JP7822593B2 - Stem cell culture method and stem cell culture medium - Google Patents

Description

The present invention relates to a method for culturing stem cells and a culture medium for stem cells.

Stem cells, including somatic stem cells such as mesenchymal stem cells (MSCs), embryonic stem cells (ES cells), and induced pluripotent stem cells (iPS cells), are promising cell sources for regenerative medicine technology. For example, mesenchymal stem cells are not only pluripotent, but also possess anti-inflammatory, angiogenic, and cell proliferation-promoting properties in an undifferentiated state, making them used as a regenerative medicine source for the treatment of various diseases around the world. However, because stem cells gradually transform into a more mature phenotype with repeated passages and lose their cellular characteristics, the number of passages that can actually be used for treatment is limited. Therefore, there is a need for a technology that allows for the efficient cultivation of stem cells while maintaining their undifferentiated state.

It is known that compositions containing nanobubbles of hydrogen, oxygen, and nitrogen have the effect of promoting cell proliferation (see, for example, Patent Document 1). Furthermore, signaling molecules such as nitric oxide, hydrogen sulfide, and carbon monoxide are known to exhibit various functions in stem cells, such as self-renewal and differentiation induction (see, for example, Non-Patent Document 1).

JP 2016-63804 A

ACS Biomater. Sci. Eng. 2020, 6, 798-812.

The present invention aims to provide a method for culturing stem cells that can promote stem cell proliferation.

The first aspect is a method for culturing stem cells, which comprises culturing stem cells in a culture medium containing bubbles containing a reducing gas.

The stem cell culture method may be a culture method that promotes stem cell proliferation. The reducing gas may contain at least one gas selected from the group consisting of hydrogen, carbon monoxide, nitric oxide, and hydrogen sulfide, and the bubbles containing the reducing gas may have an average particle size of 10 nm or more and 1000 nm or less. Furthermore, the stem cells to be cultured may contain at least one gas selected from the group consisting of somatic stem cells, embryonic stem cells, and iPS cells.

A second aspect is a method for inhibiting stem cell differentiation, which involves culturing stem cells in a culture medium containing bubbles containing a reducing gas.

The method for inhibiting stem cell differentiation may be a method for promoting stem cell proliferation. The reducing gas may contain at least one gas selected from the group consisting of hydrogen, carbon monoxide, nitric oxide, and hydrogen sulfide, and the bubbles containing the reducing gas may have an average particle size of 10 nm or more and 1000 nm or less. Furthermore, the stem cells to be cultured may contain at least one gas selected from the group consisting of somatic stem cells, embryonic stem cells, and iPS cells.

A third aspect is a stem cell culture medium containing bubbles containing a reducing gas.

The reducing gas may contain at least one gas selected from the group consisting of hydrogen, carbon monoxide, nitric oxide, and hydrogen sulfide, and the bubbles containing the reducing gas may have an average particle size of 10 nm or more and 1000 nm or less.

The present invention provides a method for culturing stem cells that can promote stem cell proliferation.

1 is a graph showing the cumulative cell number per passage in the culture of adipose tissue-derived mesenchymal stem cells (ASCs). Flow cytometry results for ASCs at passage 3. 1 shows the results of flow cytometry for ASCs at passage 9 in Example 2. 1 shows the results of flow cytometry for ASCs at passage 9 in Comparative Example 1. 1 is a graph showing the number of mesenchymal stem cells in Examples and Comparative Examples.

As used herein, the term "process" refers not only to an independent process, but also to processes that cannot be clearly distinguished from other processes, as long as the intended purpose of the process is achieved. Furthermore, when the composition contains multiple substances corresponding to each component, the content of each component in the composition refers to the total amount of those multiple substances present in the composition, unless otherwise specified. Furthermore, the upper and lower limits of the numerical ranges described herein can be arbitrarily selected and combined. Below, embodiments of the present invention are described in detail. However, the embodiments described below are intended to exemplify stem cell culture methods and stem cell culture media that embody the technical concepts of the present invention, and the present invention is not limited to the stem cell culture methods and stem cell culture media described below.

