Cell culture device and cell culture method

Abstract

To provide a device for cell culture which can be manufactured simply and at a low cost, and can efficiently proliferate a hematopoietic stem cell and a hemopoietic precursor cell in vitro in a holding state of self replication ability and pluripotency of at least either one of the cells, and a cell culture method in which the hematopoietic stem cell and the hemopoietic precursor cell can be efficiently proliferated in vitro in a holding state of self replication ability and pluripotency of at least either one of the cells.SOLUTION: A device for cell culture having a substrate equipped with a culture part used for culturing at least either one of a hematopoietic stem cell and a hemopoietic precursor cell, in which the culture part has a plurality of micro pores, and Young's modulus of the culture part which is measured in accordance with JIS K 7161-1 and JIS K 7161-2 is at least 3 GPa.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a cell culture device and a cell culture method. [Background technology]

[0002] Hematopoietic stem cells (HSCs) are cells that possess both the multipotency to differentiate into various blood cell lineages, such as leukocytes (neutrophils, eosinophils, basophils, lymphocytes, monocytes, macrophages, etc.), erythrocytes, platelets, mast cells, and dendritic cells, and the self-renewal ability to self-replicate while maintaining this multipotency. Hematopoietic stem cells are known to follow a differentiation lineage in which they first differentiate into hematopoietic progenitor cells (also called "pluripotent hematopoietic progenitor cells") and then differentiate into various blood cell lineages via various progenitor cells. Therefore, both hematopoietic stem cells and hematopoietic progenitor cells are important cells that can be applied to the treatment of blood cancers such as leukemia, malignant lymphoma, and multiple myeloma.

[0003] Hematopoietic stem cells and hematopoietic progenitor cells each have a diameter of approximately 10 μm to 15 μm and generally reside in a special microenvironment called a niche within the bone marrow. It is believed that these cells maintain a balance between quiescence, self-renewal, and differentiation through crosstalk between hematopoietic stem cells or hematopoietic progenitor cells and through humoral factors and intercellular adhesion factors from the surrounding environment. The physical space of this microenvironment is the cancellous bone within the bone marrow. Therefore, in recent years, attempts have been made to mimic the structure of cancellous bone as a scaffold for the in vitro proliferation of hematopoietic stem cells and hematopoietic progenitor cells.

[0004] For example, a method for culturing hematopoietic progenitor cells in vitro using a porous solid matrix has been proposed (see Patent Document 1). The porous solid matrix has an open-cell structure in which the internal pores are reticulated and connected. However, producing a porous solid matrix with such a unified microstructure is not only industrially difficult, but also involves high costs, making mass production difficult. Furthermore, it has been proposed to coat the porous solid matrix with a metal to reinforce the structure and improve cell adhesion to the solid matrix, but this method is difficult to achieve uniform coverage of the pores inside the porous matrix, resulting in a low yield and high costs.

[0005] Also proposed is a method for culturing undifferentiated cells such as human ES cells using a culture carrier in which a plurality of porous recesses are arranged in a matrix on the surface of a substrate made of ceramic or glass (see Patent Document 2). ES cells lose their undifferentiated state when the colony size exceeds a certain level, but the proposed culture carrier can obtain cell masses that have proliferated in an undifferentiated state by controlling the colony size using the recesses. However, the proposed culture carriers have a problem in that the ratio of depth to diameter (hereinafter sometimes referred to as "aspect ratio") is too small for application to niches (microenvironments) for hematopoietic stem cells and hematopoietic progenitor cells, resulting in sparse cell density. Furthermore, the use of ceramics or other materials as culture carrier materials is costly, making them unsuitable for mass culture.

[0006] Therefore, a cell culture device and a cell culture method that mimic the structure of cancellous bone as a scaffold for in vitro proliferation of at least one of hematopoietic stem cells and hematopoietic progenitor cells, can be produced simply and at low cost, and can efficiently proliferate at least one of hematopoietic stem cells and hematopoietic progenitor cells while maintaining their self-renewal ability and pluripotency, have not yet been provided, and there is currently a strong demand for such a device and a cell culture method. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Special Publication No. 2001-517428 [Patent Document 2] Japanese Patent Application Laid-Open No. 2008-306987 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention aims to solve the above-mentioned problems of the prior art and to achieve the following objectives: That is, the present invention aims to provide a cell culture device that can be produced easily and at low cost and that allows efficient in vitro proliferation of hematopoietic stem cells and / or hematopoietic progenitor cells while maintaining their self-renewal ability and pluripotency, and a cell culture method that allows efficient in vitro proliferation of hematopoietic stem cells and / or hematopoietic progenitor cells while maintaining their self-renewal ability and pluripotency. [Means for solving the problem]

[0009] The means for solving the above problems are as follows: <1> A cell culture device having a substrate with a culture section used for culturing at least one of hematopoietic stem cells and hematopoietic progenitor cells, The culture section has a plurality of micropores, The cell culture device is characterized in that the culture section has a Young's modulus of at least 3 GPa as measured in accordance with JIS K 7161-1 and JIS K 7161-2. <2> The material of the culture section is polystyrene. <1> 1. The cell culture device according to claim 1. <3> The ratio [H / La] of the average depth (H) of the micropores to the average length (La) of the openings of the micropores is 1.0 to 2.0. <1> from <2> 10. The cell culture device according to claim 9, wherein the cell culture device is a <4> The average length of the opening of the micropores is 30 μm to 80 μm. <1> from <3> The cell culture device according to any one of the above items. <5> The average depth of the micropores is 30 μm to 160 μm. <1> from <4> The cell culture device according to any one of the above items. <6> The aforementioned <1> from <5> The cell culture method is characterized by culturing at least one of hematopoietic stem cells and hematopoietic progenitor cells using the cell culture device according to any one of the above. [Effects of the Invention]

