medium . This allows the mesenchymal stem cells to be cultured on the carriers 40. The culture time is 40 to 60 hours.

Next, the culture medium for the serum-free supernatant is collected by the culture medium discharge nozzle 24 (step S38), while the stirring paddle 12 is stopped. The culture medium for the serum-free supernatant is collected by the culture medium discharge nozzle 24, and this is stored as a culture supernatant for mesenchymal stem cells.

The recovered serum-free supernatant culture medium (culture supernatant) is then evaluated to determine whether the mesenchymal stem cells are in a good state (step S39). The evaluation may be based on 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 threshold, this indicates that the mesenchymal stem cells are in a good state.

If the mesenchymal stem cells are in a good state, steps S36-S38 are continuously repeated. If the mesenchymal stem cells are in a bad state, the process proceeds to step S40, in which a serum-containing culture medium 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 state.

In step S39, whether the condition of mesenchymal stem cells is good or not is determined by whether cytokines, etc., are greater than a predetermined threshold, but measurement is not necessarily required every time. If alternating between culture in a serum-containing culture medium and culture in a serum-free supernatant culture medium can confirm that the secreted cytokines, etc., are greater than a predetermined threshold, culture in a serum-containing culture medium and culture in a serum-free supernatant culture medium can be alternately repeated without measuring every time. Even if culture in a serum-free supernatant culture medium is performed four times in succession, followed by one culture in a serum-containing culture medium , a cycle of four consecutive cultures in a serum-free supernatant culture medium and one culture in a serum-containing culture medium can be used, as long as it is confirmed that the secreted cytokines, etc., are greater than a predetermined threshold.

<Cultivation 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.

<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 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.

The IM-SHED, nonwoven fabric carrier 40, and FBS-DMEM were agitated with the agitation paddle 12. To allow the IM-SHED to adhere to the carrier 40, the agitation paddle 12 was programmed to rotate at 20-40 rpm for a predetermined time, then stop and rotate again for a predetermined time. After 24 hours, an additional 1000 ml of FBS-DMEM was added to the culture vessel 10.

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, allowing the carriers 40 to float 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.

216 hours after the addition of the IM-SHED, carriers 40, and FBS-DMEM, the IM-SHED cultivation was completed, and the FBS-DMEM was collected using the culture medium 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.

Next, 7,500 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 IM-SHED was cultured for 48 hours.

Next, the CM-DMEM was collected using the culture medium discharge nozzle 24 and stored refrigerated or frozen as the IM-SHED culture supernatant.

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.

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

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 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, culture supernatant containing the high concentrations of cytokines shown in Table 1 was obtained.

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 24 L of culture supernatant can be obtained per month. 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.

DESCRIPTION OF SYMBOLS 10... Cultivation tank, 12... Agitation paddle, 13... Agitation 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 holding frame, 30... Filter, 31... Opening, 33... Bottom, 40... Carrier, 100... Cultivation vessel

Claims (13)

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. The method for producing a culture supernatant according to claim 1, wherein steps (f), (g), and (h) are repeated two to four times consecutively. 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. The method for producing 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 in the nonwoven fabric is 10-100 μm. The method for producing culture supernatant according to claim 4, wherein the nonwoven fabric carrier is disk-shaped with a diameter of 3 mm to 9 mm. 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. The method for producing a culture supernatant according to any one of claims 1 to 6, wherein the mesh size of the filter is between 70 mesh and 400 mesh. 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. The method for producing a culture supernatant according to claim 1, wherein the immortalized stem cells are deciduous dental pulp stem cells. 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). The method for producing a culture supernatant according to claim 10, wherein steps (p) and (q) are performed once, then step (r) is repeated two to four times in succession, then steps (p) and (q) are performed once again, then step (r) is repeated two to four times in succession. 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. 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.