JP2024027573A - Method for selecting cells with sphere-forming ability and migration ability - Google Patents

Abstract

[Problem] To provide a new cell selection method that is simple, inexpensive, and highly accurate in cell selection. [Solution] A method for selecting cells with sphere-forming ability and migratory ability, including the following steps: (1) A cell population containing cells with sphere-forming ability and migratory ability is divided into chitin nanofibers carrying vitronectin and chitosan. A step of three-dimensionally culturing the cells in a liquid medium containing nanofibers to form spheres, (2) a step of sedimenting the spheres formed in (1), (3) a step of collecting the sedimented spheres, (4) a step of two-dimensionally culturing the collected spheres; and (5) a step of collecting cells that migrated from the spheres in the two-dimensional culture. [Selection diagram] None

Description

The present invention relates to a method for selecting cells with sphere-forming ability and migratory ability, and the use of cells obtained by the screening method.

In the field of regenerative medicine, the construction of organ regeneration means using pluripotent stem cells such as iPS cells and ES cells is being intensively studied. However, establishing ES cells involves ethical issues, and iPS cells are recognized to have problems such as the risk of canceration and the length of the culture period. Therefore, mesenchymal stem cells (MSCs) and progenitor cells are widely used as cell sources for treatment and research purposes in regenerative medicine.

For example, mesenchymal stem cells are somatic stem cells that have been shown to exist in the bone marrow, and have been identified as cells that adhere to plastic dishes and form three-dimensional spheres. Furthermore, as stem cells, they have the ability to differentiate into bone, cartilage, and fat, and are therefore attracting attention as a promising cell source for cell therapy. Recently, it has been known that cells with equivalent functions exist in mesenchymal stem cells of fetal appendages such as adipose tissue, bone marrow, dental pulp, placenta, umbilical cord, and ovarian membrane. It has also been reported that cells with similar functions exist in tissues such as the liver. Mesenchymal stem cells have attracted attention for their various functions in addition to their differentiation potential, and bone marrow-derived mesenchymal stem cells have been used to treat acute graft-versus-host disease (GVHD) in hematopoietic stem cell transplantation and inflammatory bowel disease. Trials are underway for Crohn's disease, acute myocardial infarction, liver fibrosis, ischemic disease, cerebral infarction, heart failure, and lung disease caused by the new coronavirus infection.

In recent years, efforts have been made to develop methods for recovering mesenchymal stem cells from cell populations containing mesenchymal stem cells from living tissues. For example, methods for recovering mesenchymal stem cells from dental pulp are reported in Non-Patent Document 1, Non-Patent Document 2, and the like. Furthermore, a method for collecting mesenchymal stem cells from adipose tissue is reported in Patent Document 1. The method described in Patent Document 1 proposes a method of recovering mesenchymal stem cells from adipocytes by utilizing the characteristics of mesenchymal stem cells as adhesive cells.

U.S. Patent No. 6,777,231

S. Gronthos et al., Proc Natl Acad Sci U S A. 2000 Dec 5;97(25):13625-30. M. Miura., Proc Natl Acad Sci U S A. 2003 May 13;100(10):5807-12.

As mentioned above, multiple methods have been reported for selecting and recovering mesenchymal cells from living tissues. These methods are broadly classified into methods that are relatively simple and inexpensive but have low cell selection accuracy, and methods that have high cell selection accuracy but involve complicated steps and are expensive. Therefore, under such circumstances, there is a need for the construction of a simple, inexpensive, and highly accurate cell sorting method.

Regarding selection of mesenchymal stem cells, when mesenchymal stem cells are two-dimensionally cultured (hereinafter sometimes referred to as "2D culture"), the size of the cells increases, and cells with reduced proliferation and function (so-called It has been reported that these cells transform into senescent cells (senescent cells). Young mesenchymal stem cells with high proliferation and function and aged mesenchymal stem cells with low proliferation and function have the same cell surface antigen, so it is difficult to select them with high precision even using antibodies etc. . Against this background, there is also a strong demand for the construction of a method for efficiently selecting high-quality mesenchymal stem cells.

From this background, the present invention aims to provide a new cell selection method that is simple, inexpensive, and highly accurate in cell selection. Furthermore, in the selection of mesenchymal stem cells, it is an object of the present invention to provide a selection method that can select high-quality mesenchymal stem cells simply and inexpensively.

As a result of intensive study on the above-mentioned problem, the present inventors found that
(1) Chitin nanofibers and chitosan nanofibers carrying vitronectin are mixed in a liquid medium at a specific ratio, and a cell population collected from biological tissue is cultured in three dimensions (hereinafter referred to as "3D culture") in the liquid medium. (or "suspension culture," etc.), cells with sphere-forming ability in the cell population proliferate while efficiently forming spheres, while cells without sphere-forming ability grow. not multiply;
(2) Spheres formed in the liquid medium can be easily separated and recovered based solely on the difference in sedimentation speed, without special operations such as centrifugation or FACS;
(3) By further subjecting the collected spheres to 2D culture and collecting cells that migrated and proliferated from the spheres, cells and nanofibers can be easily separated, and both sphere-forming ability and migration ability are obtained. be able to collect cells comprising;
(4) Mesenchymal stem cells prepared from living tissue using the method of the present invention have enhanced gene expression levels of HOXB7, bFGF, NANOG, OCT4, and SOX2 and are of very good quality; I found it. The present invention was completed by further research based on this knowledge. That is, the present invention is as follows.

[1]
A method for selecting cells with sphere-forming ability and migration ability, including the following steps:
(1) A step of three-dimensionally culturing a cell population containing cells having sphere-forming ability and migratory ability in a liquid medium containing chitin nanofibers and chitosan nanofibers to form spheres of the cells;
(2) a step of settling the spheres formed in (1);
(3) a step of recovering the settled spheres;
(4) a step of two-dimensionally culturing the collected spheres; and (5) a step of collecting cells that migrated from the spheres in the two-dimensional culture.
[2]
The method according to [1], wherein the chitin nanofibers carry vitronectin.
[3]
The method according to [1] or [2], wherein the liquid medium in (1) is a dedicated medium for cells having sphere-forming ability and migration ability.
[4]
The method according to any one of [1] to [3], wherein the cell population is derived from a living tissue.
[5]
The cells having sphere-forming ability and migration ability are mesenchymal stem cells, fibroblasts, neural stem cells, neural progenitor cells, dermal papilla cells, or cancer stem cells, according to any one of [1] to [4]. Method.
[6]
The method according to any one of [1] to [4], wherein the cells having sphere-forming ability and migration ability are mesenchymal stem cells.
[7]
[6] A living body transplantation agent comprising mesenchymal stem cells selected by the method described in item [6].
[8]
Method for producing mesenchymal stem cells, including the following steps:
(1) A step of three-dimensionally culturing a cell population containing mesenchymal stem cells in a liquid medium containing chitin nanofibers carrying vitronectin and chitosan nanofibers to form spheres of the mesenchymal stem cells;
(2) a step of settling the spheres formed in (1);
(3) a step of recovering the settled spheres;
(4) a step of two-dimensionally culturing the collected spheres; and (5) a step of collecting mesenchymal stem cells that migrated from the spheres in the two-dimensional culture.

According to the present invention, cells having sphere-forming ability and migratory ability contained in a cell population can be separated and sorted without performing centrifugation or separation operations using cell surface antigens. Furthermore, according to the present invention, young, high-quality mesenchymal stem cells that maintain undifferentiated properties can be efficiently prepared.

FIG. 1 is a photograph showing whether mesenchymal stem cells, bronchial epithelial cells, and renal epithelial cells form spheres in a liquid medium containing a nanofiber substrate in Test Examples 1 and 2. FIG. 2 is a diagram showing an overview of the method of the present invention. Figure 3 shows the results of mesenchymal stem cells when mesenchymal stem cells selected from a cell group derived from the SVF fraction derived from human adipose tissue were cultured in 2D using the method of the present invention in Test Example 3. It is a figure showing the state of migration and proliferation. Figure 4 shows the results of mesenchymal stem cells when mesenchymal stem cells selected from a cell group derived from a floating fraction derived from human adipose tissue were cultured in 2D using the method of the present invention in Test Example 3. It is a figure showing the state of migration and proliferation. FIG. 5 is a diagram illustrating an outline of an embodiment in which the cell sorting process through sphere formation by 3D culture and migration by 2D culture is repeated, which will be examined in Test Example 4. FIG. 6 is a diagram showing that mesenchymal stem cells selected by the method of the present invention perform a second sphere formation (FIG. 6 left) and a second migration and proliferation (FIG. 6 right) in Test Example 4. It is. FIG. 7 is a diagram showing that mouse bone marrow-derived mesenchymal stem cells form spheres in 3D culture according to the method of the present invention, and migrate and proliferate in 2D culture in Test Example 5.