Stem Cell Culturing Method The stem cell culturing method includes a culturing step of culturing stem cells in a culture medium containing bubbles containing a reducing gas (hereinafter simply referred to as a "bubble-containing culture medium"). Stem cell proliferation can be promoted by culturing stem cells in vitro in a cell culture medium containing bubbles containing a reducing gas. Furthermore, differentiation of stem cells can be suppressed, and stem cell proliferation can be promoted while maintaining the undifferentiated state. In other words, the stem cell culturing method may be a method of promoting stem cell proliferation.

Here, "promoting stem cell proliferation" means that the survival and proliferation of stem cells cultured in a culture medium containing bubbles containing a reducing gas are promoted compared to the survival and proliferation of stem cells cultured in a culture medium that does not contain bubbles containing a reducing gas. Specifically, this means that the number of stem cells after a predetermined period of culture (e.g., 9 passages) in a culture medium containing bubbles containing a reducing gas increases by, for example, 5% or more, preferably 10% or more, 20% or more, 40% or more, 60% or more, compared to the number of stem cells cultured in a culture medium that does not contain bubbles containing a reducing gas.

In the method for culturing stem cells, differentiation of stem cells can be inhibited by culturing the stem cells in a bubble-containing culture medium. That is, the method for culturing stem cells may be a method for inhibiting stem cell differentiation. Inhibition of stem cell differentiation can be determined, for example, by detecting the expression of stem cell surface markers. Examples of surface markers that are positive on stem cells include CD105, Sca-1, and CD29 in mouse mesenchymal stem cells. Examples of surface markers that are negative include CD45.

Here, "stem cell differentiation is inhibited" means that, for a cell surface marker that is positive in stem cells, the expression rate of the surface marker in stem cells cultured in bubble-containing culture medium is higher than the expression rate of the surface marker in stem cells cultured in bubble-free culture medium. Specifically, this means that the expression rate of the surface marker in stem cells after culture for a predetermined period of time (e.g., 6 passages) in bubble-containing culture medium is, for example, 5% or more, preferably 8% or more higher than the expression rate of the surface marker in stem cells cultured in bubble-free culture medium.

Stem cells to which culture methods are applicable refer to immature cells that have the ability to self-replicate 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 are stem cells that have the ability to differentiate into all tissues and cells that make up a living organism. Multipotent stem cells are stem cells that have the ability to differentiate into multiple types of tissues and cells, but not all types. Unipotent stem cells are stem cells that have the ability to differentiate into specific tissues and cells.

The species from which stem cells are derived is not particularly limited, and examples include 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. Stem cells may be derived from either humans or non-human mammals.

Specific examples of stem cells include mesenchymal stem cells that differentiate into myoblasts, vascular endothelial cells, osteoblasts, adipocytes, muscle cells, cardiomyocytes, and chondrocytes; neural stem cells that differentiate into neurons and glial cells; hematopoietic stem cells or bone marrow stem cells that differentiate into white blood cells, red blood cells, platelets, mast cells, and dendritic cells; embryonic stem cells (ES cells), which are known to proceed from a spheroid state through the formation of pseudo-embryos called embryoid bodies (EB bodies) and then differentiate and induce into various tissues; induced pluripotent stem cells (iPS cells); embryonic germ (EG) cells derived from primordial germ cells; multipotent germline stem (mGS) cells isolated during the establishment and culture process of GS cells from testicular tissue; and multipotent adult progenitor cells (MAPCs) isolated from bone marrow.

Pluripotent stem cells include, in particular, ES cells and iPS cells. Stem cells established by culturing early embryos produced by nuclear transfer of the nucleus of a somatic cell are also preferred as pluripotent stem cells.

Human ES cell lines, such as WA01 (H1) and WA09 (H9), are available from the WiCell Research Institute, and KhES-1, KhES-2, and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University (Kyoto, Japan).

Examples of iPS cells include cells that have acquired ES cell-like pluripotency and are obtained by introducing multiple genes (reprogramming factors) into somatic cells such as skin cells. Examples include iPS cells obtained by introducing the Oct3/4 gene, Klf4 gene, C-Myc gene, and Sox2 gene, and iPS cells obtained by introducing the Oct3/4 gene, Klf4 gene, and Sox2 gene.

