Method for proliferating hematopoietic stem cells and inducing differentiation into monocytes

Abstract

To proliferate hematopoietic stem cells.SOLUTION: Provided is a method for proliferating hematopoietic stem cells. The method includes: inoculating hematopoietic stem cells with a culture medium in a culture vessel; culturing the hematopoietic stem cells for at least two weeks under a condition which prevents mesenchymal stem cells from adhering to a culture surface in the culture vessel; and culturing further the hematopoietic stem cells having adhered to the culture surface in the culture vessel, thereby inducing cluster formation and proliferation of the hematopoietic stem cells under adherent conditions on the culture surface.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a method for proliferating hematopoietic stem cells and a method for inducing differentiation into monocytes, and more particularly to a method for proliferating hematopoietic stem cells and a method for inducing differentiation of the proliferated hematopoietic stem cells into monocytes. [Background technology]

[0002] Hematopoietic stem cells are cells that produce blood cells in the bone marrow, and are known to have the so-called multipotency, that is, the ability to differentiate into blood cells such as white blood cells, red blood cells, and platelets. It is expected that the use of hematopoietic stem cells with such properties will enable the treatment of various diseases, including blood diseases. For example, Patent Document 1 discusses a method for culturing hematopoietic stem cells for hematopoietic stem cell transplantation. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2011-55768 A [Patent Document 2] JP 2009-65914 A Summary of the Invention [Problem to be solved by the invention]

[0004] However, although hematopoietic stem cells are maintained in an undifferentiated state in vivo, it is difficult to maintain the undifferentiated state outside the body (in vitro). Therefore, there is a problem that hematopoietic stem cells in culture differentiate into blood cells before transplantation. To address this problem, for example, Patent Document 2 studies a method for differentiating and expanding hematopoietic stem cells by controlling gene expression.

[0005] However, the technology to proliferate hematopoietic stem cells in an undifferentiated state in vitro without genetic manipulation or the like has not yet been fully established.

[0006] The present invention has been made in view of the above-mentioned circumstances, and aims to proliferate hematopoietic stem cells and to induce differentiation of hematopoietic stem cells proliferated in vitro into monocytes. [Means for solving the problem]

[0007] In light of the above circumstances, the inventors conducted further research and made the novel discovery that hematopoietic stem cells, which are normally cultured in a floating state, can be maintained in an undifferentiated state when adhered to the culture surface of a culture vessel and proliferated therein, leading to the present invention.

[0008] The present invention has been made based on the above findings and has an object to advantageously solve the above problems. A first aspect of the present invention is Hematopoietic stem cells are seeded into a culture vessel together with a medium, Cultivate hematopoietic stem cells for at least two weeks under conditions in which mesenchymal stem cells do not adhere to the culture surface. This is a method for proliferating hematopoietic stem cells, in which the hematopoietic stem cells adhered to the culture surface in the culture vessel are further cultured to form clusters while adhered to the culture surface, thereby proliferating the hematopoietic stem cells. According to such a method for expanding hematopoietic stem cells, it is possible to expand hematopoietic stem cells while maintaining their undifferentiated state, and since it does not require complicated manipulations such as genetic manipulation, it is possible to enhance safety when transplanting the expanded hematopoietic stem cells.

[0009] Furthermore, in the first aspect above, the condition under which the mesenchymal stem cells do not adhere to the culture surface may be that the mesenchymal stem cells do not adhere to the culture surface when cultured in a medium with a serum concentration of 2% or more. In this way, it is possible to prevent mesenchymal stem cells from adhering to and proliferating on the culture surface, and to allow only hematopoietic stem cells to adhere to and proliferate on the culture surface.

[0010] In the first aspect, the water contact angle of the culture surface may be in the range of 90.5 to 91.1°. In this way, hematopoietic stem cells can be efficiently attached to the culture surface.

[0011] In the first aspect, the culture surface may be composed of a norbornene-based polymer or a hydrogenated product thereof, or may be subjected to low protein adsorption treatment. In this way, hematopoietic stem cells can be efficiently attached to the culture surface.

