JP6226545B2 - Spheroid culture method using hyaluronic acid - Google Patents

Description

  The present invention relates to a spheroid culture method and concentration technique for CD44-expressing cells.

  For the enrichment of cancer stem cells, the marker protein such as CD133 or CD44 is used as an index, and separation is performed by magnetic beads (Non-patent Document 1, Diamond CD133 Isolation Kit manufactured by Miltenyi Biotec) or by flow cytometry. There are a method of collecting (Non-patent Document 2), a laser microdissection (LMD6500 & LMD7000 manufactured by Leica), and the like.

  For the culture and maintenance of isolated and isolated cancer stem cells, a gel polymerized with hyaluronic acid or hyaluronic acid and gelatin (Non-patent Document 3, Hystem manufactured by Sigma-Aldrich), collagen gel (Nippon Becton Dickinson) Matrigel) can be used.

Jin et al. 2008. Neuroscience; 154,541-550. Griguer et al. 2008. PlosOne; 3 (11) e3655. Rehfeldt et al. 2012 Integr.Biol .; 4,422-430.

  Conventional isolation techniques for CD44-expressing cells require expensive equipment and reagents. When using equipment, space for installation and specialized techniques are required.

  In addition, since it has been necessary to maintain separated cells in a polymerized gel or on the surface of the gel, observation in the bright field is very difficult when cells that do not express fluorescent protein or staining is impossible. there were. Furthermore, it was difficult to produce a uniform gel, and it was difficult to keep the cell state constant. Furthermore, it was very difficult to collect and passage the cells after culturing and to recover factors secreted from the cells. There was also the problem that it was difficult to expose drugs, growth factors, cytokines, etc. to cells.

  An object of the present invention is to provide a technique for easily proliferating, concentrating or isolating CD44-expressing cells using a normal instrument or apparatus.

  As a result of repeated investigations in view of the above problems, the present inventor can form spheroids by culturing CD44-expressing cells in a medium supplemented with hyaluronic acid, and easily and highly reproduce a population of CD44-expressing cells. It has been found that it can be concentrated and recovered.

The present invention provides the following method for culturing CD44-expressing cells.
Item 1. A method for culturing CD44-expressing cells, comprising spheroids by non-adhesive culture of CD44-expressing cells in a medium containing hyaluronic acid.
Item 2. Item 4. The culture method according to Item 1, wherein the CD44-expressing cells are cancer cells and / or cancer stem cells.
Item 3. Item 3. The culture method according to Item 1 or 2, wherein low molecular hyaluronic acid is further used as hyaluronic acid, and CD44 high-expressing cells are obtained by binding to CD44 competitively with the low molecular hyaluronic acid.
Item 4. Item 4. The culture method according to any one of Items 1 to 3, wherein iron oxide produced by a microorganism is added to the medium.

  In order to separate the CD44-expressing cells, a very expensive apparatus is required, and it is necessary to secure an installation space and to acquire a special technique. Therefore, it is possible to obtain CD44-expressing cells, in particular, CD44-highly expressing cells at low cost without requiring special techniques.

  In the case of a gel-like culture substrate (collagen gel, hyaluronic acid polymer, etc.), it was difficult to collect cells and secreted substances that formed cell clusters, but they could be easily collected.

  When cells were treated with drugs or the like, treatment and concentration adjustment were difficult with a gel-like culture substrate, but they became easier.

  Although observation of spheroids (cell mass) over time and microscopic observation in a bright field have been very difficult, according to the present invention, they can be easily observed and maintained.

  Self-replication ability can be confirmed more easily than before.

  Highly malignant CD44 high-expressing cells (especially cancer stem cells) can be easily concentrated from cells (especially cancer stem cells) that are difficult to produce cancer bearing (low tumor-forming ability). In addition, a cell population with a high CD44 expression level can be cultured by extracting the prepared cancer-bearing cancer.