Multipotent stem cells include, in particular, somatic stem cells such as mesenchymal stem cells, hematopoietic stem cells, neural stem cells, bone marrow stem cells, and germline stem cells. Mesenchymal stem cells are preferred as multipotent stem 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, and adipblasts. More specific examples of mesenchymal stem cells include human bone marrow-derived mesenchymal stem cells (e.g., hMSC-BM; Takara), human umbilical cord matrix-derived mesenchymal stem cells (e.g., hMSC-UC; Takara), and human adipose tissue-derived mesenchymal stem cells (e.g., hMSC-AT; Takara).

Stem cells are cultured in a culture medium containing bubbles containing a reducing gas. The culture medium containing the bubbles is not particularly limited as long as it is a cell culture medium that allows stem cells to be cultured. Examples of cell culture medium for stem cells include RPMI-1640 medium, Eagle's MEM medium, Dulbecco's modified MEM medium, Glasgow's MEM medium, α-MEM medium, 199 medium, IMDM medium, DMEM medium, Hybridoma Serum-free medium, Chemically Defined Hybridoma Serum-free medium (Invitrogen), MEMα/GlutaMax (Gibco), Ham's Medium F-12, Ham's Medium F-10, and Ham's Medium. F12K, ATCC-CRCM30, DM-160, DM-201, BME, Fischer, McCoy's 5A, Leibovitz's L-15, RITC80-7, MCDB105, MCDB107, MCDB131, MCDB153, MCDB201, NCTC109, NCTC135, Waymouth's MB752/1, CMRL-1066, Williams' medium E, Brinster's BMOC-3 Medium, Essential8 Medium (all Thermo Fisher Scientific), mTeSR1 (Stem Cell Technologies), TeSR-E8 Examples of suitable medium include Stem Cell Technologies, StemSure (Fujifilm Wako Pure Chemical Industries, Ltd.), mESF medium (Fujifilm Wako Pure Chemical Industries, Ltd.), StemFit (Ajinomoto Co., Inc.), S-medium (DS Pharma Co., Ltd.), ReproXF (ReproCell, Inc.), PSGro-free Human iPSC/ESC Growth Medium (StemRD), hPSC Growth Medium (Takara Bio Inc.), ReproFF2 (ReproCell, Inc.), EX-CELL 302 medium (SAFC), EX-CELL-CD-CHO (SAFC), STEMdiff APEL Medium (Stem Cell Technologies, Inc.), and mixtures thereof. The culture medium may further contain additives such as serum, serum substitutes, plasma, serum albumin, proteins, growth factors, cytokines, hormones, amino acids, vitamins, and antibiotics, as needed.

The gas constituting the bubbles contained in the culture medium may be, for example, a reducing gas, from the perspective of promoting stem cell proliferation. Examples of reducing gases include hydrogen, carbon monoxide, nitric oxide, hydrogen sulfide, and sulfur dioxide. The gas preferably contains at least one selected from the group consisting of these, and more preferably contains at least one selected from the group consisting of hydrogen, carbon monoxide, nitric oxide, and hydrogen sulfide. The gas constituting the bubbles may contain nitrogen, oxygen, carbon dioxide, etc., or may contain low-molecular-weight hydrocarbons with 5 or fewer carbon atoms, such as methane, ethane, propane, butane, pentane, cyclopropane, cyclobutane, ethylene, propylene, propadiene, butene, acetylene, and propyne. The gas constituting the bubbles may contain only one type of gas, or a combination of two or more types. The content of the reducing gas in the gas constituting the bubbles may be, for example, 60% by volume or more, preferably 80% by volume or more, 90% by volume or more, or 95% by volume or more, and may be substantially solely a reducing gas. Here, "substantially" means that other gases besides the reducing gas that are inevitably mixed in are not excluded, and specifically means that the content of other gases is less than 5% by volume, or less than 1% by volume.

The bubbles contained in the culture solution may be ultrafine bubbles or nanobubbles, and the average particle size may be, for example, 10 nm to 1000 nm, preferably 50 nm to 500 nm, or 100 nm to 300 nm. The average particle size of the bubbles may be, for example, 10 nm to 100 nm, preferably 10 nm to 50 nm. The content (number) of bubbles contained in the culture solution may be, for example, 1 x ¹⁰ particles/ml or more, preferably 1 x ¹⁰ particles/ml or more, 5 x ¹⁰ particles/ml or more, or 1 x ¹⁰ particles/ml or more. The upper limit of the number of bubbles contained in the culture solution may be, for example, 1 x ¹⁰ particles/ml or less, preferably 1 x ¹⁰ particles/ml or less.