The present invention will be explained in detail below.

1. Method for selecting cells with sphere-forming ability and migration ability The present invention provides a method for selecting cells with sphere-forming ability and migration ability (hereinafter sometimes referred to as "the method of the present invention"), which includes the following steps. provide:
(1) A step of three-dimensionally culturing a cell population containing cells having sphere-forming ability and migratory ability in a liquid medium containing chitin nanofibers and chitosan nanofibers to form spheres of the cells;
(2) a step of settling the spheres formed in (1);
(3) a step of recovering the settled spheres;
(4) a step of two-dimensionally culturing the collected spheres; and (5) a step of collecting cells that migrated from the spheres in the two-dimensional culture.

In the method of the present invention, cells with sphere-forming ability and migration ability are selected from a cell population containing cells with sphere-forming ability and migration ability. In the present invention, the cell population may or may not be derived from living tissue. When the cell population is derived from a living tissue, the living body may be derived from any living organism, but is preferably derived from a mammalian living tissue, and more preferably from a human living tissue. In one embodiment of the method of the present invention, when selecting mesenchymal stem cells from human adipose tissue, the cell population may be a cell population derived from human adipose tissue, more preferably a mesenchymal stem cell derived from human adipose tissue. It may be a stromal vascular fraction (SVF). SVF is obtained by removing adipocytes from human subcutaneous adipose tissue by enzymatic treatment, and contains peripheral blood-derived cell groups such as macrophages and neutrophils, vascular endothelial cells, and adipose-derived mesenchymal cells. A cell population consisting of cells such as stem cells.

As used herein, the term "sphere" (also referred to as spheroid) of cells refers to a state in which cells are not in a single cell state but in a state in which they aggregate to form a three-dimensional cell mass. As used herein, a sphere can be an aggregate of cells or an aggregate of cells comprising a mixture of chitin nanofibers (which may or may not be loaded with vitronectin) and chitosan nanofibers. . Whether or not cells have the ability to form spheres can be confirmed using a method known per se.

As used herein, "migration" of cells refers to spontaneous movement of cells. Whether or not a cell has migration ability can be confirmed using a method known per se (eg, cell migration assay, etc.).

In the present invention, "cells having sphere-forming ability and migratory ability" are not particularly limited as long as they have both sphere-forming ability and migratory ability, but include, for example, mesenchymal stem cells, fibroblasts, neural stem cells, These include neural progenitor cells, dermal papilla cells, cancer stem cells, and the like. In a preferred embodiment of the present invention, "cells with sphere-forming ability and migration ability" are mesenchymal stem cells.

In the present invention, mesenchymal stem cells can be mesenchymal stem cells derived from bone marrow, umbilical cord, umbilical cord blood, placenta, adipose, dental pulp, or synovium. Preferably, the mesenchymal stem cells are adipose-derived.

(1st step)
In the first step of the method of the present invention, a cell population containing cells with sphere-forming ability and migration ability is three-dimensionally cultured in a liquid medium containing chitin nanofibers and chitosan nanofibers.

As used herein, "three-dimensional (3D) culture" refers to a method of culturing cells with increased vertical thickness, compared to two-dimensional (2D) culture, in which cells are cultured in a monolayer on a culture plate. say. 3D culture can also be referred to as "suspension culture." The first step of the method of the present invention is characterized in that 3D culture is performed using a mixture of chitin nanofibers and chitosan nanofibers as a scaffold.

In this specification, nanofibers refer to fibers having an average fiber diameter (D) of 0.001 to 1.00 μm. The average fiber diameter of the nanofibers used in the present invention is preferably 0.005 to 0.50 μm, more preferably 0.01 to 0.05 μm, and still more preferably 0.01 to 0.02 μm.

In the method of the present invention, the aspect ratio (L/D) of the nanofibers used is obtained from the average fiber length/average fiber diameter, and is not particularly limited, but is usually 2 to 500, preferably 5 to 300. , more preferably 10 to 250.

In this specification, the average fiber diameter (D) of nanofibers is determined as follows. First, a collodion support membrane manufactured by Ohken Shoji Co., Ltd. was subjected to hydrophilic treatment for 3 minutes using an ion cleaner (JIC-410) manufactured by JEOL Ltd., and several nanofiber dispersions to be evaluated (diluted with ultrapure water) were prepared. Add dropwise and dry at room temperature. This was observed using a transmission electron microscope (TEM, H-8000) manufactured by Hitachi, Ltd. (10,000 times) at an accelerating voltage of 200 kV, and using the obtained image, the number of specimens: 200 to 250 The fiber diameter of each nanofiber is measured, and the number average value is defined as the average fiber diameter (D).

Moreover, the average fiber length (L) is determined as follows. The nanofiber dispersion to be evaluated is diluted with pure water to a concentration of 100 ppm, and the nanofibers are uniformly dispersed using an ultrasonic cleaner. This nanofiber dispersion liquid is cast onto a silicon wafer whose surface has been previously treated to make it hydrophilic using concentrated sulfuric acid, and dried at 110° C. for 1 hour to prepare a sample. Using an image of the obtained sample observed with a scanning electron microscope (SEM, JSM-7400F) (2,000x) manufactured by JEOL Ltd., each nanofiber was examined one by one for the number of specimens: 150 to 250. The fiber length of the book is measured, and the number average value is defined as the average fiber length (L).

In a preferred embodiment, when the nanofibers are mixed with a liquid medium, the nanofibers are uniformly dispersed in the liquid while maintaining the primary fiber diameter, and adhere to the nanofibers without substantially increasing the viscosity of the liquid. It has the effect of substantially retaining cells and preventing their sedimentation.

The nanofibers used in the method of the present invention are chitin nanofibers and chitosan nanofibers. Chitin nanofibers are nanofibers made of chitin, and chitosan nanofibers are nanofibers made of chitosan.

The main sugar units constituting chitin and chitosan are N-acetylglucosamine and glucosamine, respectively. Generally, chitin and glucosamine have a high content of N-acetylglucosamine and are poorly soluble in acidic aqueous solutions. Chitosan has a high content and is soluble in acidic aqueous solutions. In this specification, for convenience, a substance in which the proportion of N-acetylglucosamine in the constituent sugars is 50% or more is called chitin, and a substance in which the proportion of N-acetylglucosamine is less than 50% is called chitosan.

Many biological resources can be used as raw materials for chitin, such as shrimp, crabs, insects, shellfish, and mushrooms. The chitin used in the present invention may be chitin having an α-type crystal structure, such as chitin derived from crab shell or shrimp shell, or chitin having a β-type crystal structure, such as chitin derived from the shell of a squid. Good too. Crab and shrimp shells are often treated as industrial waste and are preferred as raw materials because they are easy to obtain and can be used effectively. A process is required. Therefore, in the present invention, it is preferable to use purified chitin that has already been subjected to dematrix treatment. Purified chitin is commercially available. As a raw material for chitin nanofibers used in the present invention, chitin having either an α-type or a β-type crystal structure may be used, but α-type chitin is preferable.

Chitin nanofibers and chitosan nanofibers can be obtained by crushing the chitin and chitosan described above. The pulverization method is not limited, but in order to refine the fibers to a fiber diameter and fiber length that meet the purpose of the present invention, methods that can obtain strong shearing force, such as a high-pressure homogenizer, a grinder (stone mill), or a media stirring mill such as a bead mill, are used. is preferred.

Among these, it is preferable to micronize using a high-pressure homogenizer, and it is desirable to micronize (pulverize) using a wet pulverization method as disclosed in JP-A No. 2005-270891 and Japanese Patent No. 5232976, for example. . Specifically, the material is pulverized by injecting a dispersion liquid in which the material is dispersed at high pressure from a pair of nozzles and colliding with each other. This can be carried out by using a high-pressure crusher) or Nano Veita (high-pressure crusher manufactured by Yoshida Kikai Kogyo Co., Ltd.).

When refining (pulverizing) raw materials using the high-pressure homogenizer mentioned above, the degree of refining and homogenization depends on the pressure fed to the ultra-high pressure chamber of the high-pressure homogenizer and the number of times the raw material is passed through the ultra-high pressure chamber (number of treatments). , and the concentration of the raw materials in the aqueous dispersion. The pumping pressure (processing pressure) is not particularly limited, but is usually 50 to 250 MPa, preferably 100 to 200 MPa.