An experimental procedure in the case of using human glioma-derived U251MG cells is schematically shown. Culture in hyaluronic acid supplemented medium Examination of HA concentration Culture on non-adhesive dishes With: HA-added medium Without: HA-free medium (normal medium) Cell separation and self-replication from cell mass Comparison of CD44 gene expression level A: A172 cell B: U251MG cell C: U251MG-P1 cell Expression level of CD44 gene in U251MG-P1 cells relative to U251MG cells Tumor formation of U251MG cells cultured in medium supplemented with HA Comparison of CD44 protein expression level Detection of astrocyte marker protein HA culture of SkOv-3 cells CD44 protein is shown schematically. Spheroid production using hyaluronic acid (HA)

CD44 is a single-transmembrane glycoprotein hyaluronic acid receptor as shown in FIG. 12 , and is specifically expressed on the surface of glioma and cancer cells, such as human pancreatic cancer, prostate cancer and breast cancer. It is known to be a stem cell marker. CD44 has functions such as cell aggregation, retention of matrix around cells, signaling between matrix and cells, and control of orientation of MMPs (cell migration, invasion).

  The CD44-expressing cells are not particularly limited as long as CD44 is expressed on the cell surface, but CD44-highly expressing cells capable of detecting CD44 at the protein level such as Western blotting and immunostaining are preferred.

  CD44-expressing cells are widely expressed in various cancer cells and cancer stem cells, hematopoietic cells, erythrocytes, blood cells including T cells, gastrointestinal tract, muscle, skin, nervous system and other cells. As the cancer stem cell, a cancer stem cell highly expressing CD44 is preferably used. CD44-expressing cells can form spheroids by non-adherent (floating) culture in the presence of hyaluronic acid, and CD44-expressing cells can be concentrated, isolated and purified. Moreover, the function of CD44-expressing cells can be enhanced by culturing them in the presence of hyaluronic acid. For example, cancer stem cells cultured in the presence of hyaluronic acid have enhanced tumorigenicity.

  Hyaluronic acid is composed of a disaccharide repeating unit of N-acetylglucosamine and glucuronic acid, and has a molecular weight of about 500 to 20 million, preferably about 1000 to 1500000, more preferably about 3000 to 1000000. As hyaluronic acid, natural hyaluronic acid may be used as it is, and any of hyaluronic acid reduced in molecular weight by degradation with an enzyme (hyaluronidase) or reduced in molecular weight by acid hydrolysis may be used.

  In the present specification, the term “hyaluronic acid” simply includes both high-molecular hyaluronic acid and low-molecular hyaluronic acid. High molecular hyaluronic acid refers to hyaluronic acid having a molecular weight of 100,000 or more, preferably 300,000 or more, more preferably 500,000 or more, more preferably 700,000 or more, particularly 1,000,000 or more. This corresponds to “hyaluronic acid” having a large molecular weight (high molecular weight).

  By chemically treating hyaluronic acid with an enzyme, acid, alkali, etc., by separating hyaluronic acid having a molecular weight below a certain level from hyaluronic acid having a low molecular weight or naturally derived hyaluronic acid, the present invention The “low molecular hyaluronic acid” to be used can be obtained. The molecular weight of “low molecular hyaluronic acid” refers to hyaluronic acid having a molecular weight of 100,000 or less, the upper limit of molecular weight is about 100,000, 70,000, 30,000, 10,000, 5,000, and the lower limit of molecular weight is It is about 500, 1000, 2000, 3000.

  Hyaluronic acid may be derived from animals such as chicken and umbilical cord, or may be derived from microorganisms such as lactic acid bacteria and streptococci.

  As a medium to which hyaluronic acid is added, a medium used for cell culture can be widely used, and examples thereof include, but are not limited to, a DMEM medium. Antibiotics such as penicillin and streptomycin, serum-derived components, growth factors and the like may be added to the medium.

The amount of hyaluronic acid added is about 0.1 μg / ml to 5000 μg / ml, preferably about 1 μg / ml to 3000 μg / ml, more preferably about 5 μg / ml to 1000 μg / ml, and even more preferably 10 μg. / Ml to about 200 μg / ml.

The iron oxide produced by microorganisms and the method for producing the same are described in WO2010 / 110435 and WO2011 / 074587, which are incorporated herein in their entirety. Iron oxides produced by microorganisms are iron oxides produced by various microorganisms outside the cells, and those having various shapes are known. Iron oxide produced by microorganisms is also referred to as “biogenous iron oxide”.

In the present specification, “iron oxide” means iron oxide in a narrow sense exemplified by α-Fe ₂ O ₃ , β-Fe ₂ O ₃ , γ-Fe ₂ O ₃ , Fe ₃ O ₄ , α-FeOOH, Compounds containing iron and oxygen as main components, including iron oxyhydroxide exemplified by β-FeOOH, γ-FeOOH, etc., and iron hydroxide having a structure close to amorphous typified by ferrihydrite Is a general term.