[0010] According to the present invention, it is possible to solve the above-mentioned problems of the prior art, achieve the above-mentioned object, and provide a cell culture device that can be manufactured simply and at low cost and that allows efficient in vitro proliferation of at least one of hematopoietic stem cells and hematopoietic progenitor cells while maintaining their self-renewal ability and pluripotency, and a cell culture method that allows efficient in vitro proliferation of at least one of hematopoietic stem cells and hematopoietic progenitor cells while maintaining their self-renewal ability and pluripotency. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic explanatory diagram (perspective view) showing an example of a state in which a cell culture device 20 is placed in a well 1 of a known culture vessel (96-well plate). [Figure 2A] FIG. 2A is a schematic explanatory diagram (perspective view) showing an example of a cell culture device 20 having a culture section 21 and a removal section 22. As shown in FIG. [Figure 2B] FIG. 2B is a cross-sectional view of the cell culture device 20 of FIG. 2A taken along the line AA. [Figure 3] 3 is an enlarged top view of a portion of the cell seeding surface 21a of the culture section 21 having a plurality of micropores 30 of the cell culture device 20. X and Y each independently represent the length of one side of the opening 31 of the micropore 30, and p represents the pitch. [Figure 4] Fig. 4 is a cross-sectional view taken along line BB of the culture section 21 having a plurality of micropores 30 in Fig. 3. X represents the length of one side of the opening 31 of the micropore 30, p represents the pitch, and h represents the depth of the micropore 30. [Figure 5A]Figure 5A is a diagram showing an example of a method for forming micropores in the substrate of a cell culture device, and is a schematic explanatory diagram showing an example of a process of matching a master 40 on which the inverted shapes of multiple micropores on the surface of the culture section of the cell culture device are engraved with a resin film 41 as the substrate. [Figure 5B] Figure 5B is a diagram showing an example of a method for forming micropores in the substrate of a cell culture device, and is a schematic explanatory diagram showing an example of a process for pressing a master 40 against a resin film 41 after the process shown in Figure 5A. [Figure 5C] Figure 5C is a diagram showing an example of a method for forming micropores in the substrate of a cell culture device, and is a schematic explanatory diagram showing an example of a process in which, after the process shown in Figure 5B, the resin film 41 is peeled off from the master 40 to obtain a substrate in which the shape of the master 40 is transferred to the resin film 41. [Figure 6] FIG. 6 is a schematic explanatory diagram (top view) showing an example of a process for punching a cell culture device having a culture section 21, a removal section 22, and a connection section 23 from a resin film 41. [Figure 7] 7 shows the results of analyzing cell surface markers of hematopoietic stem cells for cells cultured using the cell culture devices of Example 1 and Comparative Example 1, and for cells cultured as a control. The vertical axis shows the number of cells in the hematopoietic stem cell fraction that is CD34-positive, CD90-positive, and CD45RA-negative (hereinafter, sometimes referred to as [CD34+, CD90+, CD45RA- cells]). [Figure 8] 8 shows the results of analyzing cell surface markers of hematopoietic stem cells for cells cultured using the cell culture devices of Examples 1 to 3. The vertical axis shows the number of cells in the hematopoietic stem cell fraction [CD34+, CD90+, CD45RA- cells]. [Figure 9A] 9A is a diagram showing an example of a photograph of cord blood-derived CD34-positive cells observed with a phase-contrast microscope after 7 days of culture using the cell culture device of Example 1. The scale bar is 100 μm. [Figure 9B]9B is an example of a photograph of cord blood-derived CD34-positive cells observed under a phase-contrast microscope after 7 days of culture using the cell culture device of Example 2. The scale bar is 50 μm. [Figure 9C] 9C is an example of a photograph of cord blood-derived CD34-positive cells observed under a phase-contrast microscope after 7 days of culture using the cell culture device of Example 3. The scale bar is 200 μm. [Figure 10] 10 shows the results of analyzing cell surface markers of hematopoietic stem cells for cells cultured using the cell culture devices of Examples 1 and 6. The vertical axis shows the number of cells in the hematopoietic stem cell fraction [CD34+, CD90+, CD45RA- cells]. [Figure 11] 11 shows the results of analyzing cell surface markers of hematopoietic stem cells for cells cultured using the cell culture devices of Examples 1, 2, 4, and 5. The vertical axis shows the number of cells in the hematopoietic stem cell fraction [CD34+, CD90+, CD45RA- cells]. [Figure 12] 12 shows the results of analyzing cell surface markers of hematopoietic stem cells for cells cultured using the cell culture devices of Examples 1, 2, 4, 5, and 6. The vertical axis shows the cell counts of hematopoietic stem cells and hematopoietic progenitor cell fractions [CD34+ cells]. [Figure 13] 13 shows the results of a methylcellulose colony assay of cells cultured using the cell culture devices of Examples 1 to 3. The vertical axis represents the number of colonies. From the bottom of the graph, dark gray represents the number of colonies of granulocytic and monocytic progenitor cells, diagonal lines represent the number of colonies of early erythroid progenitor cells, and light gray represents the number of mixed colonies containing multiple blood cell lineages. [Figure 14A] FIG. 14A is a schematic explanatory diagram showing an example of a method for calculating the average length (La1) when the shape formed by the outer edge of the opening (top view) of the micropore is polygonal. [Figure 14B] FIG. 14B is a schematic explanatory diagram showing an example of a method for calculating the average length (La2) when the shape formed by the outer edge of the opening (top view) of the micropore is not polygonal. DETAILED DESCRIPTION OF THE INVENTION

[0012] (Cell culture device) The cell culture device of the present invention has a substrate equipped with a culture area used for culturing at least one of hematopoietic stem cells and hematopoietic progenitor cells (hereinafter sometimes simply abbreviated as "cells"), and may further have other components as necessary.

[0013] The cell culture device may be used alone for culturing at least one of hematopoietic stem cells and hematopoietic progenitor cells, or may be used in the form of an insert in a known culture vessel.

[0014] When the cell culture device is used alone for culturing at least one of hematopoietic stem cells and hematopoietic progenitor cells, the shape of the device is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include shapes similar to those of known culture vessels.

[0015] As used herein, the term "insert" refers to a device that is placed inside a well or dish of a known culture vessel. When the cell culture device is used as the insert, the cell culture device may be placed in contact with or not in contact with the bottom of the well or dish of the known culture vessel, as long as it can culture at least one of hematopoietic stem cells and hematopoietic progenitor cells in the culture section.

[0016] <Base material> The substrate comprises a culture section, and may further comprise other members as required. The shape of the substrate is not particularly limited as long as it allows for the arrangement of multiple micropores in the culture area of ​​the substrate, and can be selected appropriately depending on the purpose, for example, in a sheet, film, plate, or board shape. The substrate may have a single layer structure or a laminated structure.

[0017] The average thickness of the substrate is not particularly limited and can be appropriately selected depending on the depth of the micropores, etc., but is preferably 50 μm to 300 μm, and more preferably 100 μm to 200 μm. An average thickness of the substrate of 50 μm or more is preferable in terms of warping and deflection of the substrate, and a thickness of 300 μm or less is preferable in terms of punching. The average thickness of the substrate is an average value calculated from the thicknesses of 10 arbitrary locations of the substrate measured with a micrometer MDC-25MX (No. 293-230-30, manufactured by Mitutoyo Corporation).

[0018] <<Cultivation Department>> The culture section is a member used for culturing at least one of hematopoietic stem cells and hematopoietic progenitor cells. The culture section has a plurality of micropores, and may further have other configurations as required.

[0019] The shape of the culture section is not particularly limited and can be selected appropriately depending on the purpose. Examples include circles such as a perfect circle (circle) or an ellipse; polygons such as a triangle, a square, a hexagon, or an octagon, where the sides may have different lengths; and combinations of these shapes. When the cell culture device is used as the insert, the shape can be selected appropriately depending on the shape of the culture vessel to be used.

[0020] The Young's modulus of the culture section measured in accordance with JIS K 7161-1 and JIS K 7161-2 is at least 3 GPa. If the Young's modulus of the culture section is less than 3 GPa, the in vitro proliferation efficiency of at least one of hematopoietic stem cells and hematopoietic progenitor cells will be reduced. It is important that the Young's modulus of the cultured portion is at least 3 GPa in order to make the cultured portion into a state closely resembling the intramedullary environment, i.e., into a hard state like cancellous bone. Therefore, there is no particular limit to the upper limit of the Young's modulus of the cultured portion, and it can be appropriately selected depending on the purpose.