Further, the concentration of the raw material in the aqueous dispersion during the micronization treatment is not particularly limited, but is usually 0.1% by mass to 30% by mass, preferably 1% by mass to 10% by mass. The number of times of micronization (pulverization) is not particularly limited and depends on the concentration of the raw material in the aqueous dispersion, but when the concentration of the raw material is 0.1 to 1% by mass, the number of treatments is 10 to 1%. About 100 times is enough to obtain fine particles, but if the amount is 1 to 10% by mass, about 10 to 1000 times may be necessary.

The viscosity of the aqueous dispersion during the micronization treatment is not particularly limited, but for example, in the case of α-chitin, the viscosity of the aqueous dispersion ranges from 1 to 100 mPa·S, preferably from 1 to 85 mPa·S (at 25°C Tuning fork vibration viscosity measurement (SV-1A, A&D Company Ltd.) under these conditions. In the case of chitosan, the viscosity range of the aqueous dispersion is 0.7 to 30 mPa・S, preferably 0.7 to 10 mPa・S (tuning fork vibration viscosity measurement under 25°C condition (SV-1A, A&D Company Ltd.).

The method for preparing nanofibers is described in WO2015/111686 and the like.

In one embodiment of the method of the present invention, it is preferable that chitin nanofibers support vitronectin, which is one of the extracellular matrices. As used herein, chitin nanofibers "carry" vitronectin means a state where chitin nanofibers and vitronectin are attached or adsorbed without a chemical covalent bond. Supporting of vitronectin by chitin nanofibers can be achieved by intermolecular force, electrostatic interaction, hydrogen bonding, hydrophobic interaction, etc., but is not limited to these. In addition, the state in which chitin nanofibers support vitronectin refers to a state in which chitin nanofibers and vitronectin remain in contact without a chemical covalent bond, or a state in which chitin nanofibers and vitronectin remain in contact with each other without a chemical covalent bond. This can be described as a state in which a complex is formed without any chemical covalent bond. Without wishing to be bound by theory, supporting vitronectin on chitin nanofibers may promote sphere formation. Therefore, according to this embodiment, even cells with reduced sphere-forming ability (for example, cells derived from a donor in poor health) can efficiently form spheres. Also, without wishing to be bound by theory, extracellular matrices other than vitronectin, which have a biological function similar to that of vitronectin, can also be used in the methods of the invention. Such extracellular matrices include collagen (collagen I to XIX), fibronectin, laminin (laminin-1 to 12), RGD sequence, cadherin, proteoglycan, elastin, fibrin, hyaluronic acid, selectin, etc. Not limited.

In the method of the present invention, vitronectin supported on chitin nanofibers is not particularly limited as long as the desired effect is obtained. The origin of vitronectin is not particularly limited. Vitronectin may be full-length or may be a functional fragment. As the vitronectin used in the method of the present invention, generally commercially available human-derived vitronectin fragments can be used. As a functional fragment of human-derived vitronectin, for example, vitronectin fragments such as 62-478 (SEQ ID NO: 1), 20-398 (SEQ ID NO: 2), or 20-478 (SEQ ID NO: 3) can be suitably used. . In one embodiment of the method of the invention, vitronectin of SEQ ID NO: 1 or 2 is more preferred.

In the method of the present invention, the amount of vitronectin supported by chitin nanofibers is usually 0.001 to 50 mg, preferably 0.01 to 10 mg, more preferably 0.1 to 10 mg, per 1 g of chitin nanofibers. The amount is more preferably 0.3 to 10 mg, even more preferably 1 to 10 mg, particularly preferably 2 to 10 mg, but is not limited thereto.

In the method of the present invention, chitin nanofibers carrying vitronectin are prepared by mixing a dispersion of chitin nanofibers in an aqueous solvent and an aqueous solution of vitronectin, and allowing the mixture to stand for a certain period of time if necessary. be able to. Examples of aqueous solvents in which chitin nanofibers are dispersed include, but are not limited to, water, dimethyl sulfoxide (DMSO), and the like. Water is preferred as the aqueous solvent. The aqueous solvent may contain an appropriate buffer or salt. In order to uniformly contact vitronectin with the chitin nanofibers, it is preferable to mix thoroughly by pipetting or the like. In addition, the time for leaving the mixture of the chitin nanofiber dispersion and vitronectin aqueous solution to stand is usually at least 30 minutes, preferably at least 1 hour, more preferably at least 3 hours, even more preferably at least 6 hours, and more. More preferably, it can be allowed to stand for 9 hours or more, particularly preferably for 12 hours or more. Although there is no particular upper limit to the standing time, for example, the upper limit may be set to 48 hours or less (eg, 36 hours or less, 24 hours or less, or 16 hours or less). The temperature during standing is not particularly limited, but is usually 1 to 30°C, preferably 1 to 15°C, more preferably 2 to 10°C, particularly preferably 2 to 5°C (e.g., 4°C). Can be done.

The mixing ratio of chitin nanofibers and vitronectin varies depending on the type of these substances used, but is, for example, 100:0.1 to 1, preferably 100:0.4 to 0.6 in terms of solid weight. However, it is not limited to these.

The amount of vitronectin supported on chitin nanofibers can be measured, for example, by Micro BCA method, enzyme-linked immunosorbent assay (ELISA method), etc., but is not limited thereto.

In the first step of the method of the present invention, chitosan nanofibers are further blended into the liquid medium in addition to the chitin nanofibers carrying vitronectin. By culturing a cell population in suspension using a liquid medium containing vitronectin-supported chitin nanofibers and chitosan nanofibers, it is possible to promote sphere formation of cells with sphere-forming ability.

In one embodiment, the concentration of vitronectin-loaded chitin nanofibers and chitosan nanofibers in the liquid medium used in the first step of the method of the invention satisfies the following conditions:
(1) The concentration of total nanofibers (chitin nanofibers and chitosan nanofibers carrying vitronectin) contained in the liquid medium is 0.0001 to 1.0% (w/v), and The weight ratio of chitin nanofibers carrying vitronectin contained in the medium: chitosan nanofibers is 1:0.5 to 20 (preferably 1:0.5 to 10, 1:0.7 to 9, 1: 1-8, 1:2-7, or 1:3-6);
(2) The concentration of total nanofibers (chitin nanofibers and chitosan nanofibers carrying vitronectin) contained in the liquid medium is 0.001 to 0.5% (w/v), and The weight ratio of chitin nanofibers carrying vitronectin contained in the medium: chitosan nanofibers is 1:0.5 to 20 (preferably 1:0.5 to 10, 1:0.7 to 9, 1: 1-8, 1:2-7, or 1:3-6);
(3) The concentration of total nanofibers (chitin nanofibers and chitosan nanofibers carrying vitronectin) contained in the liquid medium is 0.005 to 0.3% (w/v), and The weight ratio of chitin nanofibers carrying vitronectin contained in the medium: chitosan nanofibers is 1:0.5 to 20 (preferably 1:0.5 to 10, 1:0.7 to 9, 1: 1-8, 1:2-7, or 1:3-6);
or
(4) The concentration of total nanofibers (chitin nanofibers and chitosan nanofibers carrying vitronectin) contained in the liquid medium is 0.01 to 0.1% (w/v), and The weight ratio of chitin nanofibers carrying vitronectin contained in the medium: chitosan nanofibers is 1:0.5 to 20 (preferably 1:0.5 to 10, 1:0.7 to 9, 1: 1-8, 1:2-7, or 1:3-6).

The type of liquid medium to which chitin nanofibers and chitosan nanofibers are added in the first step of the method of the present invention can be appropriately selected depending on the type of cells to be sorted. For this purpose, media commonly used for culturing mammalian cells can be used. Examples of media for mammalian cells include Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM/F12 medium, and McCoy's 5A medium. s 5A medium), Eagle's Minimum Essential Medium; EMEM, αMEM medium (alpha Modified Eagle's Minimum Essential Medium; αMEM), MEM medium ( Minimum Essential Medium), RPMI1640 medium, Iscove's modified Dulbecco medium ( Iscove's Modified Dulbecco's Medium; IMDM), MCDB131 medium, William medium E, IPL41 medium, Fischer's medium, StemPro34 (Invitrogen), X-VIVO 10 (Cambrex), X-VIVO 15 ( HPGM (manufactured by Cambrex), StemSpan H3000 (manufactured by Stem Cell Technology), StemSpanSFEM (manufactured by Stem Cell Technology), Stemline II (manufactured by Sigma-Aldrich), QBSF-60 (manufactured by Quality Biological), Examples include StemProhESCSFM (manufactured by Invitrogen), mTeSR1 or 2 medium (manufactured by Stem Cell Technology), Sf-900II (manufactured by Invitrogen), Opti-Pro (manufactured by Invitrogen), and the like.