[0012] A second aspect of the present invention is This is a method for inducing differentiation into monocytes, in which hematopoietic stem cells proliferated by the method for proliferating hematopoietic stem cells described in the first aspect above are cultured for two weeks or more, thereby causing a portion of the adherent hematopoietic stem cells to float, thereby inducing differentiation into monocytes. As described above, when the expanded hematopoietic stem cells are cultured for 2 weeks or more, the hematopoietic stem cells that adhere to the culture surface proliferate and form clusters, and maintain the expression of CD34. Meanwhile, some of the adherent hematopoietic stem cells float. The inventors have newly discovered that such cells that do not adhere to the culture surface are induced to differentiate into monocytes. According to such a method for inducing differentiation into monocytes, it is possible to obtain a required amount of monocytes, which are considered difficult to proliferate in vitro, by inducing differentiation from proliferated hematopoietic stem cells. Furthermore, macrophages and dendritic cells can be obtained by further inducing differentiation from monocytes. Effect of the Invention

[0013] According to the present invention, it is possible to proliferate hematopoietic stem cells, and further, it is possible to induce differentiation of hematopoietic stem cells proliferated in vitro into monocytes. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 is a micrograph taken two weeks after culture. [Diagram 2] FIG. 2 is a micrograph taken after 3 weeks of culture. [Diagram 3]FIG. 3 shows micrographs of suspension cells cultured in a COP dish for four weeks. [Figure 4] FIG. 4 is a micrograph of suspension cells cultured in a control dish for 4 weeks. [Diagram 5] FIG. 5 is a micrograph of the floating cells transferred to a new dish and cultured for one week. [Figure 6] FIG. 6 is a micrograph of the cell clusters. [Figure 7] FIG. 7 shows micrographs taken before and after cell harvesting. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Hereinafter, an embodiment of the present invention will be described in detail. <Method for expanding hematopoietic stem cells> A method for expanding hematopoietic stem cells according to a first aspect of the present invention comprises the steps of: Hematopoietic stem cells are seeded into a culture vessel together with a medium, Cultivate hematopoietic stem cells for at least two weeks under conditions in which mesenchymal stem cells do not adhere to the culture surface. The method is characterized in that it is a method for proliferating hematopoietic stem cells, in which the hematopoietic stem cells attached to the culture surface in the culture vessel are further cultured to form clusters while attached to the culture surface, thereby proliferating the hematopoietic stem cells. The present inventors have newly discovered that hematopoietic stem cells can be maintained in an undifferentiated state by proliferating hematopoietic stem cells adhered to a culture surface. Furthermore, the inventors investigated the conditions for efficiently adhering hematopoietic stem cells to a culture surface and made the novel discovery that hematopoietic stem cells can be efficiently adhered to a culture surface by culturing hematopoietic stem cells on a culture surface to which general adhesive cells do not adhere, but to which mesenchymal stem cells, which have stronger adhesive strength than general adhesive cells, adhere in a medium containing less than 0.1% albumin (equivalent to 1-2% FBS), but do not adhere in a medium with an albumin concentration of 0.1% or more. This enabled hematopoietic stem cells to adhere efficiently to the culture surface, and we succeeded in maintaining the undifferentiated state of hematopoietic stem cells with high reproducibility.

[0016] <Method for inducing differentiation into monocytes> The method for inducing differentiation into monocytes according to the second aspect of the present invention comprises the steps of: The present invention is characterized in that the method for inducing differentiation into monocytes comprises culturing hematopoietic stem cells proliferated by the method for proliferating hematopoietic stem cells described in the first aspect for two weeks or more, thereby floating a portion of the adherent hematopoietic stem cells and inducing differentiation into monocytes. The inventors have newly discovered that when proliferated hematopoietic stem cells are cultured for more than two weeks, some of the hematopoietic stem cells adhering to the culture surface float, and the floating hematopoietic stem cells are induced to differentiate into monocytes. As a result, we were able to successfully proliferate monocytes, which are considered difficult to proliferate, by inducing differentiation of hematopoietic stem cells into monocytes. Furthermore, we found that the monocytes obtained by the above differentiation induction were induced to differentiate into macrophages and dendritic cells when further cultured.

[0017] (Hematopoietic stem cells) Examples of hematopoietic stem cells include, but are not limited to, umbilical cord blood-derived hematopoietic stem cells, bone marrow-derived hematopoietic stem cells, etc., and may be derived from any tissue or organ as long as hematopoietic stem cells corresponding to the above can be harvested. For example, since hematopoietic stem cells are also produced in the liver and spleen in an emergency, hematopoietic stem cells derived from the liver, spleen, etc. may be used.