The iron-oxidizing bacterium is not particularly limited as long as it forms the iron oxide containing Fe ₃ O ₄ , α-FeOOH, γ-FeOOH (repidocrocite) or the like. Examples of the iron-oxidizing bacterium that produces biogenous iron oxide include, for example, Toxothrix sp., Leptothrix sp., Crenothrix sp., And Chronosilicate. Genus bacteria (Clonothrix sp.), Galionella (Gallionella sp.), Siderocapsa (Siderocapsa sp.), Siderococus sp., Sideromonas sp Planktomyces sp.).

  As an example of a bacterium belonging to the genus Leptothrix, Leptotrix Korodini OUMS1 strain can be mentioned. The Leptotrix Korodini OUMS1 strain was founded on December 25, 2009 at the Center for Patent Microorganisms Deposits of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan) )), The deposit number is NITE P-860. This strain has now been transferred to an international deposit and its deposit number is NITE BP-860.

  The iron oxide produced by microorganisms takes various forms, and the iron oxide produced by Leptothrix ochracea has a hollow fibrous sheath structure, including Galionella ferruginea. The genus Galionella is spiral, the genus Spherotilus and Chronosrix are branched or filamentous, the genus Toxosrix is a filament (harp-like shape, fan-shaped), the genus Sideromonas is a short stem, the capsule of Siderocaps is a capsule, the siderococcus The genus produces spherical iron oxide. The size of iron oxide produced by microorganisms varies depending on the type, but is usually about 0.1 to 3000 μm. These iron oxides exist, for example, in sediments collected in natural filtration facilities of water purification plants, and can be purified by subjecting the sediments to centrifugation, drying under reduced pressure, and the like.

  Iron oxide produced by microorganisms contains trace amounts of silicon and phosphorus in addition to iron and oxygen, but even if it is iron oxide produced by the same microorganism, the types of components such as iron, silicon, and phosphorus contained in iron oxide The composition ratio varies depending on the environment in which microorganisms exist. Since iron oxide produced by microorganisms is derived from a natural product, the possibility that an antigen component is mixed from a culture supernatant using such iron oxide as a carrier is very low.

  The iron oxide produced by the microorganism of the present invention is not limited to a specific embodiment, but the iron oxide produced by Leptothrix ocracea is in the form of a hollow tube. Therefore, supply of nutrients, oxygen, etc. to the inside of the spheroid, culture solution Is preferable in that the exchange of the above is made more effective. When iron oxide produced by Leptothrix ocracea is used, the state of spheroids can be maintained better even if they grow large. Furthermore, the iron oxide produced by the microorganism of the present invention includes (i) a magnetic ceramic obtained by subjecting the iron oxide to heat treatment, and (ii) acid-treating the iron oxide to dissolve and remove the Fe component. And (iii) a portion that can be chemically modified with an organic group of iron oxide organic groups, magnetic ceramics, or porous amorphous silica produced by microorganisms (for example, Fe atoms and / or Si atoms). Organic / inorganic materials obtained by chemically modifying at least part of the bonded oxygen atoms) with organic groups are also included. Magnetic ceramics and methods for producing them are described in WO2011 / 074587, and this entire document is incorporated herein by reference. In the acid treatment, Fe component is dissolved and removed by acid treatment of biogenous iron oxide, and as a result, porous amorphous silica is obtained. Examples of the acid used for the acid treatment include hydrochloric acid and sulfuric acid. . The acid treatment time is usually 1 hour to 6 days, in particular 2 to 4 days. Before the acid treatment step, there may be a step of drying the iron oxide produced by the microorganism. After the acid treatment step, there is a step of washing and drying the porous amorphous silica obtained in the acid treatment step. You may do it. Organic / inorganic materials and methods for producing the same are described in WO2010 / 110435. CD44-expressing cells can be cultured by putting a liquid medium containing the cells and iron oxide produced by the microorganism into a culture vessel, but the cells, the iron oxide produced by the microorganism, and the liquid medium are cultured at the same timing. You may put into a container and may put into a culture container at another timing.

  The iron oxide produced by the microorganism can be added to the medium in an amount of about 0.1 to 10 mg / ml.

In the present invention, spheroids of CD44-expressing cells can be formed by culturing CD44-expressing cells under non-adherent culture conditions (FIG. 13 ).

