WO2025183119A1 - Method for producing mesenchymal stem cells - Google Patents

Abstract

The present invention addresses the problem of providing a method for producing mesenchymal stem cells, the method being capable of improving the number of cells recovered due to culturing by reducing empty microcarriers (microcarriers to which cells do not adhere). The present invention provides a method for producing mesenchymal stem cells, the method comprising culturing mesenchymal stem cells in the presence of microcarriers, wherein said method involves: performing, for at least 48 hours, a stirring/statically culturing step which includes stirring the mesenchymal stem cells and then leaving the mesenchymal stem cells alone; performing the stirring/statically culturing step at least twice during a culturing period of at least 48 hours; and performing the stirring/statically culturing step at least once every 48 hours after 24 hours from the start of the culturing.

Description

Method for producing mesenchymal stem cells

The present invention relates to a method for producing mesenchymal stem cells, which comprises culturing mesenchymal stem cells in the presence of microcarriers, and which includes an agitated static culture step that includes a step of agitating the mesenchymal stem cells and a subsequent step of allowing the mesenchymal stem cells to stand still.

In recent years, advances in regenerative medicine and cell therapy technologies have led to the active development of various cell therapies and research cell products using autologous, allogeneic, or xenogeneic cells. Among these, mesenchymal stem cells (MSCs) are expected to be a useful cell source for cell therapy. Mesenchymal stem cells can be harvested from a variety of body tissues, and it has been reported that they can be isolated from bone marrow, synovium, periosteum, fat, muscle, dental pulp, placenta, umbilical cord, and other tissues.

Patent Document 1 describes culturing adherent cells in a cell suspension containing the adherent cells, microcarriers, and a medium, and describes that the culturing may include intermittent stirring of the cell suspension.

Patent Document 2 describes a method for harvesting cultured cells from a soluble substrate, which comprises separating the cultured cells from the substrate by digesting the soluble substrate by exposing it to a chelating agent, an enzyme, or both. In Patent Document 2, mesenchymal stem cells are grown by intermittent stirring followed by continuous stirring in the step of adhering the mesenchymal stem cells to the soluble substrate.

Patent Document 3 describes cell culture using microcarriers. Patent Document 3 also describes the attachment and proliferation of MSCs to microcarriers while continuously stirring them.

JP 2023-134611 A Special Publication No. 2022-536651 Special Publication No. 2018-520662

Agitated culture technology has been used in the production of biopharmaceuticals using CHO cells (Chinese hamster ovary cells) and vaccines using Vero cells. However, in these cases, the cells themselves are not the final product, but rather the proteins secreted by the cells. On the other hand, when agitated culture technology is used to produce cell therapy products using mesenchymal stem cells, where the cells themselves are the final product, a problem has been discovered in that microcarriers to which cells do not adhere are scattered, preventing full use of the microcarriers. This has also led to the problem of low cell recovery rates.

The objective of the present invention is to provide a method for producing mesenchymal stem cells that can reduce empty microcarriers (microcarriers to which cells do not adhere), thereby increasing the number of cells recovered through culture.

As a result of extensive research by the present inventors to solve the above-mentioned problems, they have found that in a method for producing mesenchymal stem cells, which includes culturing mesenchymal stem cells in the presence of microcarriers, an agitation and static culture step, which includes a step of agitating the mesenchymal stem cells and a subsequent step of allowing the mesenchymal stem cells to stand, is carried out for 48 hours or more, and the agitation and static culture step is carried out two or more times during the 48-hour or more culture period, and that starting 24 hours after the start of culture, the agitation and static culture step is carried out at least once every 48 hours, thereby reducing empty microcarriers and improving the number of recovered cells. The present invention was completed based on the above findings.

That is, according to the present invention, the following inventions are provided.
<1> A method for producing mesenchymal stem cells, comprising culturing mesenchymal stem cells in the presence of microcarriers, the method comprising carrying out an agitation static culture step, for 48 hours or more, which step includes a step of agitating the mesenchymal stem cells and a step of allowing the mesenchymal stem cells to stand thereafter, and carrying out the agitation static culture step two or more times during the 48-hour or longer culture period, and carrying out the agitation static culture step one or more times every 48 hours starting 24 hours after the start of culture.
<2> The method for producing mesenchymal stem cells according to <1>, wherein each of the two or more stirring and static culture steps is the same step, or the two or more stirring and static culture steps include a stirring and static culture step in which either one or more of the duration of the stirring step and the duration of the static culture step are different.
<3> The method for producing mesenchymal stem cells according to <1> or <2>, wherein the stirring and static culture step is carried out from the start of culture to the end of culture.
<4> The method for producing mesenchymal stem cells according to any one of <1> to <3>, wherein the mesenchymal stem cells are derived from a human.
<5> The method for producing mesenchymal stem cells according to <4>, wherein the mesenchymal stem cells are derived from induced pluripotent stem cells, bone marrow, fat, dental pulp, umbilical cord, placenta, or synovium.
<6> The method for producing mesenchymal stem cells according to <5>, wherein the mesenchymal stem cells are derived from synovial membrane.
<7> The method for producing mesenchymal stem cells according to any one of <1> to <6>, wherein the time for the step of leaving the mesenchymal stem cells stationary in the stirring static culture step is 1 minute or more and 2,400 minutes or less.
<8> The method for producing mesenchymal stem cells according to any one of <1> to <7>, wherein the time for the step of leaving the mesenchymal stem cells standing in the stirring static culture step is 0.1 min/mL or more and 240.0 min/mL or less per volume of culture solution.
<9> The method for producing mesenchymal stem cells according to any one of <1> to <8>, wherein the time for the step of stirring the mesenchymal stem cells in the stirring static culture step is 1 minute or more and 2000 minutes or less.
<10> The method for producing mesenchymal stem cells according to any one of <1> to <9>, wherein the time for the step of stirring the mesenchymal stem cells in the stirring static culture step is 0.1 min/mL or more and 200.0 min/mL or less per volume of culture solution.
<11> The method for producing mesenchymal stem cells according to any one of <1> to <10>, wherein the stirring and static culture step is carried out 1 to 48 times per 24 hours.
<12> The method for producing mesenchymal stem cells according to any one of <1> to <11>, wherein the duration of each stirring and static culture step is 30 minutes to 1,440 minutes.
<13> The method for producing mesenchymal stem cells according to any one of <1> to <12>, wherein the ratio of the time taken for the step of stirring the mesenchymal stem cells to the time taken for the step of allowing the mesenchymal stem cells to stand in the stirring static culture step is within the range of 1:0.005 to 1:700.
<14> The method for producing mesenchymal stem cells according to any one of <1> to <13>, wherein the total time of the stirring step during the entire culture period is longer than the total time of the standing step.
<15> The method for producing mesenchymal stem cells according to any one of <1> to <14>, wherein no microcarriers are added in the stirring static culture step.
<16> The method for producing mesenchymal stem cells according to any one of <1> to <15>, wherein the stirring static culture is carried out for 48 hours or more after 24 hours from the start of the culture, and then the stirring culture is carried out continuously.
<17> The method for producing mesenchymal stem cells according to any one of <1> to <16>, wherein the stirring static culture step is carried out 24 hours or more after the start of culture, and stirring culture is carried out continuously from at least two days before the end of culture.
<18> The method for producing mesenchymal stem cells according to any one of <1> to <17>, wherein the ratio of the time of the stirring static culture step to the time of the continuous stirring step is 1:0.3 to 1:5.5.
<19> The method for producing mesenchymal stem cells according to any one of <1> to <18>, wherein the time ratios of the step (A) of leaving the mesenchymal stem cells to stand, the step (B) of stirring and leaving the mesenchymal stem cells to stand, and the step (C) of continuously stirring the mesenchymal stem cells are such that, when A=1, B=2 to 6, and C=2 to 11.
<20> The method according to any one of <1> to <19>, wherein mesenchymal stem cells are produced for use as a therapeutic agent.

The method for producing mesenchymal stem cells of the present invention promotes cell adhesion to empty carriers, thereby increasing the number of viable cells at the time of collection.

The present invention will be described in detail below. Note that in this specification, the word "to" is used to mean that the numerical values before and after it are both upper and lower limits.

The present invention relates to a method for producing mesenchymal stem cells, which comprises culturing mesenchymal stem cells in the presence of microcarriers, and which comprises carrying out an agitation-static culture step, which includes a step of agitating the mesenchymal stem cells and a subsequent step of allowing the mesenchymal stem cells to stand, for 48 hours or more, and which comprises carrying out the agitation-static culture step two or more times during the 48-hour or more culture period, and which comprises carrying out the agitation-static culture step one or more times every 48 hours starting 24 hours after the start of culture.

According to the present invention, empty microcarriers (microcarriers to which cells do not adhere) can be reduced, and the number of cells recovered from culture can be increased. Furthermore, since the present invention can ensure the differentiation ability of mesenchymal stem cells, mesenchymal stem cells are useful as therapeutic agents. According to the present invention, cells for cell therapy can be easily produced in large quantities. Furthermore, according to the present invention, the input microcarriers can be used efficiently, making it possible to culture using a minimum number of microcarriers and culturing at low cost. The method for producing mesenchymal stem cells according to the present invention can be used in industries that require large quantities of cells, such as allogeneic cell therapy products.