[0018] (Culture container) The shape and form of the culture vessel used for culturing the above-mentioned hematopoietic stem cells may be any shape and form usually used for cell culture, so long as the culture surface satisfies the "condition that mesenchymal stem cells do not adhere to the culture surface", such as, but not limited to, a flask, a dish, a multi-well plate, a culture bag, etc. Furthermore, the material of the culture vessel may be any material that is generally used for cell culture, so long as the culture surface satisfies the "condition that mesenchymal stem cells do not adhere to the culture surface." Examples of the material include, but are not limited to, flasks and dishes whose bottom surfaces have been treated to reduce protein adsorption. Alternatively, the entire culture vessel may be made of a material that satisfies the "condition that mesenchymal stem cells do not adhere to the culture surface." Examples of the culture vessel include, but are not limited to, dishes and multi-well plates whose entire culture vessel is made of norbornene-based polymers and their hydrogenated products.

[0019] (Culture medium) The medium used for seeding and culturing hematopoietic stem cells may be any medium that can be used for culturing hematopoietic stem cells, including, but not limited to, serum-free media for stationary culture such as StemPro-34 (manufactured by Thermo-Fisher). The concentration of serum added to the medium is preferably 2% or more, more preferably 10 to 20%, and even more preferably 15%. The type of serum is, for example, FBS, and is not limited thereto as long as it is a serum that can be used to culture hematopoietic stem cells. In addition, the supplemental factors added to the above-mentioned medium include, but are not limited to, SCF (stem cell factor) (hematopoietic growth factor), TPO (thrombopoietin), Flt3-Ligand (FMS-related tyrosine kinase 3 ligand), etc., and any supplemental factor that can generally be used to culture hematopoietic stem cells may be added.

[0020] (Seeding of hematopoietic stem cells) Methods for seeding hematopoietic stem cells include, for example, a method in which the number of cells is counted, the cells are concentrated by centrifugation to a predetermined concentration, or the cells are diluted by adding culture medium, and then the cells are seeded together with the culture medium in a culture vessel; however, this is not limited to this method, and any method that is typically used for seeding hematopoietic stem cells may be used. Hematopoietic stem cells are, for example, 0.01 to 1 × 10 6 It is preferable that the hematopoietic stem cells are seeded in the culture vessel at a concentration of 100 cells / ml, but this is not limiting and any concentration may be used as long as the hematopoietic stem cells can be cultured in the culture vessel used.

[0021] (Hematopoietic stem cell culture) The seeded hematopoietic stem cells are cultured in an incubator under conditions of, for example, 37°C and 5% CO2. The environment is not limited to this as long as it allows the hematopoietic stem cells to be cultured. The medium is preferably changed about once a week, which is half the frequency of medium change generally performed.

[0022] Up until two weeks after the start of culture, all hematopoietic stem cells are in a floating state, but after two weeks, cells that adhere to the culture surface of the culture vessel appear. The hematopoietic stem cells that adhere to the culture surface of the culture vessel proliferate in an adherent state and form clusters. In this way, hematopoietic stem cells that adhere to the culture surface of the culture vessel, proliferate, and form clusters continue to express CD34, and are considered to maintain the undifferentiated potential of hematopoietic stem cells. On the other hand, cells that do not adhere to the culture surface of the culture vessel and remain floating, as well as cells that have detached from the culture surface in a single cell or cluster state, express CD14, and are considered to have been induced to differentiate into monocytes without maintaining the undifferentiated potential. Here, a cluster refers to a state in which multiple cells gather to form a cell mass.

[0023] After 3 weeks from the start of culture, the culture surface reaches confluence. When some of the floating cells are transferred to a new culture vessel, some of the transferred floating cells adhere to the new vessel in the form of single cells or clusters and begin to proliferate. The adherent cells also continue to express CD34, and it is believed that the undifferentiated potential of hematopoietic stem cells is maintained. On the other hand, cells that remain floating without adhering to the culture surface of the culture vessel, and cells that have detached from the culture surface in the form of single cells or clusters, express CD14, and it is believed that they have been induced to differentiate into monocytes without maintaining their undifferentiated potential. From this, it is believed that it is important to adhere hematopoietic stem cells to the culture surface of the culture vessel in order to maintain the undifferentiated potential of hematopoietic stem cells.