[0021] The material of the culture part having a Young's modulus of at least 3 GPa is not particularly limited and can be appropriately selected from commonly used resin materials, such as thermoplastic resins that deform or expand when heated, and ultraviolet-curable resins that harden from a liquid to a solid when exposed to ultraviolet light energy.

[0022] Examples of the thermoplastic resin include polystyrene, polycarbonate, polyamide, polyvinyl alcohol, polylactic acid, and copolymers of polylactic acid and polyglycolic acid. These may be used alone or in combination of two or more. Among these, polystyrene is preferred as the thermoplastic resin.

[0023] Examples of the ultraviolet curable resin include acrylate-based resins, urethane acrylate-based resins, etc. These may be used alone or in combination of two or more.

[0024] -Micropore- It is preferable that the micropores in the culture section are non-through holes (holes) with one end connected to the outside of the culture section in the thickness direction of the culture section (thickness direction of the substrate), as this makes it easier to maintain the depth of the micropores constant.

[0025] The non-through holes may be formed as recesses or protrusions with the surface of the substrate as the reference plane, but are preferably formed as recesses in view of ease of manufacture.

[0026] In the culture section, the non-through holes are preferably provided on only one surface of the culture section. In this case, the surface of the culture section on which the non-through holes are provided is preferably used as a surface for seeding at least one of hematopoietic stem cells and hematopoietic progenitor cells (hereinafter, sometimes referred to as a "cell seeding surface").

[0027] At least one of hematopoietic stem cells and hematopoietic progenitor cells enters the interior of the micropores, whereby the cells reach an appropriate density within the micropores, and due to crosstalk between cells (e.g., paracrine factors, autocrine factors, etc.), they are efficiently proliferated in vitro while maintaining their self-renewal ability and pluripotency. At least one of hematopoietic stem cells and hematopoietic progenitor cells that have entered the interior of the micropores not only proliferate two-dimensionally on the cell-seeding surface within the micropores, but also proliferate three-dimensionally in the depth direction of the micropores, which is advantageous in that the environment within the micropores becomes more spongy bone-like. Thus, it is preferable that at least one of hematopoietic stem cells and hematopoietic progenitor cells is cultured inside the micropores.

[0028] The arrangement of the multiple micropores when the culture section is viewed from above is not particularly limited and can be selected appropriately depending on the purpose, and examples include linear, curved, broken line, concentric, lattice, honeycomb, and combinations of these shapes. Furthermore, the arrangement of the multiple micropores may be regular or irregular, but a regular arrangement is preferred in terms of ease in producing the cell culture device.

[0029] The plurality of micropores may be arranged in a part of the culture section or may be arranged throughout the culture section, but it is preferable that they are arranged throughout the culture section, as this provides good proliferation efficiency for at least one of hematopoietic stem cells and hematopoietic progenitor cells. When the plurality of micropores are arranged in a part of the culture section, there are no particular restrictions on the position and size of the plurality of micropores in the culture section, and they can be selected appropriately depending on the purpose.

[0030] The number of the micropores in the culture section is not particularly limited, and can be appropriately selected depending on the size of the culture section or the cell culture device.

[0031] The shape formed by the outer edges of the openings of the micropores when the culture section is viewed from above is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include circles such as a perfect circle (circle) or an ellipse; polygons such as a triangle, square, hexagon, or octagon, each of whose sides may have different lengths; and combinations of these shapes. Among these, a perfect circle or a regular polygon with equal side lengths is preferred as the shape formed by the outer edges of the openings of the micropores, in terms of ease in manufacturing the cell culture device. The shapes formed by the outer edges of the openings in the multiple micropores may all be the same or may be different, but it is preferable that they are all the same in terms of ease in manufacturing the cell culture device.

[0032] The opening area of ​​the micropores when the culture section is viewed from above is not particularly limited and can be appropriately selected depending on the purpose. -4 mm 2 ~0.01mm 2 is preferable, and 0.0009 mm 2 ~0.0064mm 2 is more preferable, and 0.0016 mm 2 ~0.0036mm 2 It is more preferable that the opening area of ​​the opening of the micropore when the culture section is viewed from above is the area of ​​a figure formed by the outer edge of the opening of the micropore.

[0033] Furthermore, the opening area of ​​the cross section horizontal to the opening of the micropore when the culture section is viewed from above is not particularly limited and can be appropriately selected depending on the purpose, and may or may not change from the bottom to the opening. When the opening area changes from the bottom to the opening of the micropore, it may be a shape that gradually increases.

[0034] The average length (La) of the openings of the micropores is not particularly limited and can be appropriately selected depending on the number of micropores, the average pitch of the openings, etc., but is preferably 15 μm to 100 μm, more preferably 30 μm to 80 μm, and particularly preferably 40 μm to 60 μm. Because hematopoietic stem cells and hematopoietic progenitor cells have diameters of approximately 10 μm to 15 μm, an average length (La) of 15 μm or more is preferred in that at least one of the hematopoietic stem cells and hematopoietic progenitor cells can easily enter the micropores. Furthermore, an average length (La) of 100 μm or less is preferred in that at least one of the hematopoietic stem cells and hematopoietic progenitor cells that have entered the micropores can be grown at an appropriate density, allowing them to efficiently proliferate in vitro while maintaining their self-renewal ability and pluripotency. In this specification, the term "average length (La)" refers to any of the following "average length (La1)," "average length (La2)," and "average length (La3)," depending on the shape formed by the outer edge of the opening of the micropore.

[0035] In the present specification, when the shape formed by the outer edges of the openings of the micropores is polygonal, the average length (La1) of the openings of the micropores is determined as follows. The length of each side of the polygon formed by the outer edge of the opening of one randomly selected micropore is measured, and then the average value (Sa) of all the side lengths is calculated. This is calculated for 10 randomly selected micropores, and the average values ​​are respectively (Sa1), (Sa2), (Sa3), (Sa4), (Sa5), (Sa6), (Sa7), (Sa8), (Sa9), and (Sa10). 10 ) Next, the average values ​​(Sa1) to (Sa 10 ) is calculated and designated as the "average length (La1)." However, when the number of the micropores in the cell culture device is less than 10 (n), the average values ​​(Sa1) to (Sa n ) is calculated and designated as the "average length (La1)." The length of each side of the shape formed by the outer edge of the opening can be measured using a field emission scanning electron microscope S-4700 (manufactured by Hitachi High-Technologies Corporation).