<Mesenchymal stem cells>
Stem cells refer to immature cells that have the ability to self-renew and differentiate and proliferate, and include pluripotent stem cells, multipotent stem cells, unipotent stem cells, etc., depending on their differentiation potential. Stem cells are generally defined as undifferentiated cells that have the "self-renewal ability" to proliferate while maintaining an undifferentiated state, and the "pluripotency" to differentiate into all three germ layer lineages. Pluripotent stem cells refer to cells that have the ability to differentiate into all tissues and cells that make up a living organism. Multipotent stem cells refer to cells that have the ability to differentiate into multiple types of tissues and cells, but not all types. Unipotent stem cells refer to cells that have the ability to differentiate into specific tissues or cells.

Mesenchymal stem cells broadly refer to a population of stem cells or their precursor cells that can differentiate into all or some of the mesenchymal cells, such as osteoblasts, chondroblasts, adipblasts, and muscle cells. Mesenchymal stem cells are also called stromal cells.

The origin of the mesenchymal stem cells is not particularly limited, and may be cells from, for example, rodents such as rats, mice, hamsters, and guinea pigs; lagomorphs such as rabbits; ungulates such as pigs, cows, goats, and sheep; Carnivores such as dogs and cats; and primates such as humans, monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees. It is preferable that the mesenchymal stem cells are derived from humans.

Mesenchymal stem cells are preferably derived from induced pluripotent stem cells, bone marrow, fat, dental pulp, umbilical cord, placenta, or synovial membrane. Mesenchymal stem cells are particularly preferably derived from synovial membrane.

When mesenchymal stem cells are used as a therapeutic agent, they may be autologous cells of the patient to be administered, or allogeneic cells.

Mesenchymal stem cells can be identified by detecting molecules characteristic of mesenchymal stem cells, such as enzymes, receptors, and low-molecular-weight compounds. Molecules characteristic of mesenchymal stem cells include, but are not limited to, cell surface markers (positive markers), such as CD73, CD90, CD105, and CD166. Negative markers not expressed in mesenchymal stem cells include, but are not limited to, CD19, CD34, CD45, HLA-DR, CD11b, and CD14. CD stands for Clusters of Differentiation, and HLA-DR stands for Human Leukocyte Antigen-D-Related. These positive and negative markers can be used to confirm that the cells are mesenchymal stem cells. Immunological methods can be used to detect these markers, but detection can also be performed by quantifying the amount of mRNA for each molecule.

In one example of the present invention, synovial membrane-derived mesenchymal stem cells can be used as mesenchymal stem cells. Synovial membrane-derived mesenchymal stem cells (also called synovial stem cells) are stem cells contained in the synovial membrane. Synovial membrane-derived mesenchymal stem cells can be detected, for example, by detecting CD90 positivity, CD45 negativity, and chondrogenic differentiation ability, but the detection method is not particularly limited.

Synovium-derived mesenchymal stem cells can be obtained, for example, by a method comprising step A of treating synovial tissue with an enzyme, step B of washing the mixture after enzyme treatment and culturing the synovium-derived mesenchymal stem cells contained in the mixture in a culture medium, and step C of cryopreserving the cultured synovium-derived mesenchymal stem cells.

(Step A: Treating synovial tissue with enzyme)
Synovial tissue can be harvested during arthroscopic surgery and then treated with enzymes.

The enzyme is not particularly limited as long as it contains a protease, but is preferably a mixed enzyme containing one or more types of collagenase and one or more types of neutral protease. A particularly preferred enzyme is Liberase (registered trademark). For example, Liberase MNP-S (manufactured by Roche) can be used as Liberase (registered trademark), which is an enzyme containing collagenase class I, collagenase class II, and a neutral protease (thermocillin). The enzyme reaction can be carried out in an aqueous solution containing the enzyme.

The enzymatic reaction can be carried out at a temperature of preferably 15°C to 40°C, more preferably 20°C to 40°C, and even more preferably 25°C to 40°C.
The reaction time may be 10 minutes or more, preferably 30 minutes or more, more preferably 1 hour or more, even more preferably 1.5 hours or more, and may be 2 hours or more. The upper limit of the reaction time is not particularly limited, but may be 10 hours or less, 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less, or 4 hours or less.

The enzyme-treated mixture contains synovium-derived mesenchymal stem cells.
The enzyme-treated mixture is passed through a cell strainer and transferred to a centrifuge tube, and then centrifuged to recover synovium-derived mesenchymal stem cells.

(Step B: Culturing the synovium-derived mesenchymal stem cells contained in the mixture after washing the mixture after enzyme treatment in a culture medium)
As described above, it is preferable to wash the mixture after the enzyme treatment.
Washing can be performed by resuspending the synovium-derived mesenchymal stem cells recovered by the above-mentioned centrifugation in a medium and centrifuging again. The medium can be, but is not limited to, α-modified Eagle's minimum essential medium (αMEM). Washing can be performed multiple times (two or more times) using the medium described above.

The synovium-derived mesenchymal stem cells contained in the mixture after washing the mixture following enzyme treatment are cultured in a culture medium. Examples of substrates include, but are not limited to, flat plastic substrates such as flasks and culture plates, and three-dimensional substrates such as culture bags, microcarriers, or gels.

The medium used for culturing can be prepared using a medium used for culturing ordinary animal cells as the basal medium. Examples of media used for culturing ordinary animal cells include, but are not limited to, αMEM, DMEM (Dulbecco Modified Eagle Medium), a mixed medium of DMEM and F12 (DMEM:F12=1:1), RPMI medium (GIBCO (registered trademark) RPMI 1640 medium, etc.), a mixed medium of DMEM/F12 and RPMI (DMEM/F12:RPMI=1:1), RoosterNourish ^™ -MSC-XF, etc. The medium may also contain an antibiotic or antifungal agent (e.g., amphotericin B, gentamicin, etc.).

The cell culture conditions are not particularly limited, and ordinary cell culture conditions can be used. For example, culture at a temperature of 30 to 40°C and 3 to 7% _CO2 can be used, but the conditions are not particularly limited. One example is culture at a temperature of 37°C and 5% _CO2 .

(Isolation of synovial membrane-derived mesenchymal stem cells from a substrate)
The synovium-derived mesenchymal stem cells cultured in step B are preferably separated from the substrate prior to step C. Preferably, separation from the substrate can be achieved by allowing a cell detachment solution to act on the mesenchymal stem cells for 120 minutes or less. The cell detachment solution is a solution containing a trypsin-like enzyme and EDTA. A particularly preferred enzyme is TrypLE. Examples of TrypLE that can be used include TrypLE Select Enzyme (manufactured by ThermoFisher Scientific), TrypL Express (manufactured by Gibco), and TrypLE Select (manufactured by Gibco).

The time for which the cell detachment solution is allowed to act on the mesenchymal stem cells is preferably 5 to 120 minutes, more preferably 5 to 60 minutes, and especially preferably 5 to 30 minutes.

(Step C of cryopreserving the cultured synovium-derived mesenchymal stem cells)
After cultivation, the synovium-derived mesenchymal stem cells can be cryopreserved.

<Microcarriers>
In the present invention, mesenchymal stem cells are cultured in the presence of microcarriers.
In the stirring static culture step, microcarriers may or may not be added.

Microcarriers are carriers that serve as scaffolds for cell growth in adherent cell culture. Microcarriers known as carriers for cell culture can be used. Microcarriers may be made of organic, inorganic, or composite materials of these. Microcarriers may be soluble or insoluble.

Examples of organic materials include synthetic polymers such as polystyrene, polyester, polyurethane, polyethylene, polypropylene, polyvinyl alcohol, (meth)acrylic polymers, (meth)acrylamide polymers, silicone polymers, epoxy resins, and urethane resins; and natural polymers such as cellulose, dextran, collagen, polygalacturonic acid, polyalginic acid, and gelatin.

Inorganic materials include, for example, glass, ceramic, metal, alloy, and metal oxide. From the viewpoint of cytocompatibility, the microcarrier material preferably contains an organic material, and more preferably contains a natural polymer. From the viewpoint of operability, soluble microcarriers are preferable, but are not limited to this.

Examples of microcarriers that can be used include, but are not limited to, SoloHill (registered trademark) Plastic microcarriers (Sartorius), CellBIND (Corning), SyntheMAX (Corning), Cytodex 1, and Cytodex 3 (Cytiva).

To promote cell attachment, cationic functional groups may be introduced onto the surface of the microcarrier. Examples of cationic functional groups include groups containing substituted or unsubstituted amino groups such as dimethylamino, diethylamino, and amino groups. Furthermore, to promote cell attachment, a cell adhesive polymer may be disposed on the surface of the microcarrier. The cell adhesive polymer may be a polypeptide or polysaccharide that exhibits cell adhesive properties, such as collagen, gelatin, alginic acid, Matrigel® (BD Biosciences), hyaluronic acid, laminin, fibronectin, vitronectin, elastin, heparan sulfate, dextran, dextran sulfate, and chondroitin sulfate. The cell adhesive polymer may be a partial peptide or oligosaccharide that exhibits cell adhesive properties. Furthermore, to promote cell attachment, hydrophilic functional groups may be generated by plasma treatment, corona treatment, or the like to hydrophilize the microcarrier surface. Examples of hydrophilic functional groups include groups containing hydroxyl, carbonyl, and carboxyl groups.

Microcarrier shapes include, for example, spherical, flat, cylindrical, plate-like, and prismatic. Microcarriers may be porous microcarriers with internal pores, or microcarriers without internal pores.

From the perspective of promoting cell proliferation, the average particle diameter (D50) of the microcarriers is, for example, 50 to 1,000 μm, preferably 100 to 500 μm, and more preferably 150 to 250 μm. The average particle diameter of the microcarriers is the value measured as the median diameter (D50) in physiological saline. The average particle diameter of the microcarriers can be measured using a laser diffraction/scattering particle size distribution measuring device.