Those skilled in the art may freely add sodium, potassium, calcium, magnesium, phosphorus, chlorine, various amino acids, various vitamins, antibiotics, serum, fatty acids, sugar, etc. to the above medium according to the purpose. When culturing mammalian cells, those skilled in the art can also add one or more combinations of other chemical components or biological components depending on the purpose. Components that can be added to media for mammalian cells include fetal bovine serum, human serum, horse serum, insulin, transferrin, lactoferrin, cholesterol, ethanolamine, sodium selenite, monothioglycerol, 2-mercaptoethanol, bovine serum. Examples include albumin, sodium pyruvate, polyethylene glycol, various vitamins, various amino acids, agar, agarose, collagen, methylcellulose, various cytokines, various hormones, various growth factors, various extracellular matrices, and various cell adhesion molecules. Examples of cytokines that can be added to the medium include interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), Interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), Interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-14 (IL-14), Interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), interferon-α (IFN-α), interferon-β (IFN-β), interferon- γ (IFN-γ), granulocyte colony stimulating factor (G-CSF), monocyte colony stimulating factor (M-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), stem cell factor (SCF), flk2/ Examples include, but are not limited to, flt3 ligand (FL), leukemia cell inhibitory factor (LIF), oncostatin M (OM), erythropoietin (EPO), thrombopoietin (TPO), and the like.

Hormones that may be added to the medium include melatonin, serotonin, thyroxine, triiodothyronine, epinephrine, norepinephrine, dopamine, anti-Mullerian hormone, adiponectin, adrenocorticotropic hormone, angiotensinogen and angiotensin, antidiuretic hormone, atrial Natriuretic peptide, calcitonin, cholecystokinin, corticotropin-releasing hormone, erythropoietin, follicle-stimulating hormone, gastrin, ghrelin, glucagon, gonadotropin-releasing hormone, growth hormone-releasing hormone, human chorionic gonadotropin, human placental lactogen, growth hormone, inhibin , insulin, insulin-like growth factor, leptin, luteinizing hormone, melanocyte-stimulating hormone, oxytocin, parathyroid hormone, prolactin, secretin, somatostatin, thrombopoietin, thyroid-stimulating hormone, thyrotropin-releasing hormone, cortisol, aldosterone, testosterone, dehydro Epiandrosterone, androstenedione, dihydrotestosterone, estradiol, estrone, estriol, progesterone, calcitriol, calcidiol, prostaglandins, leukotrienes, prostacyclin, thromboxane, prolactin-releasing hormone, lipotropin, brain natriuretic peptide, nerve These include, but are not limited to, peptide Y, histamine, endothelin, pancreatic polypeptide, renin, and enkephalin.

Growth factors that can be added to the medium include transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), macrophage inflammatory protein-1α (MIP-1α), epidermal growth factor ( EGF), fibroblast growth factor-1, 2, 3, 4, 5, 6, 7, 8, or 9 (FGF-1, 2, 3, 4, 5, 6, 7, 8, 9), nerve Cell growth factor (NGF), hepatocyte growth factor (HGF), leukemia inhibitory factor (LIF), protease nexin I, protease nexin II, platelet-derived growth factor (PDGF), cholinergic differentiation factor (CDF), chemokine , Notch ligand (such as Delta1), Wnt protein, angiopoietin-like protein 2, 3, 5 or 7 (Angpt2, 3, 5, 7), insulin-like growth factor (IGF), insulin-like growth factor binding protein (IGFBP), play Examples include, but are not limited to, otrophin (Pleiotrophin) and the like.

Furthermore, it is also possible to add those cytokines and growth factors whose amino acid sequences have been artificially modified by gene recombination technology. Examples include IL-6/soluble IL-6 receptor complex or Hyper IL-6 (a fusion protein of IL-6 and soluble IL-6 receptor).

Examples of antibiotics that may be added to the medium include sulfa preparations, penicillin, pheneticillin, methicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, nafcillin, ampicillin, penicillin, amoxicillin, cyclacillin, carbenicillin, ticarcillin, piperacillin, azlocillin, Mecdulocilin, mecillinam, andinocillin, cephalosporins and their derivatives, oxolinic acid, amifloxacin, temafloxacin, nalidixic acid, pyromidic acid, ciprofloxan, cinoxacin, norfloxacin, perfloxacin, rosaxacin, ofloxacin, enoxacin, pipemidic acid, sulbactam, clavuric acid , β-bromopenicillanic acid, β-chloropenicillanic acid, 6-acetylmethylene-penicillanic acid, cefoxazole, sultanepicillin, adinocillin and formaldehyde foodrate ester of sulbactam, tazobactam, aztreonam, sulfazetin, isosulfazetin, nocardicin , methyl phenylacetamidophosphonate, chlortetracycline, oxytetracycline, tetracycline, demeclocycline, doxycycline, methacycline, and minocycline.

In a preferred embodiment of the present invention, the liquid medium used in the first step is a medium suitable for maintaining and proliferating cells with sphere-forming ability and migration ability to be selected, i.e., ``sphere-forming ability to be selected''. and a dedicated medium for cells with migratory ability. Dedicated media for many cells are well known, and commercially available media may be used. Specifically, for example, if the cells to be selected are mesenchymal stem cells, a "mesenchymal stem cell growth medium" can be used as a dedicated medium, and a commercially available medium may be used. .

As used herein, floating cells (or spheres) refers to a state in which cells (or spheres) do not adhere to the culture container (non-adhesion), regardless of whether the cells are sedimented or not. . Furthermore, in the present specification, when culturing cells, the cells are dispersed in the liquid medium without applying external pressure or vibration to the liquid medium, or without shaking or rotating the liquid medium, and The state in which cells are in suspension is referred to as "stationary suspension," and culturing cells and/or tissues in this state is referred to as "stationary suspension culture." In "floating and standing", the period during which floating can be carried out is at least 5 minutes or more, preferably 1 hour or more, 24 hours or more, 48 hours or more, 6 days or more, 21 days or more, but the floating state is maintained. but are not limited to these periods.

In a preferred embodiment, the liquid medium used in the first step of the method of the present invention allows cells to remain suspended at at least one point in the temperature range where cells can be cultured (e.g., 0 to 40°C). . The liquid medium allows cells to remain suspended, preferably at at least one point in the temperature range of 25 to 37°C, most preferably at 37°C.

By culturing cells in a state attached to a mixture of chitin nanofibers (which may or may not carry vitronectin) and chitosan nanofibers, cells can be cultured in suspension. The use of chitin nanofibers (which may or may not carry vitronectin) and chitosan nanofibers allows cells to be suspended in a liquid medium and promotes sphere formation. can. Chitin nanofibers (which may or may not carry vitronectin) and chitosan nanofibers are dispersed in a liquid medium without dissolving or adhering to the culture vessel, so When cells with sphere-forming ability are cultured in a medium set, the cells adhere to nanofibers and float in the liquid medium, promoting sphere formation.

When a cell population containing cells with sphere-forming ability and migration ability is cultured in suspension (3D culture) using chitin nanofibers (which may or may not carry vitronectin) and chitosan nanofibers. , cells containing separately prepared cells with sphere-forming ability and migration ability in a liquid medium containing chitin nanofibers (which may or may not support vitronectin) and chitosan nanofibers. The mass may be added and mixed uniformly. The mixing method at this time is not particularly limited, and examples thereof include manual mixing such as pipetting, mixing using equipment such as a stirrer, vortex mixer, microplate mixer, and shaker. After mixing, the obtained cell suspension may be cultured in a stationary state, or may be cultured while being rotated, shaken, or stirred as necessary. The number of rotations and frequency may be appropriately set according to the purpose of those skilled in the art. For example, a cell population is collected from a biological tissue, dispersed to a single cell or a state close to this using an appropriate cell dissociation solution, suspended in a liquid medium, and cultured in suspension. (preferably floating stationary culture).

In the first step, the culture temperature when cells are cultured in suspension is usually 25 to 39°C, preferably 33 to 39°C (eg, 37°C). The CO ₂ concentration in the culture atmosphere is usually 4 to 10% by volume, preferably 4 to 6% by volume. The culture period may be appropriately set according to the purpose of culture.

In the first step of the method of the present invention, the 3D culture of cells is carried out using flasks, plastic bags, Teflon bags, dishes, Petri dishes, tissue culture dishes, multi-dishes, etc. commonly used for cell culture. It can be carried out using culture containers such as microplates, microwell plates, multiplates, multiwell plates, chamber slides, tubes, trays, culture bags, and roller bottles. These culture vessels must have low cell adhesion so that cells attached to chitin nanofibers (which may or may not carry vitronectin) and chitosan nanofibers do not adhere to the culture vessels. is desirable. Culture vessels with low cell adhesion are those whose surfaces have not been artificially treated (e.g., coated with extracellular matrix, etc.) for the purpose of improving adhesion with cells, or those whose surfaces are not artificially treated to improve adhesion with cells. It is possible to use a material whose surface has been artificially treated to reduce adhesion with cells.