The average particle size of the bubbles can be measured, for example, by laser diffraction/scattering, nanoparticle tracking analysis, electrical resistance, AFM (Atomic Force Microscope), or laser microscope observation. An example of a measurement device using the laser diffraction/scattering method is a flow cytometer (product name: CytoFlex) manufactured by Beckman Coulter. An example of a measurement device using the nanoparticle tracking analysis method is a nanoparticle analysis system (product name: Nonosight) manufactured by Malvern. Furthermore, an example of a device for measuring AFM is a resonant particle measurement system (product name: Archimedes) manufactured by Malvern.

Technologies for generating bubbles in the culture solution include, for example, the swirling liquid flow method, static mixer method, Venturi method, pressurized dissolution method, and pore method. Bubbles can also be generated in the culture solution by injecting the culture solution into a production vessel, sealing in the desired gas, and vibrating the production vessel.

Specifically, bubbles can be generated in the culture solution as follows. The method for generating bubbles in the culture solution may include preparing a production vessel, pouring the culture solution into the production vessel to a predetermined height, sealing the production vessel with the desired gas filled in the production vessel, and vibrating the production vessel at a predetermined rotation speed.

A production vessel is prepared, which includes a container body with an opening and containing a culture medium, and a lid capable of sealing the container body. The container body may, for example, have a cylindrical outer shape with a bottom. Specifically, for example, a vial with a capacity of approximately 0.5 ml to 20 ml can be used. The vial may have, for example, a longitudinal length X of approximately 35 mm to 60 mm and an outer diameter of approximately 10 mm to 40 mm. The lid may include a disk-shaped rubber stopper (septum) that fits tightly against the opening of the container body, and a fastening portion that secures the rubber stopper to the container body. The rubber stopper may, for example, be a silicone rubber stopper. The fastening portion is configured to cover the edge of the rubber stopper, and may have an opening approximately in the center when viewed from above.

Culture medium is poured into the container body to a predetermined height. When the container body into which culture medium has been poured is placed horizontally, the height (longitudinal length) of the container body is X [mm], and the height of the culture medium surface in the container body is Y [mm]. The relationship 0.2≦Y/X≦0.7 can be satisfied. In this state, a sufficiently large void exists above the culture medium contained in the container body. By vibrating the production container in this state, the culture medium can be caused to collide with the top, bottom, and sides of the production container with greater force. This collision generates shock waves in the culture medium, making it easy to form bubbles in the culture medium.

The container body into which the culture medium has been injected is sealed while filled with the desired gas. Specifically, the void in the container body into which the culture medium has been injected is purged with the desired gas, and then the lid is tightened onto the opening of the container body. This seals the culture medium and the desired gas inside the production container. One method for purging the void in the container body with the desired gas is to move the container body into which the culture medium has been injected into a chamber, replace the air in the chamber with the desired gas, and then tighten the lid onto the opening of the container body. The sealed production container may be pressurized with the desired gas. The sealed production container can be pressurized by adding the desired gas into the sealed production container using a syringe or the like.

Bubbles are generated in the culture solution by vibrating a production vessel in which the culture solution and desired gas are sealed. The vibration of the production vessel may be, for example, a reciprocating motion in the approximate longitudinal direction of the production vessel. This causes the culture solution to move up and down within the production vessel, repeatedly colliding with the top, bottom, and side surfaces of the production vessel. When the culture solution collides with the inner surface of the production vessel, shock waves are generated within the culture solution, and the resulting pressure causes the gas to finely disperse in the culture solution, forming bubbles. The vibration frequency may be, for example, 5,000 rpm or higher, and preferably 6,000 rpm to 20,000 rpm, or 6,000 rpm to 7,000 rpm.

The vibration amplitude in the longitudinal direction of the production vessel may be, for example, approximately 0.7X [mm] to 1.5X [mm], and preferably approximately 0.8X [mm] to 1X [mm]. The time for vibrating the production vessel may be, for example, approximately 10 seconds to 120 seconds, and preferably approximately 30 seconds to 90 seconds. By setting the vibration time within this range, the number of times the culture medium collides with the production vessel is sufficiently high, allowing a large number of bubbles to be generated in the culture medium. Note that by setting the vibration time longer within the above range, the amount of bubbles generated in the culture medium can be increased. The vibration of the production vessel may be carried out in divided vibration periods. For example, the production vessel may be vibrated by repeating vibration for 5 seconds to 30 seconds approximately 3 to 10 times.