The concentration of microcarriers in the culture medium can be adjusted appropriately based on the shape, size, surface area, etc. of the microcarriers, but may be, for example, 0.1 to 100 mg/mL, 1 to 100 mg/mL, or 5 to 100 mg/mL.

<Mesenchymal stem cell seeding>
The mesenchymal stem cells can be seeded by adding the mesenchymal stem cells to a medium containing microcarriers.

For example, a microcarrier solution is added to a container such as a centrifuge tube and allowed to stand to allow the microcarriers to settle, after which the supernatant is removed and culture medium is added. The microcarriers are thoroughly suspended, and the resulting suspension is added to a culture vessel, to which culture medium is then added. Cell seeding can then be carried out by adding a cell suspension containing a predetermined number of cells in culture medium to the culture vessel. Alternatively, it is possible to suspend the microcarriers directly in culture medium without preparing the microcarrier solution in advance, and then add the suspension to the culture vessel, followed by the cell suspension.

<Agitated stationary culture process>
The method of the present invention comprises carrying out an agitation static culture step, which includes a step of agitating mesenchymal stem cells and a subsequent step of allowing the mesenchymal stem cells to stand, for 48 hours or more, and comprises carrying out the agitation static culture step two or more times during the 48-hour or more culture period, and carrying out the agitation static culture step at least once every 48 hours starting 24 hours after the start of culture. The agitation static culture step is a combination of an agitation step and a standing step, and this set may be repeated multiple times.

In the present invention, the start of culture refers to the moment when cells and microcarriers coexist in the same liquid in the same container.

The stirring step is a step in which mesenchymal stem cells are cultured while stirring the culture solution containing mesenchymal stem cells and microcarriers. The stirring speed can be set appropriately depending on the volume of the culture solution, but is a speed at which all of the microcarriers in the culture solution are suspended. The stirring speed is not particularly limited, but is generally 50 rpm or higher, preferably 100 rpm or higher, more preferably 150 rpm or higher, and may be 200 rpm or higher, or 300 rpm or higher. There is no particular upper limit to the stirring speed, but it is generally 1000 rpm or lower, preferably 700 rpm or lower. Note that the stirring speed in the stirring step may be less than 50 rpm (for example, 10 rpm or higher but less than 50 rpm) depending on the volume of the culture solution.

The "standing step" refers to a step in which no stirring is performed, or a step in which weak stirring at less than 50 rpm is performed. Note that if the stirring speed in the stirring step is less than 50 rpm (for example, 10 rpm or more but less than 50 rpm), the weak stirring speed in the standing step will be slower than the stirring speed in the stirring step. If the stirring speed in the stirring step is less than 50 rpm, it is preferable that the weak stirring speed in the standing step be less than half the stirring speed in the stirring step.

Agitation can be achieved by placing a stirring bar, stirring blade, or stirring impeller in the culture solution (for example, a rotating shaft with stirring blades can be provided in the culture vessel) and rotating it.

In the present invention, the duration of the above-mentioned stirring and static culture process is 48 hours or more, but it may also be 3 days or more, 4 days or more, 5 days or more, 6 days or more, or 7 days or more.

In the present invention, the above-mentioned stirring and static culture step is carried out two or more times during a culture period of 48 hours or more, but the above-mentioned stirring and static culture step may also be carried out seven or more times, 42 or more times, 84 or more times, or 336 or more times during a culture period of 48 hours or more.
In the present invention, the stirring static culture step is carried out at least once every 48 hours after 24 hours from the start of culture, but the stirring static culture step may also be carried out at least twice, at least 12 times, at least 24 times, or at least 96 times every 48 hours.

The two or more stirring and static culture steps may be the same step, or the two or more stirring and static culture steps may include stirring and static culture steps in which one or more of the stirring step time and the stationary step time are different. Preferably, the two or more stirring and static culture steps are the same step.
Preferably, the stirring static culture step is carried out from the start of the culture to the end of the culture.

The time for the step of allowing the mesenchymal stem cells to stand in the stirred static culture step is preferably 1 minute or more and 2,400 minutes or less, more preferably 1 minute or more and 2,000 minutes or less, even more preferably 1 minute or more and 1,900 minutes or less, even more preferably 1 minute or more and 1,600 minutes or less, even more preferably 1 minute or more and 1,500 minutes or less, 10 minutes or more and 1,500 minutes or less, 20 minutes or more and 1,500 minutes or less, 30 minutes or more and 1,500 minutes or less, 50 minutes or more and 1,500 minutes or less, or 60 minutes or more and 1,500 minutes or less, or 1 minute or more and 240 minutes or less, 1 minute or more and 120 minutes or less, or 15 minutes or more and 120 minutes or less.
The time for the step of allowing the mesenchymal stem cells to stand in the stirred static culture step is preferably 0.1 min/mL or more and 240.0 min/mL or less, more preferably 0.1 min/mL or more and 200.0 min/mL or less, more preferably 0.1 min/mL or more and 100.0 min/mL or less, more preferably 0.1 min/mL or more and 50.0 min/mL or less, and may be 0.1 min/mL or more and 24.0 min/mL or less, 0.1 min/mL or more and 12.0 min/mL or less, or 1.5 min/mL or more and 12.0 min/mL or less, per volume of culture solution.

The time for the process of stirring the mesenchymal stem cells in the stirring static culture process is preferably 1 minute or more and 2000 minutes or less, and may be 1 minute or more and 1000 minutes or less, 1 minute or more and 500 minutes or less, 1 minute or more and 100 minutes or less, 1 minute or more and 60 minutes or less, 1 minute or more and 50 minutes or less, 1 minute or more and 30 minutes or less, 1 minute or more and 20 minutes or less, 1 minute or more and 15 minutes or less, or 1 minute or more and 10 minutes or less, or may be 5 minutes or more and 2000 minutes or less, 5 minutes or more and 1500 minutes or less, 5 minutes or more and 1440 minutes or less.
The time for the process of stirring the mesenchymal stem cells in the stirring static culture process is preferably 0.1 min/mL or more and 200.0 min/mL or less per volume of culture solution, and may be 0.1 min/mL or more and 150.0 min/mL or less, 0.1 min/mL or more and 144.0 min/mL or less, 0.5 min/mL or more and 200.0 min/mL or less, 0.5 min/mL or more and 150.0 min/mL or less, or 0.5 min/mL or more and 144.0 min/mL or less.

The stirring and static culture process can be performed preferably 1 to 48 times per 24 hours, or may be performed 1 to 24 times, 1 to 12 times, 1 to 6 times, or 6 to 12 times.

The duration of one stirring static culture step is preferably 30 to 1440 minutes, or 30 to 1000 minutes, 30 to 800 minutes, 30 to 600 minutes, 30 to 500 minutes, 30 to 400 minutes, 30 to 300 minutes, 30 to 240 minutes, 30 to 120 minutes, 50 to 1440 minutes, 50 to 1000 minutes, 50 to 800 minutes, 50 to 600 minutes, minutes, 50 to 500 minutes, 50 to 400 minutes, 50 to 300 minutes, 50 to 240 minutes, 50 to 120 minutes, 120 to 1440 minutes, 120 to 1000 minutes, 120 to 800 minutes, 120 to 600 minutes, 120 to 500 minutes, 120 to 400 minutes, 120 to 300 minutes, 120 to 240 minutes, or 240 to 1440 minutes.
The ratio of the time between the step of stirring the mesenchymal stem cells and the step of allowing the mesenchymal stem cells to stand in the stirring static culture step may be within the range of 1:0.005 to 1:700, 1:0.005 to 1:600, 1:0.005 to 1:500, 1:0.005 to 1:400, 1:0.005 to 1:380, 1:0.005 to 1:320, 1:0.005 to 1:300, 1:0.005 to 1:10, 1:0.01 to 1:10, 1:0.05 to 1:10, 1:0.08 to 1:10, 1:0.1 to 1:00, 1:0.01 to 1:1 ... The ratio may be 1 to 1:5, 1:1 to 1:700, 1:1 to 1:600, 1:1 to 1:500, 1:1 to 1:400, 1:1 to 1:300, 1:2 to 1:700, 1:2 to 1:600, 1:2 to 1:500, 1:1 to 2:400, 1:2 to 1:300, 1:5 to 1:700, 1:5 to 1:600, 1:5 to 1:500, 1:5 to 1:400, 1:5 to 1:300, 1:10 to 1:700, 1:10 to 1:600, 1:10 to 1:500, 1:10 to 1:400, or 1:10 to 1:300.

Preferably, the total time for the stirring step during the entire culture period is longer than the total time for the standing step.

In one example of the present invention, 24 hours or more after the start of the culture, stirring static culture may be carried out for 48 hours or more, and thereafter stirring culture may be carried out continuously.
In another example of the present invention, agitation static culture may be performed for 72 hours or more after 24 hours from the start of culture, followed by continuous agitation culture. In yet another example of the present invention, agitation static culture may be performed for 96 hours or more after 24 hours from the start of culture, followed by continuous agitation culture, or agitation static culture may be performed for 120 hours or more after 24 hours from the start of culture, followed by continuous agitation culture, or agitation static culture may be performed for 144 hours or more after 24 hours from the start of culture, followed by continuous agitation culture.