[0036] The average length (La1) will be explained in more detail with reference to Figure 14A. For example, when the shape formed by the outer edge of the opening of the micropore is a rectangle as shown in Figure 14A, the lengths of sides a1, b1, c1, and d1 of the rectangle formed by the outer edge of the opening of one arbitrarily selected micropore are measured, and then the average length (Sa1) of all the side lengths is calculated using the following formula (1-1). Similarly, the average values ​​(Sa2) to (Sa3) of other arbitrarily selected nine micropores are calculated using the following formulas (1-2) to (1-10), respectively. 10 ) is calculated. Next, the average values ​​(Sa1) to (Sa 10 ) can be used to determine the "average length (La1)". Average value (Sa1) = (a1 + b1 + c1 + d1) / 4 Equation (1-1) Average value (Sa2)=(a2+b2+c2+d2) / 4 ··· Formula (1-2) Average value (Sa3) = (a3 + b3 + c3 + d3) / 4 Equation (1-3) Average value (Sa4) = (a4 + b4 + c4 + d4) / 4 Equation (1-4) Average value (Sa5) = (a5 + b5 + c5 + d5) / 4 Equation (1-5) Average value (Sa6)=(a6+b6+c6+d6) / 4 ··· Formula (1-6) Average value (Sa7)=(a7+b7+c7+d7) / 4 ··· Formula (1-7) Average value (Sa8) = (a8 + b8 + c8 + d8) / 4 Equation (1-8) Average value (Sa9) = (a9 + b9 + c9 + d9) / 4 Equation (1-9) Average value (Sa 10 )=(a 10 +b 10 +c 10 +d 10 ) / 4 ··· Formula (1-10) Average length (La1) = [(Sa1) + (Sa2) + (Sa3) + (Sa4) + (Sa5) + (Sa6) + (Sa7) + (Sa8) + (Sa9) + (Sa 10 )] / 10... Equation (1-11)

[0037] In the present specification, when the shape formed by the outer edges of the openings of the micropores is not polygonal, the average length (La2) of the openings of the micropores is determined as follows. The maximum length (Ma1) of the shape formed by the outer edge of the opening of one arbitrarily selected micropore and the maximum length (Ma2) in the direction perpendicular to the maximum length (Ma1) are measured, and then the average value (Ia) of the maximum length (Ma1) and the maximum length (Ma2) is calculated. This is calculated for 10 arbitrarily selected micropores, and the average values ​​(Ia1), (Ia2), (Ia3), (Ia4), (Ia5), (Ia6), (Ia7), (Ia8), (Ia9), and (Ia10) are obtained. 10 ) Next, the average values ​​(Ia1) to (Ia 10 ) is calculated and designated as the "average length (La2)". However, when the number of the micropores in the cell culture device is less than 10 (n), the average values ​​(Ia1) to (Ia n ) is calculated and designated as the "average length (La2)". The maximum length (Ma1) of the shape formed by the outer edge of the opening and the maximum length (Ma2) in a direction perpendicular to the maximum length (Ma1) can be measured using a field emission scanning electron microscope S-4700 (manufactured by Hitachi High-Technologies Corporation).

[0038] The average length (La2) will be explained in more detail with reference to Figure 14B. For example, when the shape formed by the outer edge of the opening of the micropore is an ellipse as shown in Figure 14B, the maximum length (Ma11) (shown by a solid line in Figure 14B) of the ellipse formed by the outer edge of the opening of one arbitrarily selected micropore and the maximum length (Ma21) (shown by a dashed line in Figure 14B) in the direction perpendicular to the maximum length (Ma11) are measured, and then the average value (Ia1) of the maximum length (Ma11) and the maximum length (Ma21) is calculated using the following formula (2-1). Similarly, the average values ​​(Ia2) to (Ia 10 Next, the average values ​​(Ia1) to (Ia 10 ) can be used to determine the "average length (La2)". Average value (Ia1) = (Ma11 + Ma21) / 2 Equation (2-1) Average value (Ia2) = (Ma12 + Ma22) / 2 Equation (2-2) Average value (Ia3) = (Ma13 + Ma23) / 2 Equation (2-3) Average value (Ia4) = (Ma14 + Ma24) / 2 Equation (2-4) Average value (Ia5) = (Ma15 + Ma25) / 2 Equation (2-5) Average value (Ia6) = (Ma16 + Ma26) / 2 Equation (2-6) Average value (Ia7) = (Ma17 + Ma27) / 2 Equation (2-7) Average value (Ia8) = (Ma18 + Ma28) / 2 Equation (2-8) Average value (Ia9) = (Ma19 + Ma29) / 2 Equation (2-9) Average value (Ia 10 )=(Ma1 10 +Ma2 10 ) / 2 ··· Formula (2-10) Average length (La2) = [(Ia1) + (Ia2) + (Ia3) + (Ia4) + (Ia5) + (Ia6) + (Ia7) + (Ia8) + (Ia9) + (Ia 10 )] / 10 ··· Formula (2-11)

[0039] In addition, in the cell culture device, when the shapes formed by the outer edges of the openings of the micropores are a mixture of polygonal and non-polygonal shapes, the average length (La3) is calculated using the following formula (3). Average length (La3) = [Average length (La1) + Average length (La2)] / 2 Equation (3)

[0040] The cross-sectional shape of the micropores in the depth direction (thickness direction of the substrate) is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include a shape in which a part of the circumference of a circle such as a perfect circle or an ellipse is missing (e.g., a semicircle); a polygon such as a triangle (e.g., a V-shape), a quadrangle (e.g., a rectangle, a double-tapered shape, a single-tapered shape), a hexagon, or an octagon, each of which may have different side lengths; and a combination of these shapes (for example, in the cross-section of the micropores in the depth direction, the sides in the depth direction (sides perpendicular to the opening or bottom) are straight, and only the bottom of the micropore is U-shaped). The cross-sectional shapes in the depth direction of the multiple micropores may all be the same or may be different from each other.

[0041] The average depth (H) of the micropores is not particularly limited and can be appropriately selected depending on the thickness of the substrate, etc., but is preferably 15 μm to 160 μm, more preferably 30 μm to 160 μm, and even more preferably 40 μm to 100 μm. As described above, the diameter of hematopoietic stem cells and hematopoietic progenitor cells is approximately 10 μm to 15 μm. Therefore, an average depth (H) of the micropores of 15 μm or more is preferred because at least one of the hematopoietic stem cells and hematopoietic progenitor cells can enter the micropores. Furthermore, an average depth (H) of the micropores of 160 μm or less is preferred because at least one of the hematopoietic stem cells and hematopoietic progenitor cells that have entered the micropores can be grown at an appropriate density, allowing them to efficiently proliferate in vitro while maintaining their self-renewal ability and pluripotency.

[0042] In this specification, the depth (also referred to as "length" or "height") of the micropore from the reference plane of the substrate surface to the bottom of the micropore in the thickness direction of the substrate of the culture section (the direction perpendicular to the reference plane of the substrate surface) is referred to as the "micropore depth (h)." If the bottom of the micropore is not flat, the depth of the deepest part is referred to as the "micropore depth (h)." The micropore depth (h) is measured for 10 randomly selected micropores, and the average value of the depths (h) of the 10 micropores is referred to as the "average depth (H)." The depth (h) of the micropores can be measured using a field emission scanning electron microscope S-4700 (manufactured by Hitachi High-Technologies Corporation).