As a device capable of vibrating the production vessel, for example, a bead-type high-speed cell disruption system (homogenizer) can be used. Specific examples include Precellys® manufactured by Bertin Technologies. For details on methods for generating bubbles in the culture medium, please refer to the specification of International Publication No. 2016/163439, for example.

The method for culturing stem cells includes a culturing step in which stem cells are cultured in a bubble-containing culture medium. In the culturing step, stem cells are cultured using a bubble-containing culture medium by batch culture, fed-batch culture, continuous culture, perfusion culture, or the like. Batch culture is a culture method in which the bubble-containing culture medium is not replenished to the culture vessel during culture. Fed-batch culture is a culture method in which the bubble-containing culture medium is replenished to the culture vessel during culture. Continuous culture is a culture method in which a portion of the bubble-containing culture medium is withdrawn from the culture vessel during culture and an amount of bubble-containing culture medium equivalent to the amount withdrawn is replenished. Perfusion culture is a culture method in which only the liquid is withdrawn from the culture vessel during culture and an amount of bubble-containing culture medium equivalent to the amount withdrawn is replenished.

The culture vessel used in the culture step is not particularly limited. Examples of culture vessels include flasks, dishes, petri dishes, microwell plates, microslides, chamber slides, tubes, trays, culture bags, and culture vessels such as tanks. Examples of substrates for these culture vessels include glass, various plastics such as polypropylene and polystyrene, metals such as stainless steel, and combinations thereof.

The culture process may be either adherent culture or suspension culture. For adherent culture, a culture vessel is used in which a scaffold is formed on the substrate surface by coating the surface of the substrate with a scaffold material such as collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, partial structures of laminin, fibronectin, or a mixture thereof (e.g., Matrigel). This allows for the induction of adhesion of anchorage-dependent cells to the substrate surface. For adherent culture of stem cells, the cells may be cultured in the presence of feeder cells. Stromal cells such as fetal fibroblasts can be used as feeder cells.

Suspension culture of stem cells refers to culturing stem cells under non-adhesive conditions in a culture medium without the use of scaffold-formed culture vessels, feeder cells, etc. For suspension culture, high molecular weight polymers such as methylcellulose, MFG-E8 (Milk fat globule-EGF factor 8), or fragments of this protein can be used. Examples of suspension culture of stem cells include dissociated culture of stem cells and aggregated suspension culture of stem cells. Dissociated culture of stem cells refers to culturing suspended stem cells, and examples include dissociated culture using small cell clumps consisting of 2 to 20 stem cells. If the dissociated culture is continued, the cultured dispersed cells form larger stem cell clumps, after which aggregated suspension culture can be performed. Examples of aggregated suspension culture include embryoid body culture and sphere culture, in which cell lines are passaged by mechanical processing using a mesh filter.

The stem cells to be cultured may be dispersed or non-dispersed cells. Dispersed cells are cells that have been treated to promote cell dispersion. Dispersed cells include, for example, cells that form small cell clumps consisting of 2 to 50, 2 to 20, or 2 to 10 cells. Dispersed cells may be floating (suspended) cells or adherent cells.

The culture density of stem cells may be any density that can achieve the effect of promoting cell survival and proliferation, and may be, for example, from 1.0× ¹⁰ cells/ml to 1.0× ¹⁰ cells/ml, preferably from 1.0× ¹⁰ cells/ml to 1.0×10 cells/ml, from 1.0× ¹⁰ cells/ml to 1.0× ¹⁰ cells/ml, from 1.0× ¹⁰ cells/ml to 1.0× ¹⁰ cells/ml, or from 3.0×10 cells/ml to 1.0× ¹⁰ cells/ml.

Culture conditions such as temperature, dissolved _CO2 concentration, dissolved oxygen concentration, and pH can be appropriately set based on techniques conventionally used for culturing cells derived from animal tissues. For example, the culture temperature may be, for example, 33°C to 39°C, or 36°C to 37°C. The dissolved _CO2 concentration may be, for example, 1% to 10%, or 2% to 5%. The oxygen partial pressure may be, for example, 10% to 22%.