In one example of the present invention, the stirring static culture process may be carried out 24 hours or more after the start of culture, and stirring culture may be carried out continuously from two days or more before the end of culture. In another example of the present invention, the stirring and static culture step may be performed 24 hours or more after the start of culture and then continuous stirring culture may be performed from 3 days or more before the end of culture; the stirring and static culture step may be performed 24 hours or more after the start of culture and then continuous stirring culture may be performed from 4 days or more before the end of culture; the stirring and static culture step may be performed 24 hours or more after the start of culture and then continuous stirring culture may be performed from 5 days or more before the end of culture; the stirring and static culture step may be performed 24 hours or more after the start of culture and then continuous stirring culture may be performed from 6 days or more before the end of culture; the stirring and static culture step may be performed 24 hours or more after the start of culture and then continuous stirring culture may be performed from 7 days or more before the end of culture; the stirring and static culture step may be performed 24 hours or more after the start of culture and then continuous stirring culture may be performed from 8 days or more before the end of culture; the stirring and static culture step may be performed 24 hours or more after the start of culture and then continuous stirring culture may be performed from 9 days or more before the end of culture; or the stirring and static culture step may be performed 24 hours or more after the start of culture and then continuous stirring culture may be performed from 10 days or more before the end of culture.

The ratio of the time for the stirring static culture step to the time for the continuous stirring step may preferably be 1:0.3 to 1:5.5.
The time ratio between the step (A) of leaving the mesenchymal stem cells to stand, the step (B) of stirring and leaving the mesenchymal stem cells to stand, and the step (C) of continuously stirring the mesenchymal stem cells may preferably be A=1, B=2 to 6, and C=2 to 11.

<General conditions for culturing mesenchymal stem cells>
The culture vessel used for culturing mesenchymal stem cells is not particularly limited as long as it is capable of culturing mesenchymal stem cells, and examples include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, microslides, chamber slides, petri dishes, tubes, trays, culture bags, roller bottles, culture tanks, and bioreactors.

The culture scale (volume of culture medium) is not particularly limited, but is generally 0.01 to 3000 L, preferably 0.01 to 500 L, more preferably 0.01 to 50 L, and even more preferably 0.01 to 10 L.

As the culture medium, a basal medium containing components necessary for the survival and proliferation of mesenchymal stem cells (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, fatty acids) can be used. For example, αMEM, DMEM (Dulbecco Modified Eagle Medium), a mixed medium of DMEM and F12 (DMEM:F12=1:1), RPMI medium (GIBCO (registered trademark) RPMI1640 medium, etc.), a mixed medium of DMEM/F12 and RPMI (DMEM/F12:RPMI=1:1), RoosterNourish ^™ -MSC-XF medium (hereinafter referred to as RoosterNourish medium, RoosterBio), etc. can be used.

Culture conditions can be set appropriately. For example, the culture temperature is not particularly limited, but can be about 30 to 40°C, preferably about 37°C. The _CO2 concentration can be about 1 to 10%, preferably about 2 to 5%. The oxygen concentration can be 1 to 20%, preferably 1 to 10%.

<Positive rate of microcarriers and number of recovered viable cells>
According to the method of the present invention, the positive rate of microcarriers can be increased, and the number of recovered viable cells can also be increased.

The positivity rate of microcarriers can be measured as follows. A sample of the culture medium containing cells and microcarriers is taken, and Nucblue (ThemoFisher Scientific) is added and incubated at 37°C for 15 to 30 minutes. Nuclear staining is observed and photographed using a fluorescence microscope (KEYENCE). The image taken using ImageJ is opened and binarized using Split Channels. Based on this binarized image, the number of microcarriers to which cells are attached (nuclear stained) and the total number of microcarriers in the field of view are visually counted. The positivity rate is calculated using the following formula.
Positive rate = number of microcarriers with cells attached / total number of microcarriers × 100

The number of viable cells recovered can be determined as follows.
After culturing, the bioreactor is recovered, a 100 μm cell strainer is placed in the centrifuge tube, and the entire contents of the bioreactor are filtered. The cells and microcarriers remaining on the cell strainer are washed twice with DPBS, and then the cells and microcarriers on the cell strainer are recovered using TrypLE Select Enzyme (Gibco) and returned to the bioreactor. The bioreactor is stirred at 300 rpm for 30 minutes at 37°C. The bioreactor is recovered, and the entire contents of the bioreactor are filtered into a centrifuge tube equipped with a 100 μm cell strainer. The bioreactor and the carriers on the cell strainer are washed together with Rooster Nourish medium. The centrifuge tube is centrifuged, the supernatant is removed, and then Rooster Nourish medium is added and suspended. The suspension is mixed with AO/PI Cell Viability Kit at a ratio of 9:1, and the number of cells is counted using LUNA (Logos Biosystems).

<Therapeutic Agent>
The method of the present invention preferably produces mesenchymal stem cells for use as a therapeutic agent. That is, mesenchymal stem cells produced by the method of the present invention can be suitably used for medical purposes such as cell therapy. Mesenchymal stem cells produced by the method of the present invention can be used undifferentiated or after differentiation into osteocytes, chondrocytes, adipocytes, or the like, depending on the target disease. Target diseases include, for example, joint diseases, avascular necrosis of the lunate bone, avascular necrosis of the femoral head, osteochondritis dissecans, lumbar disc herniation, ischemic heart disease, epidermolysis bullosa, and the like. Joint diseases include, for example, meniscus injury, traumatic cartilage injury, osteochondritis dissecans, avascular necrosis of osteonecrosis, osteoarthritis (e.g., knee osteoarthritis), rheumatoid arthritis (e.g., rheumatoid arthritis), gout, reactive arthritis, psoriatic arthritis, juvenile arthritis, inflammatory arthritis, and articular cartilage defect. Mesenchymal stem cells can also be differentiated into adipocytes and then used for cosmetic surgery.

When mesenchymal stem cells are used as a therapeutic agent, the cells can be prepared into a formulation suitable for administration to an individual by, for example, mixing them with a pharmaceutically acceptable carrier using standard methods. Examples of carriers include isotonic distilled water for injection made with physiological saline, glucose, or other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.). Additionally, buffers (e.g., phosphate buffer, sodium acetate buffer), soothing agents (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives, antioxidants, etc. may also be added.

The present invention will be explained in more detail using the following examples, but the present invention is not limited to these examples.

Materials and Methods
Example 1: Production under one set of conditions: "119 minutes of stirring, 1 minute of static culture" in the stirring static culture process (1) Obtaining synovial stem cells Human clinical synovial tissue was placed on a dish and shredded with scissors. The shredded tissue was transferred to a centrifuge tube containing a mixed solution (1 mg/mL) of Liberase (Roche) and n-Liven PR (Sexton Biotechnologies) and subjected to enzyme treatment at 37°C for approximately 3 hours using a rotating mixer. After enzyme treatment, αMEM (prepared in-house, JP 2021-040551) was added to the tube, and the tissue was dispersed by pipetting. The tissue was then passed through a 70 μm cell strainer placed in a 50 mL centrifuge tube and centrifuged. After removing the supernatant, the pellet was broken up, αMEM was added, and the mixture was centrifuged. This process was repeated once. The cells were suspended in αMEM, and the suspension was mixed with AO/PI Cell Viability Kit (Logos Biosystems) at a ratio of 9:1, and the number of cells was counted using LUNA (Logos Biosystems). The cells were seeded in a flask at a density of 1000 cells/ ^cm2 and cultured for 3 to 5 days in a culture medium (hereinafter referred to as αMEM medium) prepared by mixing αMEM with n-Liven PR, amphotericin B (ThemoFisher Scientific), and gentamicin (ThemoFisher Scientific). After that, the medium was changed to RoosterNourish ^™ -MSC-XF medium (hereinafter referred to as RoosterNourish medium, RoosterBio) and cultured until the cells reached 70 to 80% confluence. The supernatant was removed from the flask, and the cells were washed twice with DPBS (ThemoFisher Scientific). TrypLE Select Enzyme (ThemoFisher Scientific) was added and the mixture was left to stand at 37°C for 10 to 30 minutes. An equal volume of αMEM medium was added, and the cell suspension was transferred to a centrifuge tube and centrifuged. The supernatant was removed, and the cells were suspended in αMEM medium. After mixing with AO/PI Cell Viability Kit at a ratio of 9:1, the cells were counted using LUNA. The suspension containing the specified number of cells was transferred to a new centrifuge tube and centrifuged. The supernatant was removed, and the cells were frozen using a DMSO-containing cryopreservation solution (P0: passage 0). P0 frozen cells were thawed in a water bath, added to a centrifuge tube containing Rooster Nourish medium, and centrifuged. The supernatant was removed, and the cells were suspended in Rooster Nourish medium. After mixing with AO/PI Cell Viability Kit at a ratio of 9:1, the number of cells was counted using LUNA. The cells were seeded into flasks at a density of 1,000-3,000 cells/ ^cm² and cultured until 70-80% confluent. The supernatant was removed from the flask, washed with DPBS, and then TrypLE Select Enzyme was added. The mixture was allowed to stand at 37°C for approximately 5 minutes. An equal volume of Rooster Nourish medium was added, and the cell suspension was transferred to a centrifuge tube and centrifuged. The supernatant was removed, and the cells were suspended in Rooster Nourish medium. After mixing with AO/PI Cell Viability Kit at a ratio of 9:1, the number of cells was counted using LUNA. The suspension containing the desired number of cells was transferred to a new centrifuge tube and centrifuged. The supernatant was removed and frozen using a DMSO-containing freezing medium (P1: passage 1).

(2) Preparation of Microcarriers SoloHill (registered trademark) Plastic microcarriers (Sartorius Cat. No. PIR-221-020) were suspended in DPBS (Gibco) at a concentration of 0.1 g/mL and stored at room temperature.