[0043] The ratio [H / La] of the average depth (H) of the micropores to the average length (La) of the openings (H) (hereinafter sometimes referred to as the "aspect ratio") is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 0.5 or greater, more preferably 1.0 or greater, and particularly preferably 1.1 or greater. An aspect ratio of 0.5 or greater is preferable because it allows at least one of hematopoietic stem cells and hematopoietic progenitor cells to easily enter the micropores, and also because at least one of the hematopoietic stem cells and hematopoietic progenitor cells that have entered the micropores can be grown at an appropriate density, allowing them to efficiently proliferate in vitro while maintaining their self-renewal and pluripotency. The lower limit of the aspect ratio is important in terms of creating a culture region that closely resembles the intrabone marrow environment. Therefore, the upper limit of the aspect ratio is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 2.0 or less in terms of ensuring a sufficient supply of oxygen and nutrients to at least one of the hematopoietic stem cells and hematopoietic progenitor cells that have entered the micropores. The aspect ratios of the plurality of micropores may all be the same or may be different from one another.

[0044] When the form of the array of the plurality of micropores is a regular array, and the shape formed by the outer edge of the opening of the micropore is a shape having a center (for example, a perfect circle, a square, etc.), there is no particular limitation on the average pitch (P) of the openings of the plurality of micropores, and it can be appropriately selected according to the purpose. However, it is preferably satisfied with the following formula (4-1), and more preferably satisfied with the following formula (4-2). When the average pitch (P) exceeds the range of the following formula (4-1), the efficiency of at least one of the hematopoietic stem cells and hematopoietic progenitor cells entering the plurality of micropores deteriorates, and it may not be possible to proliferate efficiently. P≦2La ··· Formula (4-1) 1La<P≦2La ··· Formula (4-2) In the above formula (4-1) and formula (4-2), "P" represents the average pitch, and "La" represents the average length of the opening of the micropore. In this specification, in the case of this aspect, the shortest center-to-center distance between the center of the shape formed by the outer edge of the opening of any one of the arbitrarily selected micropores and the center of the shape formed by the outer edge of the opening of another micropore adjacent to the arbitrarily selected one of the micropores is defined as "pitch (p)". Measure this shortest center-to-center distance for 10 arbitrarily selected micropores, and the average value of the 10 shortest center-to-center distances is defined as "average pitch (P)". The pitch (p) can be measured by observing the surface of the culture part with a field emission scanning electron microscope S-4700 (manufactured by Hitachi High-Technologies Corporation).

[0045] Further, when the form of the array of the plurality of micropores is an irregular array, or the shape formed by the outer edge of the opening of the micropore is a shape without a center, the shortest distance between the outer edge of the opening of any one of the arbitrarily selected micropores and the outer edge of the opening of another micropore adjacent to the arbitrarily selected one of the micropores is defined as "pitch (p)". Measure this shortest distance for 10 arbitrarily selected micropores, and the average value of the 10 shortest distances is defined as "average pitch (P)".

[0046] The pore density of the plurality of micropores in the culture section is not particularly limited and can be appropriately selected depending on the average length (La), average depth (H), average pitch (P) and the like of the openings of the micropores. 2 ~160,000 pieces / cm 2 is preferable, and 20,000 particles / cm 2 ~63,000 pieces / cm 2 It is more preferable that the pore density is 2,000 pores / cm. 2 Less than or 160,000 pieces / cm 2 If the number of cells exceeds this range, it may not be possible to efficiently proliferate at least one of hematopoietic stem cells and hematopoietic progenitor cells.

[0047] The shape formed by the outer edges of the bottoms of the micropores when the culture section is viewed from above is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include the shapes exemplified as the shapes formed by the outer edges of the openings of the micropores. In terms of ease of manufacturing the cell culture device, the shape formed by the outer edges of the bottoms of the micropores when the culture section is viewed from above is preferably the same shape as the shape formed by the outer edges of the openings of the micropores.

[0048] The shape of the bottom of the micropore in a cross section in the depth direction is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include a flat shape, a substantially flat shape, an arc shape, a substantially arc shape, a combination of these shapes, etc. The shapes of the bottoms of the plurality of micropores may all be the same or may be different from one another.

[0049] The average length (Lb) of the bottom of the micropores is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 15 μm to 100 μm, more preferably 30 μm to 80 μm, and even more preferably 40 μm to 60 μm. When the average length (Lb) of the bottom is 15 μm or more, it is preferable because at least one of hematopoietic stem cells and hematopoietic progenitor cells can easily enter the micropores. In this specification, "average length (Lb)" means any of "average length (Lb1)," "average length (Lb2)," and "average length (Lb3)," which will be described below, depending on the shape formed by the outer edge of the bottom of the micropores.

[0050] In this specification, when the shape formed by the outer edge of the bottom of the micropore is polygonal, the average length (Lb1) of the bottom of the micropore can be determined in the same manner as the average length (La1) of the opening of the micropore. Specifically, the length of each side of the polygon formed by the outer edge of the bottom of one arbitrarily selected micropore is measured, and then the average value (Sb) of all the side lengths is calculated. This is calculated for 10 arbitrarily selected micropores, and the average values ​​are respectively given as average values ​​(Sb1), average values ​​(Sb2), average values ​​(Sb3), average values ​​(Sb4), average values ​​(Sb5), average values ​​(Sb6), average values ​​(Sb7), average values ​​(Sb8), average values ​​(Sb9), and average values ​​(Sb10). 10 ) Next, the average values ​​(Sb1) to (Sb 10 ) is calculated and used as the "average length (Lb1)." However, when the number of the micropores in the cell culture device is less than 10 (n), the average values ​​(Sb1) to (Sb n ) is calculated and used as the "average length (Lb1)." The length of each side of the shape formed by the outer edge of the bottom can be measured using a field emission scanning electron microscope S-4700 (manufactured by Hitachi High-Technologies Corporation).

[0051] In this specification, when the shape formed by the outer edge of the bottom of the micropore is not polygonal, the average length (Lb2) of the bottom of the micropore can be determined in the same manner as the average length (La2) of the opening of the micropore. Specifically, the maximum length (Mb1) of the shape formed by the outer edge of the bottom of one arbitrarily selected micropore and the maximum length (Mb2) in the direction perpendicular to the maximum length (Mb1) are measured, and then the average value (Ib) of the maximum length (Mb1) and the maximum length (Mb2) is calculated. This is calculated for 10 arbitrarily selected micropores, and the average values ​​(Ib1), (Ib2), (Ib3), (Ib4), (Ib5), (Ib6), (Ib7), (Ib8), (Ib9), and (Ib10) are obtained. 10 ) Next, the average values ​​(Ib1) to (Ib 10 ) is calculated and used as the "average length (Lb2)". However, when the number of the micropores in the cell culture device is less than 10 (n), the average values ​​(Ib1) to (Ib n ) is calculated and used as the "average length (Lb2)". The maximum length (Mb1) of the shape formed by the outer edge of the bottom and the maximum length (Mb2) in a direction perpendicular to the maximum length (Mb1) can be measured using a field emission scanning electron microscope S-4700 (manufactured by Hitachi High-Technologies Corporation).

[0052] The inner surface of the micropores may be made of the material of the substrate itself or may have been subjected to a surface treatment, but is preferably surface-treated in terms of adhesiveness of at least one of hematopoietic stem cells and hematopoietic progenitor cells. At least one of hematopoietic stem cells and hematopoietic progenitor cells is normally non-adhesive, so surface treatment of the inner surface of the micropores makes them more easily adsorbed, which is advantageous in that at least one of hematopoietic stem cells and hematopoietic progenitor cells is more likely to remain inside the micropores.