[0024] After 4 weeks from the start of culture, even after some of the floating cells were transferred to a new culture vessel, the floating cells that had detached from the culture surface in the original culture vessel continued to proliferate as single cells or clusters. At this point, the floating cells do not express CD14, but express macrophage markers CD11b, CD64, CD68, and CD163, or dendritic cell markers CD11c, HLA-DR, CD83, and CD371. This suggests that the hematopoietic stem cells that did not adhere to the culture surface of the culture vessel and those that had detached from the culture surface were induced to differentiate into monocytes, and then the monocytes were induced to differentiate into macrophages and dendritic cells.

[0025] (Conditions under which mesenchymal stem cells do not adhere to the culture surface) The condition for reproducibly adhering hematopoietic stem cells to a culture surface is to culture hematopoietic stem cells on a culture surface on which mesenchymal stem cells, which have stronger adhesive strength than general adhesive cells, adhere in a medium containing less than 0.1% albumin (equivalent to 1-2% FBS), but do not adhere in a medium with an albumin concentration of 0.1% or more. Here, general adhesive cells refer to cells, such as fibroblasts, that are known to be usually cultured in a state of adhering to a culture surface.

[0026] Culture vessels are roughly divided into two types: for adherent cells and for suspension cells, and culture vessels for adherent cells are often processed to make it easier for cells to adhere by scratching the culture surface with plasma treatment, introducing functional groups, or coating, etc. On the other hand, even culture vessels for suspension cells can make it easier for cells to adhere by using a medium containing serum, since serum contains cell adhesion factors such as collagen, fibronectin, and laminin.

[0027] However, when a hydrogenated norbornene ring-opening polymer, known as a polymer containing an alicyclic structure, is used as the material for the culture surface, or when a culture vessel with a culture surface treated to reduce protein adsorption is used, it was found that general adherent cells do not adhere to these culture surfaces even in a medium containing 10% FBS, whereas mesenchymal stem cells adhere to the above culture surfaces in a medium containing less than 0.1% albumin, but do not adhere to a medium with an albumin concentration of 0.1% or more.

[0028] Taking the above into consideration, it is possible to determine the conditions under which mesenchymal stem cells do not adhere to the culture surface, depending on the culture vessel and medium used, without excessive trial and error. The condition under which mesenchymal stem cells do not adhere to the culture surface may be, for example, that mesenchymal stem cells do not adhere to the culture surface when cultured in a medium with a serum concentration of 2% or more. Culturing hematopoietic stem cells under such conditions enables the hematopoietic stem cells to adhere to the culture surface and proliferate with high reproducibility.

[0029] (Culture surface) In order to maintain the undifferentiated potential of hematopoietic stem cells, the culture surface needs to be in a state where mesenchymal stem cells do not adhere but where hematopoietic stem cells can adhere. To satisfy such conditions, for example, the culture surface may be composed of a norbornene-based polymer or a hydrogenated product thereof, or may be subjected to low protein adsorption treatment. Norbornene-based polymers and hydrogenated products thereof belong to the category of polymers containing an alicyclic structure, and examples thereof include cycloolefin polymers (COP). Norbornene polymers are obtained by polymerizing norbornene monomers, which are monomers having a norbornene skeleton, and are roughly classified into those obtained by ring-opening polymerization and those obtained by addition polymerization.

[0030] Examples of the polymers obtainable by ring-opening polymerization include ring-opening polymers of norbornene-based monomers, ring-opening polymers of norbornene-based monomers and other monomers capable of ring-opening copolymerization with the norbornene-based monomers, and hydrogenated products thereof. Examples of the polymers obtained by addition polymerization include addition polymers of norbornene-based monomers and addition polymers of norbornene-based monomers and other monomers copolymerizable therewith.

[0031] Among these, saturated norbornene polymers such as hydrogenated ring-opening polymers of norbornene monomers, addition polymers of norbornene monomers and other monomers copolymerizable therewith, and hydrogenated products of the addition polymers are preferred from the viewpoints of heat resistance, mechanical strength, etc., and those that do not have functional groups are particularly preferred from the viewpoint of ease of cell detachment. Here, functional groups refer to atoms or atomic groups that have atoms other than carbon and hydrogen. Examples of functional groups include amino groups, carboxyl groups, hydroxyl groups, acid anhydride groups, and halogen atoms.

[0032] The low protein adsorption treatment refers to a treatment capable of suppressing protein adsorption by surface treatment of the culture surface. Any known surface treatment method that can be used for culture vessels may be used as long as it can reduce protein adsorption without affecting cell culture. For example, Proteosave(R) SS suppresses non-specific adsorption of proteins and peptides by using a surface treatment that creates a hydrophilic surface using a photocrosslinked superhydrophilic polymer.