(3) Seeding of synovial mesenchymal stem cells The microcarrier solution prepared in (2) was thoroughly suspended in a 50 mL centrifuge tube and then transferred to 1.4 mL x the required number of tubes. After leaving to stand for about 5 minutes to allow the microcarriers to settle, the supernatant DPBS was removed and 2 mL x the required number of tubes of Rooster Nourish medium (RoosterBio) was added. The microcarriers were thoroughly suspended and added to Ambr15 bioreactors (Sartorius) at 2 mL/tube. Rooster Nourish medium was added to 50 mL centrifuge tubes so that 2 mL x the required number of tubes was added, and then 2 mL/tube was added to the Ambr15 bioreactor. The microcarrier concentration was 0.14 g/4 mL.

The frozen cells (P1) from (1) were thawed and adjusted to 15 x ¹⁰ cells/mL with Rooster Nourish medium to seed at a density of 3 x ¹⁰ cells/ ^cm . 1 mL of this cell suspension was added to the previously prepared Ambr15 bioreactors (microcarrier concentration per Ambr15 bioreactor: 0.14 g/5 mL). The same procedure was repeated twice on separate days.

(4) Cultivation and Evaluation (4-1) Cultivation The Ambr15 bioreactor seeded in (3) was set in the Ambr15, and a cycle of "5 minutes of stirring, 25 minutes of standing" was repeated six times, followed by the addition of 5 mL of Rooster Nourish medium. The cycle of "119 minutes of stirring, 1 minute of standing" was then repeated for 7 days, and the culture was continued (microcarrier concentration per Ambr15 bioreactor: 0.14 g/10 mL). The stirring speed was 300 rpm. Furthermore, after sampling on the fourth day of culture to evaluate the microcarrier positivity rate, medium exchange was performed using the Ambr15 program. The medium exchange was performed at the same time as the static culture step of the stirring and static culture step, and the culture was allowed to stand for about 5 minutes, allowing the carriers and cells to settle, and 5 mL of culture supernatant was withdrawn. Then, Rooster Nourish medium was added at the same time as the stirring and static culture step.

(4-2) Evaluation of Microcarrier Positivity Rate 100 μL of culture medium containing cells and microcarriers on day 4 of culture was sampled using the Ambr15 program. 400 μL of DPBS was added, and then one drop of Nucblue (ThemoFisher Scientific) was added and incubated at 37 °C for 15 to 30 minutes. Nuclear staining was observed and photographed using a fluorescence microscope (KEYENCE). The images taken with ImageJ were opened and binarized using Split Channels. Based on this binarized image, the number of microcarriers to which cells had adhered (nuclear stained) and the total number of microcarriers in the field of view were visually counted.
The positive rate was calculated using the following formula.
Positive rate = number of microcarriers with cells attached / total number of microcarriers × 100
The positive rate when cultured under one set of conditions of "119 minutes of stirring and 1 minute of standing" was 36% (average value of three tests).

(4-3) Evaluation of the number of recovered viable cells After 7 days of culture, the Ambr15 bioreactor was recovered. A 100 μm cell strainer was placed in a 50 mL centrifuge tube, and the entire contents of the Ambr15 bioreactor were filtered. The cells and microcarriers remaining on the cell strainer were washed twice with DPBS, and then the cells and microcarriers on the cell strainer were recovered using TrypLE Select Enzyme (Gibco) and returned to the Ambr15 bioreactor. The Ambr15 bioreactor was placed in the Ambr15 and stirred at 300 rpm for 30 minutes at 37 °C. The Ambr15 bioreactor was recovered, and the entire contents of the Ambr15 bioreactor were filtered into a 50 mL centrifuge tube containing a 100 μm cell strainer. The Ambr15 bioreactor and the carrier on the cell strainer were washed together with Rooster Nourish medium. The 50 mL centrifuge tube was centrifuged, the supernatant was removed, and Rooster Nourish medium was added to suspend the cells. The suspension was mixed with AO/PI Cell Viability Kit at a ratio of 9:1, and the cell number was counted using LUNA (Logos Biosystems). The cells were frozen using CP-1 High Grade (Kyokuto Pharmaceutical Industries Co., Ltd.) as needed.

When cultured under one set of conditions of "agitation for 119 minutes and standing for 1 minute," the number of viable cells recovered was 1.27 x 10 ⁶ cells (average value of two tests).

(4-4) Overall Evaluation A comprehensive evaluation was performed by combining the evaluations of (4-2) Positive Rate and (4-3) Number of Recovered Viable Cells. The evaluation criteria are as follows:

When cultured under one set of conditions, "119 minutes of agitation, 1 minute of standing," the overall rating was "D."

Example 2: Production under the condition of "225 minutes of stirring and 15 minutes of static culture" as one set in the stirring and static culture process (1) Obtaining synovial stem cells and (2) preparing microcarriers were carried out in the same manner as in Example 1.

(3) Seeding of synovial mesenchymal stem cells The microcarrier solution prepared in (2) was thoroughly suspended in a 50 mL centrifuge tube, and then 1.4 mL of the required number of tubes was transferred. After leaving the tubes to stand for about 5 minutes to allow the microcarriers to settle, the supernatant DPBS was removed, and 2 mL of Rooster Nourish medium (RoosterBio) was added in the required number of tubes. The microcarriers were thoroughly suspended, and added to Ambr15 bioreactors (Sartorius) in 2 mL/tube quantities. Rooster Nourish medium was added to 50 mL centrifuge tubes in an amount of 2 mL of the required number of tubes, and then added to Ambr15 bioreactors in 2 mL/tube quantities.

The frozen cells (P1) in (1) were thawed and prepared in Rooster Nourish medium at 15 x ¹⁰ cells/mL for seeding at a density of 3 x ¹⁰ cells/ ^cm . 1 mL of this cell suspension was added to the previously prepared Ambr15 bioreactors (microcarrier concentration per Ambr15 bioreactor: 0.14 g/5 mL).

(4) Cultivation and Evaluation (4-1) Cultivation The Ambr15 bioreactor seeded in (3) was set in the Ambr15, and a cycle of "5 minutes of agitation, 25 minutes of standing" was repeated six times, followed by the addition of 5 mL of Rooster Nourish medium. The cycle of "225 minutes of agitation, 15 minutes of standing" was then repeated for seven days, and the culture was carried out (microcarrier concentration per Ambr15 bioreactor: 0.14 g/10 mL). The agitation speed was 300 rpm.

(4-2) Evaluation of Positive Rate of Microcarriers Evaluation was carried out in the same manner as in Example 1.
The positive rate when cultured under one set of conditions of "225 minutes of stirring and 15 minutes of standing" was 80%.

(4-3) Evaluation of the Number of Recovered Viable Cells Evaluation was carried out in the same manner as in Example 1.
When cultured under one set of conditions of "225 minutes of stirring and 15 minutes of standing," the number of viable cells recovered was 0.937 x 10 ⁶ cells.

(4-4) Overall Evaluation Evaluation was carried out in the same manner as in Example 1.
The overall evaluation when the culture was performed under one set of conditions of "225 minutes of stirring and 15 minutes of standing" was "C."

Example 3: Production under the condition of "5 minutes of stirring and 25 minutes of static culture" as one set in the stirring and static culture process (1) Obtaining synovial stem cells, (2) preparing microcarriers, and (3) seeding synovial mesenchymal stem cells were carried out in the same manner as in Example 1.

(4) Cultivation and Evaluation (4-1)
The Ambr15 bioreactor seeded in (3) was set in the Ambr15, and a cycle of "5 minutes of stirring, 25 minutes of standing" was repeated six times, followed by the addition of 5 mL of Rooster Nourish medium. The cycle of "5 minutes of stirring, 25 minutes of standing" was then repeated for seven days for cultivation (microcarrier concentration per Ambr15 bioreactor: 0.14 g/10 mL). The stirring speed was 300 rpm.

(4-2) Evaluation of Positive Rate of Microcarriers Evaluation was carried out in the same manner as in Example 1.
The positive rate when cultured under one set of conditions of "5 minutes of stirring and 25 minutes of standing" was 44% (average value of three tests).

(4-3) Evaluation of the Number of Recovered Viable Cells Evaluation was carried out in the same manner as in Example 1.
When cultured under one set of conditions of "5 minutes of stirring and 25 minutes of standing," the number of viable cells recovered was 1.01 x 10 ⁶ cells (average of two tests).

(4-4) Overall Evaluation Evaluation was carried out in the same manner as in Example 1.
When cultured under one set of conditions of "5 minutes of stirring and 25 minutes of standing," the overall evaluation was "B."

Example 4: Production under the condition of "95 minutes of stirring and 25 minutes of static culture" as one set in the stirring and static culture process (1) Obtaining synovial stem cells and (2) preparing microcarriers were carried out in the same manner as in Example 1.

(3) Seeding of synovial mesenchymal stem cells The microcarrier solution prepared in (2) was thoroughly suspended in a 50 mL centrifuge tube, and then 1.4 mL of the required number of tubes was transferred. After leaving the tubes to stand for about 5 minutes to allow the microcarriers to settle, the supernatant DPBS was removed, and 2 mL of Rooster Nourish medium (RoosterBio) was added in the required number of tubes. The microcarriers were thoroughly suspended, and added to Ambr15 bioreactors (Sartorius) in 2 mL/tube quantities. Rooster Nourish medium was added to 50 mL centrifuge tubes in an amount of 2 mL of the required number of tubes, and then added to Ambr15 bioreactors in 2 mL/tube quantities.
The frozen cells (P1) in (1) were thawed and adjusted to 15 x ¹⁰ cells/mL with Rooster Nourish medium to seed at a density of 3 x ¹⁰ cells/ ^cm . 1 mL of this cell suspension was added to the previously prepared Ambr15 bioreactors (microcarrier concentration per Ambr15 bioreactor: 0.14 g/5 mL).
The same procedure was repeated four times on different days.