1. Method for producing a cell composition containing stem cells The method for culturing stem cells can be applied to a method for producing a cell composition containing stem cells. The method for producing a cell composition containing stem cells includes a culture step of culturing stem cells in a culture medium containing bubbles containing a reducing gas, and may include a passaging step of passaging the stem cells as needed.

The cell composition can be used for further culturing of stem cells or as a cell source for regenerative medicine. The cell composition may also be a composition containing dispersed stem cells, such as small cell clumps. The cell composition may be used, for example, for preserving, transporting, and subculturing stem cells by cryopreservation. When used for preservation, such as cryopreservation, the cell composition may further contain serum or a substitute therefor, or an organic solvent such as DMSO. The cell composition may also contain feeder cells, etc.

Stem Cell Culture Medium The stem cell culture medium is a cell culture medium containing bubbles containing a reducing gas. The bubbles contained in the stem cell culture medium may be ultrafine bubbles or nanobubbles. Culturing stem cells in the stem cell culture medium can promote the proliferation of the cultured stem cells. Furthermore, differentiation of the stem cells can be suppressed, and proliferation of the stem cells can be promoted while maintaining the undifferentiated state.

Stem cell culture medium can be prepared by generating bubbles containing a reducing gas in a commonly used cell culture medium. Details of the gas that makes up the bubbles and the method for generating the bubbles have been described above. The cell culture medium in which bubbles are generated may be a cell culture medium that is suitable for culturing stem cells, and specific examples of cell culture medium have been described above.

The gas that constitutes the bubbles contained in the stem cell culture medium may be, for example, a reducing gas, from the perspective of promoting stem cell proliferation. The reducing gas may include, for example, at least one gas selected from the group consisting of hydrogen, carbon monoxide, nitric oxide, and hydrogen sulfide. The gas that constitutes the bubbles may further include nitrogen, oxygen, carbon dioxide, etc.

The average particle size of the bubbles contained in the stem cell culture medium may be, for example, 10 nm to 1000 nm, preferably 50 nm to 500 nm, or 100 nm to 300 nm. The average particle size of the bubbles may be, for example, 10 nm to 100 nm, preferably 10 nm to 50 nm. The content (number) of bubbles contained in the cell culture medium may be, for example, 1 x ¹⁰ particles/ml or more, preferably 1 x ¹⁰ particles/ml or more, 5 x ¹⁰ particles/ml or more, or 1 x 10 particles ^/ ml or more. The upper limit of the number of bubbles contained in the culture medium may be, for example, 1 x ¹⁰ particles/ml or less, preferably 1 x ¹⁰ particles/ml or less.

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

Reference Example: Preparation of Adipose Tissue-Derived Mesenchymal Stem Cells Mouse adipose tissue was collected subcutaneously from both limbs of an ICR mouse (female, 5 weeks old). 10 ml of 0.2% collagenase solution (GIBCO 17100-017) was added to 1 to 2 g of collected adipose tissue, and the adipose tissue was then immersed and shredded with scissors. 20 ml of 0.2% collagenase solution was added to the shredded adipose tissue and shaken at 120 rpm at 37°C for 1 hour. The tissue was then filtered through a cell strainer (FALCON, REF 352360), and the filtrate was centrifuged at 400 G for 5 minutes. The supernatant was removed, and the resulting pellet was suspended in 10 ml of phosphate-buffered saline (PBS) and centrifuged at 400 G for 5 minutes, repeating this process three times to obtain a cell pellet. The resulting cell pellet was designated as mouse ADRCs. Mouse ADRCs were cultured in MEMα/GlutaMax (Gibco) medium at 37°C and 5% CO for ₅ days, and adipose tissue-derived mesenchymal stem cells (ASCs) were obtained after three passages. The medium was changed every 3 days.

Example 1
MEMα/GlutaMAX was prepared as a stem cell culture medium. 1 ml of MEMα/GlutaMAX was placed in a 2 ml vial, purged with carbon monoxide, and then the vial was sealed with a cap. Next, 1 ml of carbon monoxide was added to the vial using a syringe. The sealed vial was subjected to six cycles of 10-second vibration at 6,500 rpm using ^{a Precellys®} (high-speed cell disruption system) manufactured by Bertin Technologies, to obtain a culture medium containing carbon monoxide bubbles. A 20-second rest period was provided between each cycle to prevent temperature rise.