[0033] (water contact angle) The water contact angle is measured using a known fully automatic contact angle meter (for example, the LCD-400S manufactured by Kyowa Interface Science Co., Ltd.). The surface to be measured is cut out with a circle cutter having a diameter of 30 mm, and five measurement points are taken: the center of the sample and the four vertices of a square with sides of 20 mm each having the center at that point. The radius r and height h of the droplet are calculated, and θ is calculated by tan θ1 = h / r, θ = 2θ1 → θ = 2arctan(h / r) (θ / 2 method). The water contact angle of the culture surface may be in the range of 90.5 to 91.1°. This prevents adhesion of mesenchymal stem cells to the culture surface, and allows hematopoietic stem cells to efficiently adhere to and proliferate on the culture surface. The water contact angle is more preferably 91.0°. The water contact angle can be controlled within the above range by a surface modification treatment such as atmospheric pressure plasma treatment, reduced pressure plasma treatment, vacuum ultraviolet ray treatment, corona treatment, or ozone treatment. EXAMPLES

[0034] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0035] Example 1: Cultivation of hematopoietic stem cells As hematopoietic stem cells (HSCs), umbilical cord blood-derived hematopoietic stem cells (PromoCell, product number: C-12921) were used at a concentration of 2 × 10 4 The cells were cultured in a culture vessel containing 5 ml of hematopoietic stem cell culture medium (PromoCell, product number: C-28021) to which 50 μl of a cytokine mix (PromoCell, product number: C-39890) had been added so as to achieve a concentration of cells / ml.

[0036] The culture vessels used were a 6 cm diameter culture dish (hereinafter referred to as the "COP dish") (water contact angle 91.0°) obtained by injection molding Zeonex (registered trademark) 790R (manufactured by Zeon Corporation), and a surface-treated 6 cm dish for adherent cells (manufactured by SARSTEDT, product number: 83.3901) (hereinafter referred to as the "control dish").

[0037] Up to 2 weeks after cell seeding, cells in both COP and control dishes expressed CD34 (CD34 + ) and were in a floating state (Figure 1). Whether or not the cells expressed CD34 was determined by staining with anti-CD34-FITC (Miltenyi Biotec, product number: 130-081-001) and observing under a fluorescent microscope.

[0038] FIG. 1 is a micrograph taken two weeks after culture. Panel A of Figure 1 is a phase-contrast micrograph of cells cultured in a COP dish. B is a fluorescent micrograph of cells cultured in a COP dish stained with anti-CD34-FITC antibody. C is a phase-contrast micrograph of cells cultured in a control dish; D is a fluorescence micrograph of cells cultured in a control dish stained with anti-CD34-FITC antibody. In Figure 1B and D, the cells stained green are CD34 positive cells (CD34 + ), indicating that they retain the undifferentiated potential of HSCs.

[0039] FIG. 2 is a phase contrast micrograph (×160) taken after 3 weeks of culture. A1 in Figure 2 is a phase-contrast micrograph of hematopoietic stem cells cultured in a COP dish, with the focus on the floating cells. A2 is a phase contrast micrograph of hematopoietic stem cells cultured in a COP dish, with the focus on the adherent cells. B1 is a phase contrast micrograph of hematopoietic stem cells cultured in a control dish, with the focus on the floating cells. B2 is a phase-contrast micrograph of hematopoietic stem cells cultured in a control dish, with the focus focused on the adherent cells. Two weeks after cell seeding, adherent cells appeared in the COP dishes, but few adherent cells appeared in the control dishes.

[0040] The floating cells cultured for 4 weeks after cell seeding were stained with anti-CD34-FITC to determine whether they expressed CD34, and with anti-CD14-PE antibody (Miltenyi Biotec, product number: 130-096-628) to determine whether they expressed CD14, and then observed under a fluorescent microscope.

[0041] FIG. 3 shows micrographs of suspension cells cultured in a COP dish for four weeks. FIG. 3A is a phase contrast micrograph. B is a fluorescence micrograph of cells stained with anti-CD14-PE antibody; C is a fluorescence micrograph of cells stained with anti-CD34-FITC antibody.