[0053] The material used for surface treatment inside the micropores is not particularly limited and can be appropriately selected depending on the purpose from those commonly used in cell culture, and examples include hydrophilic polymers such as MPC polymer (2-methacryloyloxyethyl phosphorylcholine), polyethylene glycol (PEG), polyvinyl alcohol (PVA), etc.; natural polymers such as collagen, fibronectin, vitronectin, proteoglycan, gelatin, lectin, polylysine, etc.; inorganic materials such as hydroxyapatite, etc. These may be used alone or in combination of two or more.

[0054] The method for performing a surface treatment on the inside of the micropores is not particularly limited, and can be appropriately selected from known surface treatment methods depending on the purpose, such as dip coating, spray coating, and graft polymerization.

[0055] The surface treatment is preferably applied to the inner surface of the micropores in terms of adhesiveness of at least one of hematopoietic stem cells and hematopoietic progenitor cells, but may also be applied to the entire culture section or the entire cell culture device.

[0056] <<Other materials>> The other members of the cell culture device are not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include an extraction part, a connection part, and a joining part.

[0057] -Removal section- The removal part is a member used to remove the cell culture device from a culture vessel. The cell culture device has the removal section, which is advantageous in that the cell culture device can be easily removed from the culture vessel and is easy to operate.

[0058] The material of the extraction section is not particularly limited as long as it does not impair the effects of the present invention and can be appropriately selected depending on the purpose, but it is preferable that the material be the same as that of the culture section, as this simplifies the production of the cell culture device. Therefore, the surface of the extraction section may be one in which the plurality of micropores are arranged, similar to the culture section. Furthermore, the surface of the extraction section may be one that has been subjected to the surface treatment, similar to the culture section.

[0059] The shape of the removal part is not particularly limited as long as it allows the cell culture device to be removed from the culture vessel, and can be appropriately selected depending on the purpose. Examples of the shape include a cylindrical shape or a polygonal shape such as a triangular prism, a square prism, a hexagonal prism, or an octagonal prism, where the lengths of the sides may be different; a conical shape or a pyramidal shape such as a triangular pyramid, a square prism, a hexagonal pyramid, or an octagonal pyramid, where the lengths of the sides may be different; a circular shape such as a perfect circle (circle) or an ellipse, or a sheet or plate shape having a polygonal surface such as a triangle, a square, a hexagon, or an octagon, where the lengths of the sides may be different; and a combination of these shapes.

[0060] The size of the removal portion is not particularly limited as long as it allows the cell culture device to be removed from the culture vessel, and can be appropriately selected depending on the purpose.

[0061] The position of the removal portion in the cell culture device is not particularly limited as long as it allows the cell culture device to be removed from the culture vessel, and can be selected appropriately depending on the purpose. Examples include the entire end of the side or diameter of the culture portion in the cell culture device (a shape that surrounds the outer edge of the culture portion), the end of part of the side or diameter of the culture portion in the cell culture device, or any part surrounded by the side or diameter of the culture portion in the cell culture device (for example, the center of the culture portion).

[0062] Furthermore, the removal section is preferably provided on the surface of the culture section having the micropores, which makes it possible to easily identify the surface of the cell culture device having the micropores, i.e., the cell seeding surface, and is advantageous in that it provides excellent operability when the cell culture device is used as an insert.

[0063] The angle of the removal section relative to the cell seeding surface of the culture section is not particularly limited as long as it allows the cell culture device to be removed from the culture vessel, and can be selected appropriately depending on the purpose. For example, when the cell culture device is used as an insert, it can be designed appropriately depending on the shape of the culture vessel to which the cell culture device is applied.

[0064] The number of removal sections in the cell culture device is not particularly limited as long as the cell culture device can be removed from the culture vessel, and can be selected appropriately depending on the purpose, and may be one or more.

[0065] -Connection- The connection part is a member that connects the culture part and the removal part. The cell culture device having the connection part is advantageous in that a step for connecting the culture part and the removal part is not required when manufacturing the cell culture device. Furthermore, if the connection part has a bendable shape or structure, it is also advantageous in that a cell culture device having the culture part and the removal part can be easily manufactured by simply bending the connection part.

[0066] The material of the connection part is not particularly limited as long as it does not impair the effects of the present invention and can be appropriately selected depending on the purpose, but it is preferable that the material be the same as that of the culture part, as this simplifies the production of the cell culture device. Therefore, the surface of the connection part may be one in which the plurality of micropores are arranged, similar to the culture part. Furthermore, the surface of the connection part may be one that has been subjected to the surface treatment, similar to the culture part.

[0067] The shape of the connection part is not particularly limited as long as it can connect the culture part and the removal part, and can be appropriately selected depending on the purpose. Examples include cylindrical shapes, or polygonal shapes such as triangular, square, hexagonal, and octagonal prisms, where the lengths of each side may be different; conical shapes, or pyramidal shapes such as triangular, square, hexagonal, and octagonal pyramids, where the lengths of each side may be different; circular shapes such as perfect circles (circles) and ellipses, or sheet or plate shapes having polygonal surfaces such as triangular, square, hexagonal, and octagonal, where the lengths of each side may be different; and combinations of these shapes.

[0068] The size of the connection part is not particularly limited as long as it can connect the culture part and the removal part, and can be appropriately selected depending on the purpose, but it is preferable that the size of the connection part is such that the area occupied by the culture part is as small as possible, which is advantageous in that a large area can be secured in the culture part for culturing at least one of hematopoietic stem cells and hematopoietic progenitor cells, and at least one of hematopoietic stem cells and hematopoietic progenitor cells can be efficiently proliferated.

[0069] The position of the connection part in the cell culture device is not particularly limited as long as it can connect the culture part and the removal part, and can be selected appropriately depending on the purpose. Examples include the entire end of the side or diameter of the culture part in the cell culture device (a shape that surrounds the outer edge of the culture part), the end of part of the side or diameter of the culture part in the cell culture device, or any part surrounded by the side or diameter of the culture part in the cell culture device (for example, the center of the culture part).

[0070] The number of connection parts in the cell culture device is not particularly limited as long as it can connect the culture part and the removal part, and can be selected appropriately depending on the purpose, and may be one or more.

[0071] -Joining part- The connecting portion is a member that connects one culture portion to another culture portion, or one connecting portion to another connecting portion. When the cell culture device is used as the insert and applied to a culture vessel having multiple wells, if the cell culture device does not have the connecting portion, the cell culture device must be inserted into the multiple wells one by one and then removed one by one after culture, making the operation complicated. On the other hand, if the cell culture device has the connecting portion, the cell culture device can be inserted into the multiple wells in a single operation and can be removed from the multiple wells in a single operation after culture, which is advantageous in terms of excellent operability.

[0072] The material of the connecting portion is not particularly limited as long as it does not impair the effects of the present invention, and can be selected appropriately depending on the purpose. However, it is preferable that the connecting portion be made of the same material as the culture portion, as this makes it easier to manufacture the cell culture device.