(4) Cultivation and Evaluation (4-1) Cultivation The Ambr15 bioreactor seeded in (3) was set in the Ambr15, and a cycle of "5 minutes of stirring, 25 minutes of standing" was repeated six times, followed by the addition of 5 mL of Rooster Nourish medium. Subsequently, a cycle of "95 minutes of stirring, 25 minutes of standing" was repeated for seven days, and the culture was carried out (microcarrier concentration per Ambr15 bioreactor: 0.14 g/10 mL). The stirring speed was 300 rpm.

(4-2) Evaluation of Positive Rate of Microcarriers Evaluation was carried out in the same manner as in Example 1.
The positive rate when cultured under one set of conditions of "95 minutes of stirring and 25 minutes of standing" was 78% (average value of five tests).

(4-3) Evaluation of the Number of Recovered Viable Cells Evaluation was carried out in the same manner as in Example 1.
When cultured under one set of conditions of "95 minutes of stirring and 25 minutes of standing," the number of viable cells recovered was 1.70 x 10 ⁶ cells (average value of four tests).

(4-4) Overall Evaluation Evaluation was carried out in the same manner as in Example 1.
When cultured under one set of conditions of "95 minutes of stirring and 25 minutes of standing," the overall evaluation was "A."

Example 5: Production under one set of conditions in the stirring and static culture process: "215 minutes of stirring, 25 minutes of static culture" (1) Obtaining synovial stem cells, (2) preparing microcarriers, and (3) seeding synovial mesenchymal stem cells were carried out in the same manner as in Example 1.

(4) Cultivation and Evaluation (4-1) Cultivation The Ambr15 bioreactor seeded in (3) was set in the Ambr15, and a cycle of "5 minutes of agitation, 25 minutes of standing" was repeated six times, followed by the addition of 5 mL of Rooster Nourish medium. The cycle of "215 minutes of agitation, 25 minutes of standing" was then repeated for 7 days, and the culture was carried out (microcarrier concentration per Ambr15 bioreactor: 0.14 g/10 mL). The agitation speed was 300 rpm.

(4-2) Evaluation of Positive Rate of Microcarriers Evaluation was carried out in the same manner as in Example 1.
The positive rate when cultured under one set of conditions of "215 minutes of stirring and 25 minutes of standing" was 86% (average value of three tests).

(4-3) Evaluation of the Number of Recovered Viable Cells Evaluation was carried out in the same manner as in Example 1.
When cultured under one set of conditions of "215 minutes of stirring and 25 minutes of standing," the number of viable cells recovered was 1.75 x 10 ⁶ cells (average value of three tests).

(4-4) Overall Evaluation Evaluation was carried out in the same manner as in Example 1.
The overall evaluation when the culture was performed under one set of conditions of "215 minutes of stirring and 25 minutes of standing" was "A."

Example 6: Production under the condition of "1415 minutes of stirring and 25 minutes of static culture" as one set in the stirring and static culture process (1) Obtaining synovial stem cells and (2) preparing microcarriers were carried out in the same manner as in Example 1.

(3) Seeding of synovial mesenchymal stem cells The microcarrier solution prepared in (2) was thoroughly suspended in a 50 mL centrifuge tube, and then 1.4 mL of the required number of tubes was transferred. After leaving the tubes to stand for about 5 minutes to allow the microcarriers to settle, the supernatant DPBS was removed, and 2 mL of Rooster Nourish medium (RoosterBio) was added in the required number of tubes. The microcarriers were thoroughly suspended, and added to Ambr15 bioreactors (Sartorius) in 2 mL/tube quantities. Rooster Nourish medium was added to 50 mL centrifuge tubes in an amount of 2 mL of the required number of tubes, and then added to Ambr15 bioreactors in 2 mL/tube quantities.
The frozen cells (P1) in (1) were thawed and adjusted to 15 x ¹⁰ cells/mL with Rooster Nourish medium to seed at a density of 3 x ¹⁰ cells/ ^cm . 1 mL of this cell suspension was added to the previously prepared Ambr15 bioreactors (microcarrier concentration per Ambr15 bioreactor: 0.14 g/5 mL).
The same procedure was repeated once on another day.

(4) Cultivation and Evaluation (4-1) Cultivation The Ambr15 bioreactor seeded in (3) was set in the Ambr15, and a cycle of "5 minutes of agitation, 25 minutes of standing" was repeated six times, followed by the addition of 5 mL of Rooster Nourish medium. The cycle of "1415 minutes of agitation, 25 minutes of standing" was then repeated for seven days, and the culture was carried out (microcarrier concentration per Ambr15 bioreactor: 0.14 g/10 mL). The agitation speed was 300 rpm.

(4-2) Evaluation of Positive Rate of Microcarriers Evaluation was carried out in the same manner as in Example 1.
The positive rate when cultured under one set of conditions of "1415 minutes of stirring and 25 minutes of standing" was 83% (average value of two tests).

(4-3) Evaluation of the Number of Recovered Viable Cells Evaluation was carried out in the same manner as in Example 1.
When cultured under one set of conditions of "1415 minutes of stirring and 25 minutes of standing," the number of viable cells recovered was 1.95 x 10 ⁶ cells (average of two tests).

(4-4) Overall Evaluation Evaluation was carried out in the same manner as in Example 1.
The overall evaluation when the culture was performed under one set of conditions of "1415 minutes of stirring and 25 minutes of standing" was "A."

Example 7: Production under conditions where one set of "60 minutes of stirring, 60 minutes of standing" was used in the static culture process (1) Obtaining synovial stem cells and (2) preparing microcarriers were carried out in the same manner as in Example 1.

(3) Seeding of synovial mesenchymal stem cells The microcarrier solution prepared in (2) was thoroughly suspended in a 50 mL centrifuge tube, and then 1.4 mL of the required number of tubes was transferred. After leaving the tubes to stand for about 5 minutes to allow the microcarriers to settle, the supernatant DPBS was removed, and 2 mL of Rooster Nourish medium (RoosterBio) was added in the required number of tubes. The microcarriers were thoroughly suspended, and added to Ambr15 bioreactors (Sartorius) in 2 mL/tube quantities. Rooster Nourish medium was added to 50 mL centrifuge tubes in an amount of 2 mL of the required number of tubes, and then added to Ambr15 bioreactors in 2 mL/tube quantities.
The frozen cells (P1) in (1) were thawed and adjusted to 15 x ¹⁰ cells/mL with Rooster Nourish medium to seed at a density of 3 x ¹⁰ cells/ ^cm . 1 mL of this cell suspension was added to the previously prepared Ambr15 bioreactors (microcarrier concentration per Ambr15 bioreactor: 0.14 g/5 mL).
The same procedure was repeated once on another day.

(4) Cultivation and Evaluation (4-1) Cultivation The Ambr15 bioreactor seeded in (3) was set in the Ambr15, and a cycle of "5 minutes of agitation, 25 minutes of standing" was repeated six times, followed by the addition of 5 mL of Rooster Nourish medium. Subsequently, a cycle of "60 minutes of agitation, 60 minutes of standing" was repeated for seven days, and the culture was carried out (microcarrier concentration per Ambr15 bioreactor: 0.14 g/10 mL). The agitation speed was 300 rpm.

(4-2) Evaluation of Positive Rate of Microcarriers Evaluation was carried out in the same manner as in Example 1.
The positive rate when cultured under one set of conditions of "60 minutes of stirring and 60 minutes of standing" was 89% (average of two tests).

(4-3) Evaluation of the Number of Recovered Viable Cells Evaluation was carried out in the same manner as in Example 1.
When cultured under one set of conditions of "agitation for 60 minutes and standing for 60 minutes," the number of viable cells recovered was 1.33 x 10 ⁶ cells (average of two tests).

(4-4) Overall Evaluation Evaluation was carried out in the same manner as in Example 1.
When the culture was performed under one set of conditions, "agitation for 60 minutes, standing for 60 minutes," the overall evaluation was "B."

Example 8: Production under one set of conditions in the static culture process: "180 minutes of stirring, 60 minutes of static culture" (1) Obtaining synovial stem cells and (2) preparing microcarriers were carried out in the same manner as in Example 1.

(3) Seeding of synovial mesenchymal stem cells The microcarrier solution prepared in (2) was thoroughly suspended in a 50 mL centrifuge tube, and then 1.4 mL of the required number of tubes was transferred. After leaving the tubes to stand for about 5 minutes to allow the microcarriers to settle, the supernatant DPBS was removed, and 2 mL of Rooster Nourish medium (RoosterBio) was added in the required number of tubes. The microcarriers were thoroughly suspended, and added to Ambr15 bioreactors (Sartorius) in 2 mL/tube quantities. Rooster Nourish medium was added to 50 mL centrifuge tubes in an amount of 2 mL of the required number of tubes, and then added to Ambr15 bioreactors in 2 mL/tube quantities.
The frozen cells (P1) in (1) were thawed and adjusted to 15 x ¹⁰ cells/mL with Rooster Nourish medium to seed at a density of 3 x ¹⁰ cells/ ^cm . 1 mL of this cell suspension was added to the previously prepared Ambr15 bioreactors (microcarrier concentration per Ambr15 bioreactor: 0.14 g/5 mL).

(4) Cultivation and Evaluation (4-1) Cultivation The Ambr15 bioreactor seeded in (3) was set in the Ambr15, and a cycle of "5 minutes of agitation, 25 minutes of standing" was repeated six times, followed by the addition of 5 mL of Rooster Nourish medium. Subsequently, a cycle of "180 minutes of agitation, 60 minutes of standing" was repeated for seven days, and the culture was carried out (microcarrier concentration per Ambr15 bioreactor: 0.14 g/10 mL). The agitation speed was 300 rpm.