In the first step of the present invention, the period of 3D culture is not particularly limited as long as sphere formation is achieved, but it is usually 1 day or more, preferably 2 days or more, 3 days or more, 4 days or more, 5 days or more. It can be at least 1 day, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days. The upper limit of the culture period is not particularly limited, but is usually 30 days or less, preferably 29 days or less, 28 days or less, 27 days or less, 26 days or less, 25 days or less, 24 days or less, 23 days or less, 22 days or less. The period may be 21 days or less, 20 days or less, 19 days or less, 18 days or less, or 17 days or less. In one embodiment, the culture period is 1 to 30 days, 2 to 29 days, 3 to 28 days, 4 to 27 days, 5 to 26 days, 6 to 25 days, 7 to 24 days, May be from 8th to 23rd, from 9th to 22nd, from 10th to 21st, from 11th to 20th, from 12th to 19th, from 13th to 18th, from 14th to 17th, but is not limited to these days. .

If it takes a relatively long time (for example, 2 days or more) to form spheres, the medium may be replaced as necessary. The method of medium exchange is not particularly limited, but for example, after centrifuging the culture container to sediment the spheres and chitin nanofibers/chitosan nanofibers to the bottom of the container, remove the old liquid medium in the supernatant portion using a pipette. This can be done by adding fresh liquid medium to the container. The frequency of medium exchange and the amount of medium exchange during one medium exchange may be set as appropriate.

(Second process)
The second step of the present invention is characterized in that the spheres obtained in the first step are allowed to settle on the bottom of the culture container. Compared to chitin nanofibers/chitosan nanofibers, which exist in a free state without single cells or spheres attached, spheres have a faster sedimentation rate. By utilizing this difference in sedimentation speed, spheres can be easily separated from single cells and free chitin nanofibers/chitosan nanofibers by simply leaving the sample still.

The step of settling the spheres is preferably carried out by adding a large amount of liquid such as a buffer to the liquid medium containing the spheres so that the difference in sedimentation speed becomes significant. The liquid that can be added in the second step is not particularly limited as long as it does not adversely affect the survival of the cells, and a fresh liquid medium or a buffer such as PBS can be used. The amount of liquid to be added is not particularly limited as long as only the spheres settle and chitin nanofibers/chitosan nanofibers existing in a single cell or free state are suspended. For example, the amount may be usually 1.5 times or more, preferably 2 times or more, 3 times or more, 4 times or more, or 5 times or more the amount of the liquid medium in the first step. Further, although the conditions are not particularly limited, it may be usually 10 times or less, preferably 9 times or less, 8 times or less, 7 times or less, or 6 times or less.

The time period for which the sample is allowed to stand in order to allow the spheres to settle is not particularly limited as long as only the spheres settle and single cells and chitin nanofibers/chitosan nanofibers are suspended. The time the sample is allowed to stand may be, for example, usually 1 minute or more, preferably 2 minutes or more, 3 minutes or more, 4 minutes or more, or 5 minutes or more. The upper limit of the standing time is not particularly limited, but may be usually 1 hour or less, preferably 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, or 10 minutes or less.

The temperature conditions under which the sample is allowed to stand in order to precipitate the spheres are also not particularly limited as long as they do not adversely affect the survival of the cells. The temperature conditions for allowing the sample to stand still are, for example, usually 25°C or higher, 30°C or higher, 35°C or higher, or 37°C or higher. Further, the upper limit of the temperature condition is not particularly limited, but is usually 42°C or lower, preferably 41°C or lower, 40°C or lower, 39°C or lower, or 38°C or lower.

In the second step of the method of the present invention, the differences in sedimentation velocity are used to separate the spheres from other components. In other words, separation of spheres from chitin nanofibers/chitosan nanofibers existing in a single cell or free state involves separation operations such as centrifugation or separation operations using cell surface antigens (e.g., FACS). do not have. That is, the second step of the method of the present invention is characterized in that the spheres and other components are separated only by a standing separation operation that utilizes the difference in sedimentation speed.

(3rd step)
In the third step of the method of the invention, the spheres precipitated in the second step are recovered.

Spheres may be recovered using a method known per se. For example, in a state where the spheres have settled to the bottom of the culture container and single cells and chitin nanofibers/chitosan nanofibers are floating in the liquid medium, the spheres can be removed by removing the liquid medium using a pipette or the like. can only be collected. A fresh liquid medium (preferably a dedicated medium) may or may not be added to the collected spheres as necessary.

(4th step)
In the fourth step of the method of the present invention, the spheres collected in the third step are subjected to two-dimensional culture (2D culture). By subjecting the sphere to 2D culture, cells constituting the sphere are allowed to migrate from the sphere.

Two-dimensional culture (adhesive culture) is not particularly limited as long as the cells to be selected have conditions that allow them to survive and proliferate, and methods known per se can be used.

The medium used in the 2D culture in the fourth step is not particularly limited as long as the cells constituting the sphere can proliferate. Examples of the culture medium that can be used include, but are not limited to, the culture medium described in the above (first step). In a preferred embodiment, the medium used in the 2D culture in the fourth step is a medium exclusively for cells having sphere-forming and migratory abilities to be selected.

The culture conditions for carrying out the 2D culture in the fourth step are not particularly limited as long as the cells constituting the sphere migrate from the sphere and subsequently proliferate. For example, the culture period of the 2D culture in the fourth step is usually one day, preferably 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more. Or for more than 10 days. The upper limit of the period is not particularly limited, but is usually 30 days or less, preferably 29 days or less, 28 days or less, 27 days or less, 26 days or less, 25 days or less, 24 days or less, 23 days or less, 22 days or less. or 21 days or less. The culture temperature is not particularly limited, but is usually 25°C or higher, 30°C or higher, 35°C or higher, or 37°C or higher. Further, the upper limit of the temperature condition is not particularly limited, but is usually 42°C or lower, preferably 41°C or lower, 40°C or lower, 39°C or lower, or 38°C or lower.

(5th step)
In the fifth step of the method of the present invention, cells that migrated and proliferated from the spheres as a result of the 2D culture of the spheres in the fourth step are collected.

Proliferated cells may be collected using a method known per se. Cells that have migrated and proliferated from the spheres, which are the target of recovery, adhere to the surface of the culture container, so they can be easily recovered by detaching them from the container surface using a cell detachment agent such as trypsin. .

In one embodiment of the method of the present invention, the cells that have been sorted through the fifth step described above are again subjected to the first to fifth steps of the present invention, and the cells are repeatedly sorted to improve the accuracy of the sorting. It can also be increased.

In addition, when the cells to be selected are mesenchymal stem cells, the mesenchymal stem cells selected by the method of the present invention may be young, high-quality mesenchymal stem cells that maintain good undifferentiation. . As used herein, "high quality mesenchymal stem cells" are defined as follows:
(1) High expression of at least one (preferably at least two, more preferably all three) of CD73, CD90, and CD105 (mesenchymal stem cell markers);
(2) high expression of at least one (preferably at least two, more preferably all three) of NANOG, OCT4, and SOX2 (undifferentiated markers of mesenchymal stem cells);
(3) High expression of bFGF (factor that maintains undifferentiated state of mesenchymal stem cells), and
(4) High expression of HOXB7 (senescence suppressor of mesenchymal stem cells).
Note that "high expression" means that the expression level of these genes is higher than the expression level of these genes in mesenchymal stem cells obtained by conventional 2D culture (or Explant culture).
(For genes that serve as standards for mesenchymal stem cell quality, see Olivia Candini et al. Stem Cells. 2015 Mar;33(3):939-50 and Elisabetta Manuela Foppiani et al. Stem Cell Res Ther. 2019 Mar 19; 10(1):101 etc.)

2. Agent for living body transplantation The present invention also provides a agent for living body transplantation (hereinafter sometimes referred to as "the agent of the present invention"), which contains mesenchymal stem cells selected by the method of the present invention as an active ingredient.

As described above, mesenchymal stem cells selected using the method of the present invention are young, high-quality mesenchymal stem cells that maintain well undifferentiated properties. Therefore, a living body transplantation agent containing this as an active ingredient can have a higher therapeutic effect than ordinary mesenchymal stem cells.

The amount of mesenchymal stem cells incorporated into the agent of the present invention is not particularly limited as long as it is a therapeutically effective amount.