The bubble size distribution of the bubbles contained in the bubble-containing culture medium was measured using a nanoparticle analysis system (NanoSight; manufactured by Malvern). The average bubble size was 164.2 nm, and the bubble content was 5.12 × 10 ⁹ bubbles/ml.

Example 2
Using the culture medium containing carbon monoxide bubbles obtained in Example 1, 5 x ¹⁰⁵ ASCs were seeded in a 100 mm dish, and the medium was replaced and passaged every two days. The number of cells was counted at each passage, and the cumulative cell number was calculated. Culture was started with ASCs from the third passage and continued for six passages up to the ninth passage.

The cumulative cell number at each passage is shown as NB in Figure 1. The cumulative cell number of ASCs at passage 9 was 2.19 x 10 ^cells . Significance testing was performed using the Mann-Whitney U test.

Comparative Example 1
ASCs were cultured in the same manner as in Example 2, except that a MEM/GlutaMAX culture medium (containing 20% FBS) not containing carbon monoxide bubbles was used as the culture medium.

The cumulative number of cells at each passage is shown as a control in Figure 1. The cumulative number of ASCs at passage 9 was 1.17 x 10 ⁷ cells.

Evaluation The ASCs at the 3rd and 9th passages used in Example 2 and Comparative Example 1 above were stained with fluorescent dyes for surface markers of mouse mesenchymal stem cells (MSCs) (positive: CD105, Sca-1, CD29; negative: CD45), and then subjected to flow cytometry to calculate the proportion of MSCs contained in each ASC.

The results are shown in Figures 2A to 2C. Figure 2A shows the results of flow cytometry of ASCs at passage 3, at the start of culture. Figure 2B shows the results of flow cytometry of ASCs at passage 9 in Example 2. Figure 2C shows the results of flow cytometry of ASCs at passage 9 in Comparative Example 1. The MSC content of the ASCs at passage 3, at the start of culture, was 50.7%. The MSC content of the control group at passage 9 in Comparative Example 1 decreased to 31.7%, whereas the MSC content in Example 2, when MEM/GlutaMAX medium containing nanobubbles carrying carbon monoxide was used, remained at 40.2%.

Next, the number of ASCs was multiplied by the MSC content rate to compare the absolute number of MSCs. The results are shown in Figure 3. The number of MSCs among the ASCs at passage 3 (P3) was 2.5 x ^10. The number of MSCs among the ASCs at passage 9 was 3.6 x ¹⁰ in the control group (Control-P3) of Comparative Example 1 and 8.8 x ¹⁰ in the nanobubble group (NB-P3) of Example 2.

These results demonstrate that the use of a culture medium containing bubbles containing a reducing gas in the culture of ASCs can promote the proliferation of stem cells and suppress differentiation from MSCs.

Claims (7)

Culturing stem cells in a culture medium containing bubbles containing a reducing gas,
the bubbles have an average particle size of 100 nm or more and 300 nm or less;
the content of the reducing gas in the gas constituting the bubbles is 60% by volume or more;
The method for culturing stem cells, wherein the reducing gas contains carbon monoxide . The culture method of claim 1, which promotes stem cell proliferation. 3. The culture method according to claim 1, wherein the content of the reducing gas in the gas constituting the bubbles is 90% by volume or more. The culture method according to any one of claims 1 to 3 , wherein the stem cells comprise at least one type selected from the group consisting of somatic stem cells, embryonic stem cells, and iPS cells. Culturing stem cells in a culture medium containing bubbles containing a reducing gas,
the bubbles have an average particle size of 100 nm or more and 300 nm or less;
the content of the reducing gas in the gas constituting the bubbles is 60% by volume or more;
The method for inhibiting stem cell differentiation, wherein the reducing gas comprises carbon monoxide . bubbles containing a reducing gas;
the bubbles have an average particle size of 100 nm or more and 300 nm or less;
the content of the reducing gas in the gas constituting the bubbles is 60% by volume or more;
The stem cell culture medium contains carbon monoxide as the reducing gas . 7. The stem cell culture medium according to claim 6 , wherein the content of the reducing gas in the gas constituting the bubbles is 90% by volume or more.