(4-2) Evaluation of Positive Rate of Microcarriers Evaluation was carried out in the same manner as in Example 1.
The positive rate when cultured under one set of conditions of "180 minutes of stirring and 60 minutes of standing" was 84%.

(4-3) Evaluation of the Number of Recovered Viable Cells Evaluation was carried out in the same manner as in Example 1.
When cultured under one set of conditions of "180 minutes of stirring and 60 minutes of standing," the number of viable cells recovered was 1.22 x 10 ⁶ cells.

(4-4) Overall Evaluation Evaluation was carried out in the same manner as in Example 1.
The overall evaluation when the culture was performed under one set of conditions of "180 minutes of stirring and 60 minutes of standing" was "B."

Example 9: Production under conditions where one set of "120 minutes of stirring, 120 minutes of standing" was used in the static culture process (1) Obtaining synovial stem cells and (2) preparing microcarriers were carried out in the same manner as in Example 1.

(3) Seeding of synovial mesenchymal stem cells The microcarrier solution prepared in (2) was thoroughly suspended in a 50 mL centrifuge tube, and then 1.4 mL of the required number of tubes was transferred. After leaving the tubes to stand for about 5 minutes to allow the microcarriers to settle, the supernatant DPBS was removed, and 2 mL of Rooster Nourish medium (RoosterBio) was added in the required number of tubes. The microcarriers were thoroughly suspended, and added to Ambr15 bioreactors (Sartorius) in 2 mL/tube quantities. Rooster Nourish medium was added to 50 mL centrifuge tubes in an amount of 2 mL of the required number of tubes, and then added to Ambr15 bioreactors in 2 mL/tube quantities.
The frozen cells (P1) in (1) were thawed and adjusted to 15 x ¹⁰ cells/mL with Rooster Nourish medium to seed at a density of 3 x ¹⁰ cells/ ^cm . 1 mL of this cell suspension was added to the previously prepared Ambr15 bioreactors (microcarrier concentration per Ambr15 bioreactor: 0.14 g/5 mL).

(4) Cultivation and Evaluation (4-1) Cultivation The Ambr15 bioreactor seeded in (3) was set in the Ambr15, and a cycle of "5 minutes of agitation, 25 minutes of standing" was repeated six times, followed by the addition of 5 mL of Rooster Nourish medium. Subsequently, a cycle of "120 minutes of agitation, 120 minutes of standing" was repeated for 7 days, and the culture was carried out (microcarrier concentration per Ambr15 bioreactor: 0.14 g/10 mL). The agitation speed was 300 rpm.

(4-2) Evaluation of Positive Rate of Microcarriers Evaluation was carried out in the same manner as in Example 1.
The positive rate when cultured under one set of conditions of "120 minutes of stirring and 120 minutes of standing" was 91%.

(4-3) Evaluation of the Number of Recovered Viable Cells Evaluation was carried out in the same manner as in Example 1.
When cultured under one set of conditions of "120 minutes of stirring and 120 minutes of standing," the number of viable cells recovered was 1.11 x 10 ⁶ cells.

(4-4) Overall Evaluation Evaluation was carried out in the same manner as in Example 1.
When the culture was performed under one set of conditions, "120 minutes of stirring and 120 minutes of standing," the overall evaluation was "B."

Comparative Example 1: Production under the condition of "stirring only, no standing" (1) Obtaining synovial stem cells, (2) preparing microcarriers, and (3) seeding synovial mesenchymal stem cells were carried out in the same manner as in Example 1.

(4) Cultivation and Evaluation (4-1) Cultivation The Ambr15 bioreactor seeded in (3) was set in the Ambr15, and the "5 minutes of agitation, 25 minutes of standing" cycle was repeated six times, after which 5 mL of Rooster Nourish medium was added. The cells were then cultured for 7 days under "stirring only, no standing," i.e., without static agitation culture (microcarrier concentration per Ambr15 bioreactor: 0.14 g/10 mL). The agitation speed was 300 rpm. Furthermore, after sampling on the fourth day of culture to evaluate the microcarrier positivity rate, medium exchange was performed using the Ambr15 program. The medium exchange was performed by standing for approximately 5 minutes, allowing the carriers and cells to settle, and 5 mL of culture supernatant was withdrawn. Then, Rooster Nourish medium was added when agitation was resumed.

(4-2) Evaluation of Positive Rate of Microcarriers Evaluation was carried out in the same manner as in Example 1.
The positive rate when cultured with "agitation only, no standing" was 24% (average of three tests).

(4-3) Evaluation of the Number of Recovered Viable Cells Evaluation was carried out in the same manner as in Example 1.
The number of viable cells recovered when the culture was performed with "stirring only, without standing" was 0.976 x 10 ⁶ cells (average value of two tests).

(4-4) Overall Evaluation Evaluation was carried out in the same manner as in Example 1.
The overall evaluation when the culture was performed with "stirring only, without standing" was "E".

Example 10: Production under the conditions of "5 minutes of stirring and 1435 minutes of static culture" as one set in the stirring static culture process. (1) Obtaining synovial stem cells and (2) preparing microcarriers were carried out in the same manner as in Example 1.

(3) Seeding of synovial mesenchymal stem cells The amount to be used in Example 10 and Comparative Example 2 was prepared together.
The microcarrier solution prepared in (2) was thoroughly suspended and then 280 mL was transferred to a 500 mL storage bottle. After allowing the microcarriers to settle for approximately 10 minutes, the supernatant DPBS was removed and 200 mL of Rooster Nourish medium (RoosterBio) was added. The microcarriers were thoroughly suspended and divided equally among four suspension cell culture flasks 225 (Sumitomo Bakelite). 32 mL of Rooster Nourish medium was added to the 500 mL storage bottle and divided equally among the four suspension cell culture flasks.
The frozen cells (P1) in (1) were thawed and adjusted to 0.5 x ¹⁰ cells/2 mL with Rooster Nourish medium to seed at a density of 2 x ¹⁰ cells/ ^cm . 2 mL of this cell suspension was added to the previously prepared suspension cell culture flasks (microcarrier concentration per suspension cell culture flask: 7 g/60 mL).

(4) Cultivation and Evaluation (4-1) Cultivation The suspension cell culture flasks seeded in (3) were placed in an incubator and cultured for approximately 24 hours. Subsequently, two suspensions of cells and carriers cultured in the suspension cell culture flasks were transferred into UniVessel SU 2L containers (Sartorius Stedim) that had previously been equilibrated with 800 mL of Rooster Nourish medium for approximately 3 hours (37°C, 5% CO ₂ (aeration from above), 120 rpm). The two suspension cell culture flasks were washed together with Rooster Nourish medium, and the solution was added to the 2L container, ultimately filling it to 1L with medium (microcarrier concentration of 14 g/1L per 2L container). This was set in a Biostat B (Sartorius Stedim) and the "5 minutes of stirring, 1435 minutes of static" setting was repeated for three days. On the third day, 120 minutes after the start of static culture, the culture was switched to stirring only, and culture was continued for six days. The stirring speed was 120 rpm. On the second day, 60 minutes after the start of static culture, 14 g of microcarriers were added (microcarrier concentration 28 g/1 L per 2 L container). Immediately after adding the 14 g of microcarriers, the microcarriers were mixed by stirring at 120 rpm for approximately 2 minutes. Medium changes were performed on the fourth and seventh days of culture. The medium was left to stand for approximately 10 minutes, the carriers and cells were allowed to settle, and 700 mL of culture supernatant was removed. Then, 700 mL of Rooster Nourish medium was added, and stirring was resumed.

(4-2) Evaluation of the positive rate of microcarriers On the fourth day of culture, 10 mL of the culture medium containing cells and microcarriers was sampled from the sample port of a 2 L container using a syringe. Approximately 100 μL of this was taken, and 400 μL of DPBS was added. One drop of Nucblue (ThermoFisher Scientific) was then added, and the mixture was incubated at 37°C for 15 to 30 minutes. Thereafter, evaluation was carried out in the same manner as in Example 1.
The positive rate was 80% when cultured under one set of conditions: "5 minutes of stirring, 1435 minutes of standing."

(4-3) Evaluation of the number of recovered viable cells After 7 days of culture, 10 mL of culture medium containing cells and microcarriers was collected from the sample port of a 2 L container using a syringe. A 100 μm cell strainer was placed on a 50 mL centrifuge tube, and the entire volume of the sampling solution was filtered. The cells and microcarriers remaining on the cell strainer were washed once with DPBS, and then the cells and microcarriers on the cell strainer were collected into a new 50 mL centrifuge tube 1 using TrypLE Select Enzyme (Gibco). The 50 mL centrifuge tube 1 was placed on a half-turn rotator and shaken at 37 °C for 30 minutes. The 50 mL centrifuge tube 1 was collected, and the entire contents of the 50 mL centrifuge tube 1 were filtered into a new 50 mL centrifuge tube (2) equipped with a 100 μm cell strainer. The 50 mL centrifuge tube 1 and the carrier on the cell strainer were washed together with Rooster Nourish medium. The 50 mL centrifuge tube (2) was centrifuged, the supernatant was removed, and Rooster Nourish medium was added to suspend the cells. The suspension was filled into a dedicated cassette, and the cell number was counted using a NucleoCounter NC-202 (MSESS Techno Systems). The cells were frozen using CTS ^™ Synth-a-Freeze ^™ Medium (Gibco) as needed.
When cultured under one set of conditions of "agitation for 5 minutes and standing for 1435 minutes," the number of viable cells recovered was 2.01 x 10 ⁶ cells.