[0042] FIG. 4 is a micrograph of suspension cells cultured in a control dish for 4 weeks. FIG. 4A is a phase contrast micrograph. B is a fluorescence micrograph of cells stained with anti-CD14-PE antibody; C is a fluorescence micrograph of cells stained with anti-CD34-FITC antibody. In both the COP dish (FIG. 3) and the control dish (FIG. 4), the number of CD34-expressing cells decreased and the number of CD14-expressing cells increased in the floating cells.

[0043] (Example 2) Subculture of hematopoietic stem cells In Example 1, the cells that adhered to the culture surface, grew, and then floated were transferred in their entirety to new dishes of the same type every week from 3 weeks after the culture. Figure 5 shows phase contrast micrographs of cells transferred to new dishes 3 weeks after seeding in the first dish and cultured for 1 week, where A is the cell cultured in the COP dish and B is the cell cultured in the control dish. Furthermore, if new medium is added to the adherent cells and the culture is continued, floating single cells or cluster cells will grow. These proliferated floating cells can also be passaged in the same way.

[0044] Similar to the dishes in which the cells were initially seeded (Fig. 2), adherent cells appeared in the COP dishes (Fig. 5A), whereas few adherent cells appeared in the control dishes (Fig. 5B). Such passage could be repeated three times at weekly intervals in the COP dishes (from 3 to 5 weeks after seeding in the initial dish), whereas it could be repeated only once in the control dishes.

[0045] The cells adhered to the COP dish shown in Figure 5B above formed clusters. Figure 6A is a phase-contrast micrograph of the clusters. These clusters were stained with anti-CD34-FITC antibody (Figure 6B), but not with anti-CD14-PE antibody (Figure 6C). This indicates that the cells forming the clusters continue to express CD34 and maintain their undifferentiated potential.

[0046] Of the cells that proliferated in the COP dish and the control dish, the number of floating cells at the start of culture in the new dish after subculture and 3 weeks after the start of culture (6 weeks after the start of culture in the first dish) was counted by pipetting to break up the clusters and measuring with a hemocytometer, and the proliferation numbers were compared with those at the start of culture. As shown in Table 1, the cells proliferated about 13 times in the COP dish and about twice in the control dish, meaning that the proliferation rate in the COP dish was more than 6 times higher.

[0047] [Table 1]

[0048] (Example 3) Flow cytometry (FCM) analysis As hematopoietic stem cells (HSCs), bone marrow-derived hematopoietic stem cells (Lonza, product number: 2M-101C / 4Y-101C) were used at 2 × 10 4 The cells were seeded into two types of 6 cm diameter culture dishes (Zeonex (registered trademark) 790R, manufactured by Zeon Corporation, and PrimeSurface (registered trademark) (Proteosave SS), product number: MS-90600, manufactured by Sumitomo Bakelite Co., Ltd.) containing 5 ml of hematopoietic stem cell culture medium StemPro-34 (manufactured by Thermo-Fisher, product number: 10639011) supplemented with 10% FBS to achieve a cell density of 100 cells / ml, and then cultured (hereinafter referred to as "COP dish" and "PS dish", respectively).

[0049] As in Example 1, the cells were in a floating state and hardly proliferated in both the COP dish and the PS dish until 2 weeks after the start of culture. After 2 weeks after the start of culture, adherent cells appeared in both the COP dish and the PS dish. Four weeks after the start of culture, cells were collected from the COP dishes and analyzed by a flow cytometer (FCM). The cells were collected in two ways: (1) by spraying the medium onto the adherent cells (collecting suspended cells + adherent cells), and (2) by not spraying the medium onto the adherent cells (collecting only suspended cells).

[0050] Figure 7 shows phase-contrast micrographs (160x) of floating cells (A1-A4) and adherent cells (B1-B4) in COP dishes (A1-A3 / B1-B3) and PS dishes (A4 / B4) before and after cell recovery. In the dishes A1(COP) and A2(COP) in Figure 7, floating cells were collected without spraying medium. Phase contrast micrographs after collection are shown in B1 and B2 in Figure 7, respectively. In the dishes A3 (COP) and A4 (PS) in Figure 7, the medium was sprayed onto the dishes to recover the floating and adherent cells. Phase contrast micrographs after recovery are shown in B3 and B4 in Figure 7, respectively. As shown in B3 and B4 in Figure 7, almost no adherent cells remain on the dishes that were recovered by spraying. This shows that most of the adherent cells can be recovered by spraying the medium.