[0073] The shape of the connecting portion is not particularly limited as long as it can connect one culture portion to another culture portion, or one connecting portion to another connecting portion, and can be selected appropriately depending on the purpose. Examples include cylindrical shapes, or polygonal shapes such as triangular, square, hexagonal, and octagonal prisms, where the lengths of each side may be different; conical shapes, or pyramidal shapes such as triangular, square, hexagonal, and octagonal pyramids, where the lengths of each side may be different; circular shapes such as perfect circles (circles) and ellipses, or sheet or plate shapes having polygonal surfaces such as triangular, square, hexagonal, and octagonal, where the lengths of each side may be different; and combinations of these shapes.

[0074] The position, size, and number of the connecting portions are not particularly limited as long as they can connect one culture portion to another culture portion, or one connecting portion to another connecting portion, and can be appropriately selected depending on the purpose. When the cell culture device is used as the insert, the connecting portions can be appropriately designed depending on the shape of the culture vessel to which the cell culture device is applied, etc. The cell culture device having the connecting portion may have a shape that can cover the wells of a culture vessel having multiple wells, which is advantageous in that the culture vessel can be recycled by removing the cell culture device from the culture vessel after the culture is completed.

[0075] One embodiment of the cell culture device of the present invention, when used as an insert for a 96-well plate, will be specifically described below with reference to Figures 1 to 4, but the present invention is not limited to this embodiment.

[0076] FIG. 1 is a perspective view showing a cell culture device 20 having a circular culture section 21 that is substantially the same shape as the bottom of the well 1 inserted into one well 1 of a known 96-well plate, with the culture section 21 in contact with the bottom surface of the well 1. FIG. 2A is a perspective view showing an overall image of the cell culture device 20 placed in the well of FIG. 1. The cell culture device 20 has a removal section 22, and the cell culture device 20 can be inserted into or removed from the well 1 by grasping the removal section 22 with tweezers or the like. FIG. 2B is a cross-sectional view taken along line AA in FIG. 2A. The culture section 21 and the removal section 22 in the cell culture device 20 are bent at the connection section 23 so that the cell seeding surface 21a (the surface on which non-through holes are provided as micropores) of the culture section 21 is on the surface opposite to the surface that contacts the bottom of the well 1, and the angle T formed between the cell seeding surface 21a and the removal section 22 is less than 180°, preferably about 90°. By adopting such a shape, the cell seeding surface 21a of the cell culture device 20 can be placed in the well 1 without any mistake.

[0077] FIG. 3 is an enlarged view (top view) of a portion of the cell seeding surface 21a of the culture section 21, showing an embodiment in which a plurality of micropores 30 with rectangular openings are provided on one surface of the culture section 21. FIG. 4 is a cross-sectional view taken along line BB in FIG. 3, showing an embodiment in which the cell seeding surface 21a has openings 31. FIG. 4 shows an embodiment in which the openings 31 and the bottom 32 have substantially the same shape and size. In this embodiment, the length X of one side of the opening 31 and the length Y of the other side of the same micropore 30 may be the same or different. The preferred ranges for the average length (La), average pitch P, average depth H, and aspect ratio [H / La] of the openings of the plurality of micropores 30 are as described above. 1, with the cell culture device 20 placed in the well 1, at least one of hematopoietic stem cells and hematopoietic progenitor cells suspended in a culture medium is seeded into the well 1, whereby at least one of the hematopoietic stem cells and hematopoietic progenitor cells settle in the culture medium, enter the micropores 30 of the culture section 20, and are adsorbed to the interior of the micropores 30. At this time, at least one of the hematopoietic stem cells and hematopoietic progenitor cells can enter the interior of the micropores 30 at an appropriate density, so that the cells influence each other and are amplified while maintaining an undifferentiated state.

[0078] <Manufacturing method> The method for manufacturing the cell culture device is not particularly limited and can be appropriately selected depending on the purpose. For example, there can be mentioned a method in which a plurality of micropores are formed on the surface of a substrate having a Young's modulus of at least 3 GPa measured in accordance with JIS K 7161-1 and JIS K 7161-2, and if necessary, an extraction section and a connection section are further formed.

[0079] The method for creating multiple micropores on the surface of the substrate is not particularly limited, and can be appropriately selected from known molding methods depending on the material of the substrate to be used, etc., and examples include heat compression molding, transfer molding, injection molding, extrusion molding (T-die method, etc.), laminate molding, and vacuum molding.

[0080] An example of a method for manufacturing a cell culture device of the present invention will be specifically described below with reference to FIGS. 5A to 6, but the method for manufacturing a cell culture device is not limited to this method.

[0081] First, a master 40 is prepared, which has a surface structure in which the shape of the plurality of micropores on the surface of the culture section is inverted. The material of the master 40 is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include metal, glass, silicon, resin, etc. These may be used alone or in combination of two or more. Examples of the metal include iron-based, aluminum-based, copper-based, and stainless steel such as SUS. These may be used alone or in combination of two or more. Among these, iron-based or stainless steel surfaces coated with electroless Ni-P, and oxygen-free copper are preferred. The resin preferably has a higher glass transition temperature (Tg) than the material of the substrate.

[0082] The method for processing the surface structure on the master 40 is not particularly limited and can be appropriately selected from known methods, such as mechanical cutting, laser drawing, laser interference drawing, electron beam drawing, etching, etc. Among these, mechanical cutting is preferred, and precision machining using single crystal diamond is more preferred.

[0083] When a film-like thermoplastic resin (hereinafter sometimes referred to as "resin film") is used as the material of the substrate, as shown in FIG. 5A, a resin film 41 is placed against a master 40 having a surface structure (convex shape) that is the inverse of the shape of the concave portions of the multiple micropores on the surface of the culture section, and pressure is applied while heating. As a result, the convex surface structure of the master 40 is transferred to the resin film 41 as a pressure-bonded pattern. There are no particular restrictions on the heating temperature during the transfer and it can be selected appropriately depending on the purpose, but a temperature higher than the glass transition temperature (Tg) of the resin film 41 is preferred. 5B, the resin film 41 is pressed sufficiently against the master 40. There are no particular restrictions on the pressure during the transfer, and it can be selected appropriately depending on the purpose. Next, as shown in Figure 5C, once the convex surface structure of the master 40 has been transferred to the resin film 41, the mold is cooled and the resin film 41 is peeled off from the master 40, thereby forming multiple micropores 30 on the surface of the resin film 41, which can be used as a culture section.

[0084] Although not shown in the figure, a specific description will be given of a case where the ultraviolet curable resin is used as the material of the base material instead of the resin film 41. First, a UV-curable resin is applied to a master plate having a surface structure in which the shape of the multiple micropores on the surface of the culture section is inverted. Next, a transparent resin film (e.g., a PET film; Cosmoshine A4300 manufactured by Toyobo Co., Ltd.) is attached to the surface of the UV-curable resin opposite to the surface that contacts the master plate, and the UV-curable resin is cured by irradiating it with UV light through the transparent resin film. Next, the cured UV-curable resin is peeled off from the master plate, thereby forming multiple micropores on the surface of the UV-curable resin, which can be used as a culture section.