On the 9th day of culture, the 2L container was collected. The 2L container was left to stand for approximately 10 minutes to allow the carriers and cells to settle, after which the culture supernatant was removed and the cells + microcarriers were washed once with DPBS. The carriers and cells were allowed to settle, the DPBS was removed, a detachment agent was added, the cells were detached, and the cell number was counted (cell count 2).

When the above cell number 1 and cell number 2 were cultured under one set of conditions of "agitation for 5 minutes, standing for 1435 minutes", the number of viable cells recovered was 5.65 x 10 ⁸ cells.

(4-4) Overall Evaluation The overall evaluation was carried out in the same manner as in Example 1.
The overall evaluation when cultured under one set of conditions of "5 minutes of stirring and 1435 minutes of standing" was "A."

Comparative Example 2: Production under the condition of "stirring only, no standing" (1) Obtaining synovial stem cells, (2) preparing microcarriers, and (3) seeding synovial mesenchymal stem cells were carried out in the same manner as in Example 10.

(4) Cultivation and Evaluation (4-1) Cultivation The suspension cell culture flasks seeded in (3) were placed in an incubator and cultured for approximately 24 hours. Subsequently, two suspensions of cells and carriers cultured in the suspension cell culture flasks were transferred into UniVessel SU 2L containers (Sartorius Stedim) that had previously been equilibrated with 800 mL of Rooster Nourish medium for approximately 3 hours (37°C, 5% CO ₂ (aeration from above), 120 rpm). The two suspension cell culture flasks were washed together with Rooster Nourish medium, and the solution was added to the 2L container, ultimately filling it to 1L with medium (microcarrier concentration of 14 g/1L per 2L container). This was placed in a Biostat B (Sartorius Stedim) and cultured for 9 days under "agitation only, no standing" conditions, i.e., without static agitation culture. The agitation speed was 120 rpm. 14 g of microcarriers were added during the second day of culture (microcarrier concentration 28 g/1 L per 2 L container). The 14 g of microcarriers was added by stopping agitation and moving the 2 L container into a safety cabinet. After the microcarrier addition, the container was immediately placed in the Biostat B and agitation was resumed. The medium was replaced on the fourth and seventh days of culture. The container was left to stand for approximately 10 minutes, the carriers and cells were allowed to settle, and 700 mL of culture supernatant was removed. Then, 700 mL of Rooster Nourish medium was added, and agitation was resumed.

(4-2) Evaluation of Positive Rate of Microcarriers Evaluation was carried out in the same manner as in Example 10.
The positive rate when cultured with "agitation only, no standing" was 24%.

(4-3) Evaluation of the number of recovered viable cells Evaluation was carried out in the same manner as in Example 10.
The number of viable cells recovered when the culture was performed with "stirring only, without standing" was 1.61 x 10 ⁶ cells.

As in Example 10, on the 9th day of culture, the 2 L container was harvested.
When the cells 1 and 2 were cultured together under "stirring only, no standing", the number of viable cells recovered was 4.21 x 10 ⁸ cells.

(4-4) Overall Evaluation Evaluation was carried out in the same manner as in Example 1.
The overall evaluation when the culture was performed with "stirring only, without standing" was "E".

The results of Examples 1 to 10 and Comparative Examples 1 and 2 described above are summarized in the table below.

<Evaluation of cartilage differentiation>
The frozen cells from Examples 4 and 6 were thawed in a water bath and transferred to a centrifuge tube containing a culture medium (non-differentiation medium) prepared by mixing αMEM with 20% FBS (ThemoFisher Scientific) and 1% Antibiotic-Antimycotic (x100) (ThemoFisher Scientific). After centrifugation, the cells were suspended in non-differentiation medium and mixed with AO/PI Cell Viability Kit at a ratio of 9:1. The cell number was counted using LUNA. The suspension was dispensed into two 1.5 mL centrifuge tubes at 2.5 x ¹⁰ cells each and centrifuged. After removing the supernatant, one 1.5 mL centrifuge tube was filled with 1 mL of chondrogenic differentiation medium (DMEM high glucose (ThemoFisher Scientific), 10 ng/mL TGF-β3 (Fujifilm Wako Pure Chemical Industries), 3.92 μg/mL Dexamethasone (Fujifilm Wako Pure Chemical Industries), 50 μg/mL L-Asorbic Acid 2-Phosphate (Cayman Chemical), 40 μg/mL L-Proline (MP Biomedicals), 1 mmol/L Sodium Pyruvate (Invitrogen), 100-fold diluted ITS-X supplement (x100) (Fujifilm Wako Pure Chemical Industries), 500 ng/mL To one tube, 1 mL of a BMP-2 (R&D Systems) mixed solution was added, and 1 mL of non-differentiation medium was added to the other. After centrifugation to settle the cells, culture was initiated. Culture medium was replaced on days 2 and 7 of culture, and evaluation was performed on day 14. Each spheroid was removed from the 1.5 mL centrifuge tube using a microspatula and transferred to a 1.5 mL tube containing DPBS. The spheroid was removed, placed on a dish, and weighed using an analytical balance (A&D). A microscope microruler (Kennis) was placed near the spheroid, and images were taken with a compact digital camera (OLYMPUS). The major axis was measured from the captured images.

The results of measuring weight and major axis are shown in the table below. In both Example 4 (95 minutes of stirring, 25 minutes of standing) and Example 6 (1415 minutes of stirring, 25 minutes of standing), the differentiated specimens were larger in both weight and major axis than the undifferentiated specimens, and had differentiated into a cartilage-like structure.

The above results show that static agitation culture increases the positive rate and the number of recovered viable cells compared to the "agitation only, no standing" condition, which does not involve static agitation culture. It was also shown that static agitation culture allows the cultured cells to differentiate into cartilage.

Claims (20)

A method for producing mesenchymal stem cells, comprising culturing mesenchymal stem cells in the presence of microcarriers, wherein an agitation static culture step, which includes a step of agitating the mesenchymal stem cells and a subsequent step of allowing the mesenchymal stem cells to stand, is carried out for 48 hours or more, the agitation static culture step is carried out two or more times during the 48-hour or more culture period, and the agitation static culture step is carried out one or more times every 48 hours starting 24 hours after the start of culture. The method for producing mesenchymal stem cells according to claim 1, wherein each of the two or more stirring and static culture steps is the same step, or the two or more stirring and static culture steps include stirring and static culture steps in which one or more of the stirring step duration and the static step duration differ. The method for producing mesenchymal stem cells according to claim 1 or 2, wherein the stirring and static culture step is carried out from the start of culture to the end of culture. The method for producing mesenchymal stem cells according to claim 1 or 2, wherein the mesenchymal stem cells are derived from humans. The method for producing mesenchymal stem cells according to claim 4, wherein the mesenchymal stem cells are derived from induced pluripotent stem cells, bone marrow, fat, dental pulp, umbilical cord, placenta, or synovium. The method for producing mesenchymal stem cells according to claim 5, wherein the mesenchymal stem cells are derived from synovial membrane. The method for producing mesenchymal stem cells according to claim 1 or 2, wherein the time for leaving the mesenchymal stem cells stationary in the stirring and static culture step is 1 minute or more and 2,400 minutes or less. The method for producing mesenchymal stem cells according to claim 1 or 2, wherein the time for allowing the mesenchymal stem cells to stand in the stirring and static culture step is 0.1 minutes/mL or more and 240.0 minutes/mL or less per volume of culture medium. The method for producing mesenchymal stem cells according to claim 1 or 2, wherein the time for the step of agitating the mesenchymal stem cells in the agitated static culture step is from 1 minute to 2000 minutes. The method for producing mesenchymal stem cells according to claim 1 or 2, wherein the time for stirring the mesenchymal stem cells in the stirring static culture step is 0.1 min/mL or more and 200.0 min/mL or less per volume of culture medium. The method for producing mesenchymal stem cells according to claim 1 or 2, wherein the stirring and static culture step is performed 1 to 48 times per 24 hours. The method for producing mesenchymal stem cells according to claim 1 or 2, wherein the duration of each stirring and static culture step is 30 to 1,440 minutes. The method for producing mesenchymal stem cells according to claim 1 or 2, wherein the ratio of the time spent stirring the mesenchymal stem cells to the time spent leaving the mesenchymal stem cells standing in the stirring and static culture step is within the range of 1:0.005 to 1:700. The method for producing mesenchymal stem cells according to claim 1 or 2, wherein the total time of the stirring steps during the entire culture period is longer than the total time of the standing steps. The method for producing mesenchymal stem cells according to claim 1 or 2, wherein no microcarriers are added during the stirring and static culture step. The method for producing mesenchymal stem cells according to claim 1 or 2, wherein agitated static culture is carried out for 48 hours or more 24 hours or more after the start of culture, and then agitated culture is carried out continuously thereafter. The method for producing mesenchymal stem cells according to claim 1 or 2, wherein the agitated static culture step is carried out 24 hours or more after the start of culture, and the agitated culture is continued for at least two days before the end of culture. The method for producing mesenchymal stem cells according to claim 1 or 2, wherein the time ratio between the agitation static culture step and the continuous agitation step is 1:0.3 to 1:5.5. The method for producing mesenchymal stem cells according to claim 1 or 2, wherein the time ratios for step (A) of leaving the mesenchymal stem cells to step (B) of stirring and leaving the mesenchymal stem cells to step (C) of continuously stirring the mesenchymal stem cells are A = 1, B = 2 to 6, and C = 2 to 11. The method of claim 1 or 2, wherein mesenchymal stem cells are produced for use as a therapeutic agent.