  As a culture method and culture conditions for CD44-expressing cells, general culture methods and conditions can be adopted depending on the type of each cell. As the medium, any medium for cell culture can be used except that hyaluronic acid is added. For example, Dulbecco's modified Eagle medium (DMEM), Glasgow MEM (GMEM), EMEM, MEMα, RPMI-1640, Ham F- 12, MCDB medium and the like, but are not limited thereto. The culture method of the present invention can be carried out by adding hyaluronic acid to these media. Furthermore, serum or various growth factors or differentiation-inducing factors may be added to these media.

  The culture container may be any culture container or culture dish such as a flask, petri dish, petri dish, plate, tissue culture tube, tray, culture bag, roller bottle, or hollow fiber suitable for spheroid culture. In one embodiment, the culture container is a culture container having a non-cell-adherent inner bottom surface. Here, cell non-adhesiveness means that cultured cells may or may not adhere at all to such an extent that spheroid formation is not inhibited.

  The culture container having a cell non-adhesive inner bottom surface may be any known or commercially available cell non-adhesive culture container, for example, a special surface treatment is performed by injection molding a general-purpose plastic such as polystyrene. Highly hydrophilic such as non-culture containers, non-woven fabric or porous film scaffolds, plastic molded culture containers with fine irregular surfaces, polyethylene glycol, polyhydroxyethyl methacrylate, ethylene vinyl alcohol copolymer A culture container formed from a substance, a culture container in which the surface of the container is coated with a substance capable of making the surface performance hydrophilic, such as a surfactant or phospholipid, and a culture container imparted with hydrophilicity by surface treatment such as plasma treatment Including, but not limited to.

The number capacity of the culture vessel of the culture cells, type of cells, but is appropriately determined in consideration of the final size of the spheroids or the like, for example, ⁴ to 10 ⁷ medium 1mL Atari 10, preferably 10 ⁴ Seed in culture vessel at ~ 10 ⁶ final concentration. The culture may be shake culture or stationary culture. The replacement of the culture solution is not particularly limited as long as spheroids are formed, but may be performed once or twice a day, for example, or may be replaced once every 2 to 4 days. It is also possible to perform a treatment such as centrifugation before exchanging the culture solution to precipitate the cells and then exchange the culture solution. The culture period is about 2 to 20 days. The culture temperature is preferably around 37 ° C. The size of the spheroid (cell mass) is usually about 10 to 500 μm in diameter, preferably about 50 to 200 μm. If the spheroids become too large, nutrients will not easily penetrate into the spheroids. Therefore, it is desirable to separate the spheroids when they reach an appropriate size. The obtained spheroids can form spheroids repeatedly by separating cells and then performing non-adherent culture again. CD44-expressing cells can be purified by spheroid formation, and by purifying spheroid formation, purer (homogeneous trait) CD44-expressing cells can be obtained.

  CD44-expressing cells are derived from animals, preferably from vertebrates, particularly from mammals. Examples of mammals include humans, mice, rats, hamsters, rabbits, dogs, cats, cows, horses, sheep, monkeys, etc., and particularly humans. The CD44-expressing cells are not particularly limited as long as they are cells derived from these animals (preferably vertebrates, particularly humans), and may be, for example, primary cultured cells, subcultured cells, or established cells. Also, ES cells, iPS cells, various stem cells, or cells derived from these pluripotent cells / stem cells may be used.

  According to the culture method of the present invention, CD44-expressing cells in which spheroids are formed have an increased CD44 expression level, further promoted cell proliferation, and enhanced cell function.

  Cancer stem cells are known to have many cells that highly express CD44. Highly expressing cells of CD44 are enhanced in their proliferation by adding hyaluronic acid, which is a ligand of CD44, to the medium, and in the case of cancer stem cells, their malignancy is increased.

  The CD44-expressing cells before culturing in the method of the present invention may be almost a single type of cell, or may be a group of two or more mixed cells in which CD44 is expressed. Even if two or more types of cells are contained, a plurality of spheroids having similar properties / functions are formed by using spheroids. By separating these spheroids, desired spheroids of CD44-expressing cells can be obtained.

  Hereinafter, the present invention will be described in more detail based on examples.

Example 1
1) Outline FIG. 1 shows an outline when human glioma-derived U251MG cells are used.