The agent of the present invention may or may not contain components other than mesenchymal stem cells. Components other than mesenchymal stem cells include, for example, a medium that can maintain the survival of mesenchymal stem cells such as DMEM and RPMI1640, human serum albumin, dimethyl sulfoxide, dextran, calcium chloride hydrate, potassium chloride, and sodium chloride. , sodium L-lactate, serum derived from the subject to be administered, and the like, but are not limited to these.

The subject to which the agent of the present invention is applied is not particularly limited, but it is preferable that the species from which the mesenchymal stem cells are derived and the species to which the agent of the present invention is applied are the same. Specifically, when the mesenchymal stem cells contained in the agent of the present invention are of human origin, the agent of the present invention is preferably applied to humans.

The dosage of the agent of the present invention to be administered to a subject may be appropriately determined in consideration of the species, age, sex, body weight, type of disease to be treated, etc. of the subject. Furthermore, the route of administration of the agent of the present invention to a subject is not particularly limited as long as a therapeutic effect is obtained, but parenteral administration is preferred. In one embodiment, the agent of the present invention can be locally administered to the treatment area using a syringe or the like.

The dosage form of the agent of the present invention is not particularly limited as long as the mesenchymal stem cells, which are the active ingredients, remain viable, but a liquid form is preferred. Furthermore, the agent of the present invention may be prepared in a liquid form and then cryopreserved by adding an appropriate cryopreservative.

3. Method for producing mesenchymal stem cells The present invention also provides a method for producing mesenchymal stem cells (hereinafter sometimes referred to as "the production method of the present invention"), which includes the following steps:
(1) A step of three-dimensionally culturing a cell population containing mesenchymal stem cells in a liquid medium containing chitin nanofibers carrying vitronectin and chitosan nanofibers to form spheres of the mesenchymal stem cells;
(2) a step of settling the spheres formed in (1);
(3) a step of recovering the settled spheres;
(4) a step of two-dimensionally culturing the collected spheres; and (5) a step of collecting mesenchymal stem cells that migrated from the spheres in the two-dimensional culture.

The production method of the present invention is synonymous with the aspect of selecting mesenchymal stem cells in the method of the present invention. Therefore, in the production method of the present invention, mesenchymal stem cells, cell populations containing them, their origins, nanofibers used, etc. are the same as those described in the method of the present invention.

Note that mesenchymal stem cells produced by the production method of the present invention can also be used as an active ingredient of the agent of the present invention.

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

[Preparation example 1]
(Preparation of aqueous dispersion containing chitin nanofibers)
A 2% by mass aqueous chitin nanofiber dispersion prepared according to the description in WO2015/111686 was sterilized in an autoclave at 121°C for 20 minutes. Thereafter, this aqueous dispersion was mixed and suspended in sterile distilled water (Otsuka Distilled Water, manufactured by Otsuka Pharmaceutical Factory Co., Ltd.) to a concentration of 1% (w/v), thereby containing sterile chitin nanofibers. An aqueous dispersion was prepared.

[Preparation example 2]
(Preparation of aqueous dispersion containing chitosan nanofibers)
A 2% by mass chitosan nanofiber aqueous dispersion prepared according to the description in WO2015/111686 was sterilized in an autoclave at 121°C for 20 minutes. Thereafter, this aqueous dispersion was mixed and suspended in sterile distilled water (Otsuka Distilled Water, manufactured by Otsuka Pharmaceutical Factory Co., Ltd.) to a concentration of 1% (w/v), thereby containing sterile chitosan nanofibers. An aqueous dispersion was prepared.

[Preparation example 3]
(Preparation of aqueous dispersion containing vitronectin-supported chitin nanofibers)
A 500 μg/mL vitronectin aqueous solution (Gibco Vitronectin (VTN-N) Recombinant Human Protein, Truncated, Thermo Fisher Scientific) was added to the 1% (w/v) chitin nanofiber aqueous dispersion prepared in Preparation Example 1. (manufactured by company c), and After mixing by petting, the mixture was stored at 4° C. overnight to prepare an aqueous dispersion containing vitronectin-supported chitin nanofibers. The composition of the aqueous dispersion containing the produced vitronectin-supported chitin nanofibers is shown below.

1% (w/v) chitin nanofiber aqueous dispersion (5 mL)
500μg/mL vitronectin aqueous solution (0.5mL)

[Preparation example 4]
(Preparation of aqueous dispersion containing vitronectin-supported chitin nanofibers and chitosan nanofibers)
The chitosan nanofiber aqueous dispersion prepared in Preparation Example 2 (8 mL) was added to the vitronectin-supported chitin nanofiber aqueous dispersion (2 mL) prepared in Preparation Example 3, and the mixture was mixed by pipetting to obtain the vitronectin-supported chitin. An aqueous dispersion (10 mL) containing a mixture of nanofibers and chitosan nanofibers (hereinafter sometimes simply referred to as "the nanofiber material") was prepared.

[Test Example 1] Cell selection 1 by 3D culture (dedicated medium)
Evaluation of proliferation and morphology of three types of cells: mesenchymal stem cells, bronchial epithelial cells, and renal epithelial cells, when cultured in 3D using a dedicated medium for each cell containing nanofiber substrate. did.
(1) Mesenchymal stem cells Nanofiber base material was added to mesenchymal stem cell growth medium (C-28009, Takara Bio Inc., serum medium) to a final concentration of 0.05% (w/v). , prepared a 3D culture medium for mesenchymal stem cells. Subsequently, precultured human adipose-derived mesenchymal stem cells (C-12977, manufactured by Takara Bio Inc.) were suspended in the 3D culture medium at 10,000 cells/mL, and placed in a 24-well flat bottom ultra-low attachment surface microplate ( (manufactured by Corning, #3473) at 1 mL/well.
(2) Bronchial epithelial cells Nanofiber base material was added to airway epithelial cell growth medium (C-21160, manufactured by Takara Bio Inc.) to a final concentration of 0.05% (w/v). 3D culture medium was prepared. Subsequently, precultured human bronchial epithelial cells (C-12640, manufactured by Takara Bio Inc.) were suspended in the 3D culture medium at 10,000 cells/mL, and placed in a 24-well flat bottom ultra-low attachment surface microplate (manufactured by Corning Inc.). , #3473) at 1 mL/well.
(3) Renal epithelial cells Nanofiber base material was added to renal epithelial cell growth medium (C-26130, manufactured by Takara Bio Inc.) to a final concentration of 0.05% (w/v). 3D culture medium was prepared. Subsequently, precultured human renal epithelial cells (C-12665, manufactured by Takara Bio Inc.) were suspended at 10,000 cells/mL and placed in a 24-well flat bottom ultra-low attachment surface microplate (manufactured by Corning Inc., #3473). It was seeded at 1 mL/well.

Each cell was 3D cultured in a static state in a CO ₂ incubator (37° C., 5% CO ₂ ) for 14 days.

100 μL of ATP reagent (CellTiter-Glo ^™ Luminescent Cell Viability Assay, manufactured by Promega) was added to and suspended in the culture solution (100 μL) at the time of seeding (0 days), 7 days and 14 days after seeding. . After allowing the mixture to stand at room temperature for about 10 minutes, the luminescence intensity (RLU value) was measured using FlexStation 3 (manufactured by Molecular Devices), and the amount of viable cells (2 The average value of the points) was calculated. Table 1 shows the converted RLU value (ATP measurement, luminescence intensity) in each cell.

As shown in Table 1, mesenchymal stem cells proliferated efficiently in 3D culture using a nanofiber substrate and a dedicated medium for each cell. On the other hand, the two types of epithelial cells did not proliferate and the number of cells decreased.

Furthermore, when the morphology of each cell was observed, the mesenchymal stem cells formed spheres, but the two types of epithelial cells did not form spheres (Fig. 1, upper row).

[Test Example 2] Cell selection 2 by 3D culture (non-dedicated medium)
The proliferation and morphology of each cell were evaluated under the same conditions as in Test Example 1 except that 10% FBS-containing IMDM medium (12440053, manufactured by Thermo Fisher) was used as the medium. The results are shown in Table 2 and the lower part of FIG.

As shown in Table 2, mesenchymal stem cells proliferated in 3D culture using nanofiber substrates and non-specific medium (ie, IMDM medium containing 10% FBS). On the other hand, the two types of epithelial cells did not proliferate and the number of cells decreased.

Furthermore, when the morphology of each cell was observed, mesenchymal stem cells formed spheres, but two types of epithelial cells did not form spheres (Figure 1, bottom row).

From the results of Test Examples 1 and 2, by performing 3D culture using a medium containing nanofiber substrates, cells with sphere-forming ability can be grown, while cells without sphere-forming ability can be grown. was shown to be able to suppress proliferation.