[0051] Next, the collected cells were reacted with anti-CD34-PE antibody (Miltenyi Biotec, product number: 130-113-179), anti-CD14-APC antibody (Miltenyi Biotec, product number: 130-110-577), anti-CD64-PE antibody (Miltenyi Biotec, product number: 130-116-197), anti-CD83-FITC antibody (Miltenyi Biotec, product number: 130-104-473), and anti-CD371-APC antibody (Miltenyi Biotec, product number: 130-124-878), and FCM analysis was performed. The results are shown in Table 2. No. 1 to 4 in Figure 7 correspond to A1·B1, A2·B2, A3·B3, and A4·B4 in Figure 7, respectively. The expression rate in Table 2 is the ratio (%) of cells to which the fluorescently labeled antibody bound when the recovered cells were reacted with the fluorescently labeled antibody.

[0052] [Table 2] CD34 + :Human CD34 + hematopoietic stem cells Mo: Monocyte Mφ: Macrophage DC: Dendritic cell

[0053] As shown in Table 2, the percentage of floating cells stained with anti-CD34 antibody in No. 1 and No. 2, where adherent cells remained on the COP dish, was 9% and 3%, respectively, while the percentage of floating cells stained with anti-CD34 antibody in No. 3 and No. 4, where few adherent cells remained, was 32% and 28%, respectively. This result shows that there are many cells that react with anti-CD34 antibody among the cells adherent to the dish.

[0054] Comparative Example 1 The same HSC as in Example 3 was added to 2 × 10 4The cells were seeded into a 6 cm control dish containing 5 ml of hematopoietic stem cell culture medium StemPro-34 (Thermo-Fisher, product number: 10639011) supplemented with 10% FBS to give a cell density of 100 cells / ml, and three types of cytokines, Human SCF (Cat. #300-07-10UG), Human TPO (Cat. #300-18-10UG), and Human Flt3-Ligand (Cat. #300-19-10UG), and cultured for 3 weeks.

[0055] Next, the collected cells were reacted with anti-CD3-FITC antibody (Miltenyi Biotec, product number: 130-113-700), anti-CD11a-APC antibody (Miltenyi Biotec, product number: 130-105-480), anti-CD56-PE antibody (Miltenyi Biotec, product number: 130-113-874), and anti-CD72-PE antibody (Miltenyi Biotec, product number: 130-101-336), and FCM analysis was performed. The results are shown in Table 3.

[0056] [Table 3]

[0057] Even after 3 weeks of culture, the HSCs did not adhere to the dish. As shown in Table 3, the expression rates of CD34 and CD14 in the HSCs were almost the same as those of the adherent cells in Example 2. However, the number of proliferated cells was only about twice as high as that in the previous experiment, indicating a low proliferation rate, as shown in Table 4. Table 4 shows the cell numbers (floating cells counted) after the cells in Table 3 were cultured for an additional three weeks (six weeks after the start of culture).

[0058] [Table 4]

[0059] These results suggest that it is possible to proliferate HSCs in COP and PS dishes while maintaining their undifferentiated state.

Claims

1. Hematopoietic stem cells are seeded into a culture vessel together with a medium, Cultivating hematopoietic stem cells for at least two weeks under conditions in which mesenchymal stem cells do not adhere to a culture surface; The method for proliferating hematopoietic stem cells comprises further culturing the hematopoietic stem cells adhered to the culture surface in the culture vessel, thereby forming clusters while adhered to the culture surface, thereby proliferating the hematopoietic stem cells.

2. The method for expanding hematopoietic stem cells described in claim 1, wherein the condition under which the mesenchymal stem cells do not adhere to the culture surface is that the mesenchymal stem cells do not adhere to the culture surface when cultured in a medium with a serum concentration of 2% or more.

3. 2. The method for expanding hematopoietic stem cells according to claim 1, wherein the water contact angle of the culture surface is in the range of 90.5 to 91.1°.

4. 2. The method for proliferating hematopoietic stem cells according to claim 1, wherein the culture surface is composed of a norbornene-based polymer or a hydrogenated product thereof, or is treated to reduce protein adsorption.

5. A method for inducing differentiation into monocytes, comprising culturing hematopoietic stem cells proliferated by the hematopoietic stem cell proliferation method described in claim 1 for two weeks or more, thereby floating a portion of the adherent hematopoietic stem cells and inducing differentiation into monocytes.

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