  When subcultured cells cultured on a normal adhesive dish, transfer to a medium supplemented with hyaluronic acid (HA), culture in a non-adhesive dish, and further subculture to obtain a cell mass on the non-adhesive dish Was made. After inoculating cell masses into nude mice to produce cancer-bearing cells, primary culture (referred to as U251MG-P1) was performed on an adhesive dish. Primary cultured cells were subcultured again on HA-added medium and cultured on non-adhesive dishes.

2) Culture using hyaluronic acid-added medium Human glioma-derived A172 cells, U251MG cells, and U251MG-P1 cells were each added with HA on a non-adhesive dish (100 μg / ml sodium hyaluronate (Hyaluron manufactured by Wako Pure Chemical Industries, Ltd.) Acid medium, chicken crown), 10% FBS, 50 U / ml penicillin, and DMEM with 50 μg / ml streptomycin) plus normal medium (10% FBS, 50 U / ml penicillin, and 50 μg) / ml on DMEM containing streptomycin) for 2-5 days. When HA was not added, the cells hardly proliferated (FIG. 2 shows only the result of U251MG-P1), but when HA was added, a dense cell part was formed, and partly Then, a cell mass was formed (Fig. 2, each right end). In addition, since these cells grew on a normal adhesive dish, it was considered that the cells used were in good condition (FIG. 2, each left end).

  A172 cells and U251MG cells on non-adherent dishes each containing HA (0-100 μg / ml sodium hyaluronate, 10% FBS, 50 U / ml penicillin, and DMEM containing 50 μg / ml streptomycin) Cultured for 5 days (FIG. 3). Although it was confirmed that a cell mass was formed at any concentration, the cell mass could be observed uniformly at a concentration of 100 μg / ml. Since no difference was observed between this addition concentration and the case where 200 μg / ml was added, 100 μg / ml was used as the HA addition concentration in the subsequent tests.

  Similarly, as a result of culturing on a non-adhesive dish for 2 weeks, when HA was added, any cell formed a cell mass, and cells adhering to the dish surface were observed around the cell mass ( Figure 4. With). When HA was not added, the formation of cell clusters was observed, but the number of cell clusters was smaller than when HA was added, and when the diameter grew to about 200 μm, the clusters collapsed or stopped growing. The situation was observed, especially in A172 (Fig. 4. Without).

3) Confirmation of cell mass formation ability A172, U251MG, and U251MG-P1 cell masses prepared in HA-added medium were suspended in Accutase solution (Sigma-Aldrich) to disperse the cells, and then each HA was added again. The cells were cultured for 2 weeks on a medium and a non-adhesive dish (FIG. 5). Each cell formed a cell mass again, and cells adhered to the dish surface were observed around the cell mass, indicating that the cell mass has a self-replicating ability.

4) Comparison of CD44 gene expression level
RNA was extracted from cells cultured on A172 cells, U251MG cells, and U251MG-P1 cells in adherent dishes, normal and non-adhesive dishes, and HA-added media, and the expression level of CD44 gene was analyzed by qPCR ( FIG. 6). U251MG cells and U251MG-P1 cells were also compared with RNA extraction from non-adhesive dishes, cells cultured on normal media.

  Compared to the normal culture in the adhesive dish, the normal culture in the non-adhesive dish has increased CD44 gene expression, but the expression level is higher by culturing in the HA-added medium on the non-adhesive dish. Thus, in U251MG cells, the number was about 2.2 times that of the culture in the adhesion system or normal medium.

  U251MG cells and U251MG-P1 cells were cultured in an adhesive dish and a normal medium, and the expression levels of CD44 gene were compared between these two cells (FIG. 7). U251MG-P1 was about 3.9 times higher in expression than U251MG. From these results, it is possible to selectively grow cells that induce CD44 gene expression or have high expression levels by this culture method, that is, spheroid culture on non-adhesive dishes of U251MG cells on HA. It turned out that. Furthermore, when the cells are transplanted into nude mice, cells that are excised from the tumor-bearing cells of the nude mice are primary cultured, so that cells with a constant CD44 gene expression level can be obtained compared to the parent strain cells. I understood.