[Test Example 3] Selection of mesenchymal stem cells contained in adipose tissue We examined whether mesenchymal stem cells could be selected from living tissue by performing 3D culture using a medium containing a nanofiber substrate. This research protocol was approved by the Toho University Medical Center Sakura Hospital Ethics Committee and was conducted in compliance with the ethical guidelines for medical research involving human subjects. The human specimen used in this study was residual subcutaneous adipose tissue generated during plastic surgery. Consent was obtained in advance from the patient regarding the use of the remaining subcutaneous adipose tissue in research.

Subcutaneous adipose tissue (#1; male, 46 years old; #2; female, 71 years old; #3; male, 41 years old) was washed with PBS solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., 045-2979). The subcutaneous adipose tissue was shredded with scissors and transferred to a 50 mL centrifuge tube. To this was added 4 mg/mL of collagenase (Collagenase NB 4G Proved Grade S1746501 manufactured by Nordmark Biochemicals) (the solvent was HBSS solution (H9269, manufactured by SIGMA)), and the extracellular matrix was removed by shaking at 37°C for 1 hour. The cells were disrupted and dispersed into single cells. Next, DMEM-HAM medium (GIBCO 11330-032) containing 20% FBS was added to the collagenase/HBSS solution containing this single cell, centrifuged at 400 g for 1 minute, the supernatant was removed, and the precipitated pellet was collected. Recovered. After repeating these operations three times, a pellet containing stromal vascular fraction (SVF) was obtained, and the pellet was suspended in DMEM-HAM medium containing 20% FBS to obtain a precipitate fraction. Subsequently, a 500 μm mesh (CORNING 2211007) was placed on a new 50 mL centrifuge tube, and the precipitate fraction was filtered to collect filtrate 1 (SVF fraction). Further, only DMEM-HAM medium containing 20% FBS was added to the mesh in that state, and filtration was performed again, and the filtered liquid was mixed with filtrate 1. The mixed filtrate was seeded in a 10 cm petri dish, and then cultured for an average of 4 days at 37°C and 5% CO2 in DMEM-HAM medium containing 20% FBS or mesenchymal stem cell growth medium (C-28009, manufactured by Takara Bio). did. After 4 days, the culture was washed with PBS solution (045-2979, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to remove residual blood cells and adipose tissue contained in the culture solution, and then adhered to a plastic container. A cell population derived from the adipose tissue SVF fraction was obtained. Regarding specimen #1, a cell population derived from the floating fraction of human adipose tissue was also obtained, which did not adhere to the plastic container and was floating together with the remaining adipose tissue.

A 3D culture medium was prepared by adding nanofiber substrate to a mesenchymal stem cell growth medium (C-28009, Takara Bio Inc., serum medium) to a final concentration of 0.05% (w/v). . Subsequently, a cell population derived from the SVF fraction derived from adipose tissue (#1; male, 46 years old, #2; female, 71 years old, #3; male, 41 years old) and a cell population derived from the floating fraction derived from adipose tissue ( #1; male, 46 years old) was suspended in the above-mentioned 3D culture medium, and then seeded at 5 mL/well in a 6-well flat-bottom ultra-low attachment surface microplate (Corning, #3471). The cells were left standing in a CO ₂ incubator (37° C., 5% CO ₂ ) and cultured for up to 26 days. The frequency of medium exchange was once a week. Medium exchange was performed by removing the upper half (approximately 2.5 mL) of the culture medium in the well and adding 2.5 mL of fresh mesenchymal stem cell growth medium (without nanofiber substrate). . After adding fresh medium, the medium was thoroughly mixed by pipetting, and 3D culture in a static state was continued.

At 14 days of culture, it was confirmed that cell spheres were formed on the nanofiber substrate in all of specimens #1 to #3.

Only the formed spheres were recovered by the process shown in FIG. 2. The collection process will be briefly explained below. The culture solution containing spheres etc. was transferred from the well to a 15 mL tube, and about 7.5 mL of PBS (045-2979, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added thereto and mixed well by pipetting. PBS contains spheres, single cells, and nanofiber substrates, which have different self-weights and therefore different settling rates. Spheres have a relatively heavy weight and have a fast settling rate, whereas single cells and nanofiber substrates have a light weight and have a slow settling rate. By utilizing this difference in sedimentation speed, the spheres can be separated from the single cells and the substrate simply by standing for several minutes without performing separation operations such as centrifugation or FACS. After allowing only the spheres to settle on the bottom of the tube by allowing the tube to stand for several minutes, only the spheres were recovered by removing the PBS supernatant using a Pasteur pipette. Mesenchymal stem cell growth medium was added to the collected spheres.

It is generally known that mesenchymal stem cells migrate and proliferate when 2D culture is performed. Therefore, 2D culture was performed to confirm that mesenchymal stem cells were contained in the collected spheres. Specifically, the collected spheres were seeded in a petri dish, and 2D cultured in a stationary state in a CO ₂ incubator (37° C., 5% CO ₂ ) for up to 10 days. The frequency of medium exchange was once every 3 to 4 days. Medium exchange was performed with fresh mesenchymal stem cell growth medium (not containing nanofiber substrate). Only cells that migrated from the sphere and proliferated were collected by trypsin treatment.

The results of migration and proliferation of a cell population derived from the SVF fraction derived from human adipose tissue are shown in FIG. 3, and the results of migration and proliferation of a cell population derived from a floating fraction derived from human adipose tissue are shown in FIG. 4, respectively.

Formation of spheres was confirmed in 3D culture using nanofiber substrates in the SVF fraction of all samples. Furthermore, it was confirmed that cells migrated and proliferated from the spheres by 2D culture. Furthermore, spheres were also formed by subjecting the cell population derived from the floating fraction (specimen #1) to 3D culture using a nanofiber substrate. Furthermore, it was confirmed that cells migrated and proliferated from the spheres by 2D culture. The above results showed that by combining 3D culture using a nanofiber substrate and 2D culture, mesenchymal stem cells can be selected and recovered from a cell population that constitutes a living tissue.

[Test Example 4] Selection of mesenchymal stem cells by repeating 3D culture and 2D culture We examined whether the cell selection process by sphere formation in 3D culture and migration in 2D culture could be repeated. An overview of this test is shown in Figure 5.

Nanofiber base material was added to mesenchymal stem cell proliferation medium (C-28009, Takara Bio Inc., serum medium) to a final concentration of 0.05% (w/v), and a 3D culture medium was prepared. Subsequently, mesenchymal stem cells (#1; male, 46 years old) derived from the adipose tissue-derived SVF fraction obtained in Test Example 3 were suspended in a 3D culture medium for mesenchymal stem cells, and placed in a 6-well flat-bottomed cell culture medium. The cells were seeded at 5 mL/well in a low-adhesion surface microplate (#3471, manufactured by Corning).

The cells were 3D cultured in a static state in a CO ₂ incubator (37° C., 5% CO ₂ ) for up to 14 days. The frequency of medium exchange was once a week. Medium exchange was performed by removing the upper half (approximately 2.5 mL) of the culture medium in the well and adding 2.5 mL of fresh mesenchymal stem cell growth medium (without nanofiber substrate). . After adding fresh medium, the medium was thoroughly mixed by pipetting, and 3D culture in a static state was continued. Mesenchymal stem cells subjected to the second 3D culture formed spheres (Fig. 6, left).

Spheres formed by 3D culture were collected using a process similar to that used in Test Example 3.

The collected spheres were subjected to 2D culture under the same conditions as Test Example 3. Mesenchymal stem cells migrated and proliferated again from the spheres subjected to 2D culture (Fig. 6, right). These results showed that the screening method of the present invention allows the screening process to be carried out repeatedly.

[Test Example 5] Selection of mesenchymal stem cells from mouse bone marrow-derived cell population
A bone marrow-derived cell population was obtained by collecting bone marrow from the femur of a C57BL/6J mouse (female). Red blood cells in the bone marrow-derived cell population were hemolyzed using ACK buffer (150mM NH ₄ CI, 10mM KHCO ₃ , 0.1mM Na ₂ EDTA, pH 7.2-7.4). This cell population was cryopreserved at -80°C using a cell cryopreservation solution (Stem Cell Banker (registered trademark) (CB061, manufactured by Takara Bio Inc.).

Nanofiber base material was added to mesenchymal stem cell proliferation medium (Y-50200, Takara Bio Inc., serum medium) to a final concentration of 0.05% (w/v), and a 3D culture medium was prepared. Subsequently, a frozen and thawed mouse bone marrow-derived cell population (approximately 2x10 ⁶ cells) was suspended in the 3D culture medium and placed in a 6-well flat-bottom ultra-low attachment surface microplate (Corning, #3471) at 5 mL/well. Sowed.

The cells were 3D cultured in a static state in a CO ₂ incubator (37° C., 5% CO ₂ ) for up to 16 days.

The frequency of medium exchange was not for the first week, and then once a week. Medium exchange was performed by removing the upper half (approximately 2.5 mL) of the culture medium in the well and adding 2.5 mL of fresh mesenchymal stem cell growth medium (without nanofiber substrate). . After adding fresh medium, the medium was thoroughly mixed by pipetting, and 3D culture in a static state was continued.

At 16 days of culture, it was confirmed that spheres were formed (Fig. 7, left).

The formed spheres were collected as follows. Transfer the 3D culture (approximately 2.5 mL) containing spheres from the well to a 15 mL tube, add approximately 7.5 mL of PBS (045-2979, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and pipette. Mixed well. After allowing only the spheres to settle on the bottom of the tube by allowing the tube to stand for several minutes, only the spheres were recovered by removing the PBS supernatant using a Pasteur pipette. Mesenchymal stem cell growth medium was added to the collected spheres.

Furthermore, the collected spheres were subjected to 2D culture. Specifically, the collected spheres were seeded in a petri dish, and cultured in a 2D manner for 2 days in a stationary state in a CO ₂ incubator (37° C., 5% CO ₂ ). The frequency of medium exchange was once every 3 to 4 days. Medium exchange was performed with fresh mesenchymal stem cell growth medium (not containing nanofiber substrate). Mesenchymal stem cells migrated and proliferated from the spheres subjected to 2D culture (Fig. 7, right). Only cells that migrated from the sphere and proliferated were collected by trypsin treatment.

The above results showed that mesenchymal stem cells can be selected and recovered from a cell population derived from mouse bone marrow by combining 3D culture using a nanofiber substrate and 2D culture.

[Test Example 6] Comparison of characteristics of mesenchymal stem cells obtained by the selection method of the present invention and mesenchymal stem cells obtained by the conventional method Mesenchymal stem cells obtained by the selection method of the present invention were obtained by the conventional method (2D culture) We investigated whether they are superior to mesenchymal stem cells obtained in

(1) Preparation of mesenchymal stem cells using conventional methods Subcutaneous adipose tissue obtained from donors (#2; female, 71 years old, #3; male, 41 years old) was treated with enzymes to remove adipocytes, and The quality vascular cell population (SVF) was obtained. The obtained SVF was subjected to 2D culture, and only the migrated and proliferated cells were treated with trypsin to recover a cell population containing mesenchymal stem cells. The collected cell population was suspended in a mesenchymal stem cell growth medium (C-28009, Takara Bio Inc., serum medium) and seeded in a cell-adhesive petri dish. The cells were cultured for 2D in a static state in a CO ₂ incubator (37° C., 5% CO ₂ ) for 5 days. At day 5, mesenchymal stem cells proliferating in a monolayer were obtained (MSCs obtained by conventional methods).

(2) Preparation of mesenchymal stem cells using the method of the present invention Among the mesenchymal stem cells obtained in Test Example 3 above, mesenchymal stem cells obtained from donors #2 and #3 were used. .

(3) Culture Mesenchymal stem cells obtained by the conventional method and mesenchymal stem cells obtained by the method of the present invention were suspended in a mesenchymal stem cell growth medium (C-28009, manufactured by Takara Bio Inc., serum medium). The mixture was clouded and seeded in a cell-adhesive petri dish. Each cell was 2D cultured for 6 days in a static state in a CO ₂ incubator (37° C., 5% CO ₂ ).

A portion of each cell was collected on the 6th day, and induced to differentiate into adipocytes by 2D culture using human adipocyte differentiation medium (#CAS11D250, manufactured by Toyobo Co., Ltd.). Differentiation induction was performed for 14 days.

Total RNA was extracted from cells that were not induced to differentiate on day 6 and cells that were induced to differentiate for 14 days. Total RNA was extracted using RNeasy Mini kit (manufactured by QIAGEN). A reverse transcription reaction was performed on GeneAmp PCR System 9700 (manufactured by Applied Biosystems) using PrimeScript ^™ RT Master Mix (manufactured by Takara Bio), and cDNA was synthesized from the extracted total RNA. The obtained cDNA sample was diluted 1/10 with sterile water and used as a template for real-time PCR. In addition, a cDNA sample was prepared by collecting and mixing a portion of all the cDNA samples, and the cDNA sample was used as a sample for preparing a calibration curve. The calibration curve was set in the quantitative range from 1/3 to 1/243 dilution using a 3-fold common ratio. Real-time PCR was performed using a 7500 Real Time PCR System (Applied Biosystems) using each cDNA sample, calibration sample, Premix Ex Taq ^TM (manufactured by Takara Bio), and various Taqman probes (manufactured by Applied Biosystems). (manufactured by S.A.). The probes used (manufactured by Applied Biosystems) are shown below.

18s rRNA: Applied Biosystems REF 4319413E
HOXB7: Hs04187556
bFGF: Hs00266645
NANOG: Hs04399610
OCT4: Hs04260367
SOX2: Hs04234836
CD73: Hs00159686
CD90: Hs06633377
CD105: Hs00923996

The results of real-time PCR on day 6 from the start of 2D culture are shown in Table 3 (donor #2) and Table 4 (donor #3).

As shown in Tables 3 and 4, for both #2 and #3, the expression of surface antigen markers (CD73, CD90, CD105) of mesenchymal stem cells was higher than that of mesenchymal stem cells selected by the method of the present invention. it was high. We also investigated the expression of undifferentiated markers (NANOG, OCT4, SOX2), the expression of bFGF that maintains undifferentiated state, and the expression of HOXB7, which contributes to the suppression of aging of mesenchymal stem cells and controls the expression of bFGF. Both #2 and #3 were higher in mesenchymal stem cells selected by the method of the present invention. Therefore, the mesenchymal stem cells selected by the method of the present invention were shown to have better quality than mesenchymal stem cells selected by the conventional method.

Furthermore, the results of real-time PCR on day 14 from the start of differentiation induction into adipocytes are shown in Table 5 (donor #2) and Table 6 (donor #3).

As shown in Tables 5 and 6, both mesenchymal stem cells sorted by the method of the present invention and mesenchymal stem cells sorted by the conventional method showed mesenchymal stem cells after being subjected to differentiation induction for 14 days. The expression levels of stem cell markers (CD73, CD90, CD105), undifferentiated markers (NANOG, OCT4, SOX2), bFGF, and HOXB7 mRNA were decreased.

According to the present invention, cells having sphere-forming ability and migratory ability contained in a cell population can be separated and sorted without performing centrifugation or separation operations using cell surface antigens. Furthermore, according to the present invention, young, high-quality mesenchymal stem cells that maintain undifferentiated properties can be efficiently prepared. Therefore, the present invention is very useful, for example, in the field of regenerative medicine.

Claims (8)

A method for selecting cells with sphere-forming ability and migration ability, including the following steps:
(1) A step of three-dimensionally culturing a cell population containing cells having sphere-forming ability and migratory ability in a liquid medium containing chitin nanofibers and chitosan nanofibers to form spheres of the cells;
(2) a step of settling the spheres formed in (1);
(3) a step of recovering the settled spheres;
(4) a step of two-dimensionally culturing the collected spheres; and (5) a step of collecting cells that migrated from the spheres in the two-dimensional culture. The method according to claim 1, characterized in that the chitin nanofibers carry vitronectin. 3. The method according to claim 1, wherein the liquid medium in (1) is a medium exclusively for cells having sphere-forming ability and migration ability. 2. The method according to claim 1, wherein the cell population is derived from living tissue. 2. The method according to claim 1, wherein the cells having sphere-forming ability and migration ability are mesenchymal stem cells, fibroblasts, neural stem cells, neural progenitor cells, dermal papilla cells, or cancer stem cells. 2. The method according to claim 1, wherein the cells having sphere-forming ability and migration ability are mesenchymal stem cells. A preparation for living body transplantation, comprising mesenchymal stem cells selected by the method according to claim 6. Method for producing mesenchymal stem cells, including the following steps:
(1) A step of three-dimensionally culturing a cell population containing mesenchymal stem cells in a liquid medium containing chitin nanofibers carrying vitronectin and chitosan nanofibers to form spheres of the mesenchymal stem cells;
(2) a step of settling the spheres formed in (1);
(3) a step of recovering the settled spheres;
(4) a step of two-dimensionally culturing the collected spheres; and (5) a step of collecting mesenchymal stem cells that migrated from the spheres in the two-dimensional culture.