5) Tumor-forming ability of U251MG cells cultured in spheroid culture (ITS +, FBS-) in HA-added medium After culturing in HA-added medium for 5 days, spheroid culture (100 μg / ml sodium hyaluronate and ITS (manufactured by Sigma-Aldrich)) For 2 weeks in a non-adhesive dish (diameter 10 mm, medium volume 10 ml), and U251MG cells were transplanted into nude mice Balb / c-nu / nu, female, 4 weeks old For 4 weeks (FIG. 8). Cells collected from 1 to 3 dishes were transplanted to the left abdomen of each mouse. The right abdomen was transplanted with 10 ⁷ U251MG cells cultured normally. Cancer bearing was formed by transplanting cells collected from two or more dishes.

6) CD44 protein expression level of cells spheroid-cultured in HA-added medium A172 cells and U251MG cells in normal medium (A) on non-adhesive dishes, normal medium (N) on non-adhesive dishes and HA-added medium, respectively. Culture (H) was performed, and the CD44 protein expression levels were compared by Western blotting (FIG. 9). 10 μg of total protein extracted from the cells was migrated, and anti-CD44 mouse monoclonal antibody (Katayama Chemical Co., Ltd.) and HRP-labeled anti-mouse IgG antibody (CST Japan Co., Ltd.) were used as primary antibodies. Moreover, (beta) -actin was detected as an internal parameter | index of the protein constitutively expressed using the anti- (beta) -actin antibody (made by CST Japan) and the HRP labeled anti-rabbit IgG antibody (made by CST Japan).

  In both cell lines, the expression level was increased by culturing on a non-adhesive dish, and the expression level was further increased by culturing in a medium supplemented with HA.

7) Detection of astrocyte marker protein in U251MG-P1 In order to confirm that U251MG-P1 cells are glioma-derived cells, U251MG cells, U251MG-P1 cells, and U251MG-P2 cells were used in normal medium on an adhesive dish. After culturing, GFAP protein which is a marker protein of astrocytes was detected by Western blotting (FIG. 10). U251MG-P1 cells were transplanted into nude mice, the tumor-bearing cells were removed, and the primary cultured cells were designated as U251MG-P2. Moreover, the protein extracted from the cell (human ovarian cancer origin SkOv-3 cell) which is not expressing GFAP was used as negative control. 10 μg of total protein extracted from each cell was run and detected using an anti-GFAP antibody (Santa Cruz Biotechnology) and an HRP-labeled anti-mouse IgG antibody (CST Japan). Β-actin was detected as an internal indicator of the constitutively expressed protein.

  For each cell line, U251MG cells are represented as 0, U251MG-P1 cells as P1, U251MG-P2 cells as P2, and SkOv-3 cells as SkOv-3 in FIG. It was confirmed that U251MG-P1 and U251MG-P2 cells expressed GFAP when cultured in an adhesive dish.

8) HA culture of SkOv-3 cells Each of SkOv-3 cells and SkOv-3-P1 cells (cells obtained by inoculating SkOv-3 cells and then removing primary tumors after primary inoculation) were the same as U251MG cells. Medium (100 μg / ml sodium hyaluronate (manufactured by Wako Pure Chemical Industries, Co., Ltd.), 10% FBS, 50 U / ml penicillin, and 50 μg / ml streptomycin containing HA on an adhesion dish) ) And on normal medium (RPMI 1640 containing 10% FBS, 50 U / ml penicillin, and 50 μg / ml streptomycin) for 4 days (FIG. 11).

  In the SkOv-3 cells, cell clumps were observed in the HA-added medium, but no cell clumps were observed in the medium to which HA was not added (HA-, normal medium). In the SkOv-3-P1 cells, the cell mass increased in the presence of HA, and many cells adhering to the dish surface were observed in the surrounding area. In the medium without addition of HA (normal medium), the cells slightly adhered to the dish surface formed a small cell mass, but the growth was slower and the cell mass was small compared to the culture in the medium with HA added. It was. On the adhesive dish, there was almost no difference due to the presence or absence of HA, and growth was normal.

Claims (4)

A method for culturing CD44-expressing cells, comprising spheroids by non-adhesive culture of CD44-expressing cells in a medium containing hyaluronic acid. The culture method according to claim 1, wherein the CD44-expressing cells are cancer cells and / or cancer stem cells. The culture method according to claim 1 or 2, wherein low molecular hyaluronic acid is further used as hyaluronic acid, and CD44 highly expressing cells are obtained by binding to CD44 competitively with the low molecular hyaluronic acid. The culture method according to any one of claims 1 to 3, wherein iron oxide produced by a microorganism is added to the medium.