[0085] FIG. 6 is a top view (a view showing the surface having the micropores) of the resin film 41 as a substrate to which the culture section having the micropores arranged therein, obtained as described above, is attached. The culture section 21 having the micropores arranged therein in a desired shape can be obtained by punching the culture section 21 from the resin film 41. At this time, if necessary, the extraction section 22 having the desired shape can also be obtained by punching. In this case, the culture section 21 and the extraction section 22 may be punched separately or simultaneously. However, when the culture section 21 and the extraction section 22 are punched simultaneously in a form having the connection section 23, it is advantageous in that a cell culture device having the culture section 21 and the extraction section 22 can be easily manufactured by bending the connection section 23. Figure 6 illustrates a cell culture device having a roughly rectangular extraction section 22 at a portion of the end of the diameter of a circular culture section 21, but the size, position, shape, etc. of the culture section 21 and extraction section 22 are not limited to this.

[0086] Furthermore, Figure 6 shows a method for simultaneously manufacturing a cell culture device having a culture section 21, an extraction section 22, and a connection section 23 from a single resin film 41 having a plurality of micropores. Therefore, not only the culture section 21 but also the extraction section 22 and the connection section 23 may have a surface structure similar to that of the culture section 21 (i.e., a plurality of micropores), but the cell culture device of the present invention may also have the extraction section and the connection section manufactured from a substrate different from that of the culture section.

[0087] <Application> The cell culture device can be easily produced at low cost and can efficiently grow hematopoietic stem cells and / or hematopoietic progenitor cells in vitro while maintaining their self-renewal ability and pluripotency. Therefore, the cell culture device can be suitably used as a scaffold for culturing and growing hematopoietic stem cells and / or hematopoietic progenitor cells in vitro. The device can also be suitably used in the cell culture method of the present invention described below. Furthermore, the device can also be suitably used in research on the maintenance, proliferation, differentiation, etc. of hematopoietic stem cells.

[0088] Furthermore, after the cell culture device has been used to culture at least one of hematopoietic stem cells and hematopoietic progenitor cells, if necessary, a factor or the like that differentiates at least one of hematopoietic stem cells and hematopoietic progenitor cells into desired hematopoietic cells may be added to the cell culture device, and the cell culture device may then be used as is to culture the differentiated hematopoietic cells. The factor that induces differentiation into blood cells is not particularly limited and can be appropriately selected from known factors, such as granulocyte-monocyte colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), macrophage-colony-stimulating factor (M-CSF), thrombopoietin (TPO), erythropoietin (EPO), oncostatin M, and various interleukins (IL).

[0089] The culture vessel is not particularly limited and can be appropriately selected from known culture vessels depending on the purpose. Examples include multi-well microplates with 384 wells, 192 wells, 96 wells, 48 ​​wells, 24 wells, 12 wells, 6 wells, etc.; multi-well chambers or square dishes with 8 wells, 4 wells, 2 wells, etc.; round cell culture dishes with diameters of 35 mm, 60 mm, 100 mm, 150 mm, etc.; and flask-type culture vessels.

[0090] The material of the culture vessel is not particularly limited and can be appropriately selected from known culture vessel materials depending on the purpose, and examples thereof include glass, polystyrene, polypropylene, and polycarbonate.

[0091] <<Hematopoietic stem cells and hematopoietic progenitor cells>> Hematopoietic stem cells are cells that possess both the pluripotency, which allows them to differentiate into all blood cell types, such as leukocytes (neutrophils, eosinophils, basophils, lymphocytes, monocytes, macrophages, etc.), erythrocytes, platelets, mast cells, and dendritic cells, and the self-renewal ability, which allows them to self-replicate while maintaining this pluripotency, and are also called "long-term hematopoietic stem cells." Hematopoietic progenitor cells do not have the self-renewal capacity of hematopoietic stem cells, but are multipotent cells that can differentiate into various blood cell lineage cells.

[0092] The origin of at least one of the hematopoietic stem cells and hematopoietic progenitor cells cultured in the cell culture device is not particularly limited and can be appropriately selected depending on the intended use after culture, and examples include primates such as humans, monkeys, and marmosets; rodents such as mice, rats, and hamsters; birds such as chickens; lagomorphs such as rabbits; ungulates such as pigs, sheep, cattle, goats, and horses; and carnivores such as dogs and cats. Among these, at least one of human-derived hematopoietic stem cells and hematopoietic progenitor cells is preferred from the viewpoint of application to the treatment of blood cancers such as leukemia, malignant lymphoma, and multiple myeloma.

[0093] The tissue from which at least one of hematopoietic stem cells and hematopoietic progenitor cells cultured in the cell culture device originates is not particularly limited and can be appropriately selected depending on the purpose, and examples include bone marrow, umbilical cord blood, peripheral blood, liver, etc. At least one of hematopoietic stem cells and hematopoietic progenitor cells derived from these tissues may be used alone or in combination of two or more types.

[0094] At least one of hematopoietic stem cells and hematopoietic progenitor cells cultured in the cell culture device may be primary cultured cells isolated from the tissue, or may be subcultured cells. Alternatively, the primary cultured cells or subcultured cells may be cryopreserved and then thawed. These may be used alone or in combination of two or more.

[0095] The method for isolating at least one of hematopoietic stem cells and hematopoietic progenitor cells from the tissue is not particularly limited and can be appropriately selected depending on the purpose. For example, a method for isolating at least one of hematopoietic stem cells and hematopoietic progenitor cells from the tissue can be exemplified by using the expression of a cell surface marker specific to at least one of hematopoietic stem cells and hematopoietic progenitor cells (a hematopoietic stem cell-specific surface marker or a hematopoietic progenitor cell-specific surface marker) as an indicator.

[0096] The method for isolating hematopoietic stem cells and / or hematopoietic progenitor cells from the tissue using the expression of a cell surface marker specific to hematopoietic stem cells and / or hematopoietic progenitor cells as an index is not particularly limited and can be appropriately selected from known techniques. For example, there is a method in which an antibody against a hematopoietic stem cell-specific surface marker or a hematopoietic progenitor cell-specific surface marker is used, and cells having the properties of the hematopoietic stem cell-specific surface marker or the hematopoietic progenitor cell-specific surface marker are isolated using a cell sorter, magnetic beads, or the like.

[0097] The cell surface marker specific to at least one of hematopoietic stem cells and hematopoietic progenitor cells can be appropriately selected depending on the animal species from which at least one of the hematopoietic stem cells and hematopoietic progenitor cells is derived. Human hematopoietic stem cells are typically CD34 positive (+), CD90 (Thy1) positive (+), and CD45RA negative (-). In addition to these cell surface markers, other cell surface markers such as CD38 negative (-) and CD49 (CD49f) positive (+) may also be used. Human-derived hematopoietic progenitor cells are typically CD34 positive (+), CD38 negative (-), CD45RA negative (-), and CD90 (Thy1) negative (-). Mouse-derived hematopoietic stem cells include long-term hematopoietic stem cells (LT-HSCs), intermediate-term hematopoietic stem cells (IT-HSCs), and short-term hematopoietic stem cells (ST-HSCs). Markers of long-term hematopoietic stem cells (LT-HSCs) are Sca-1 positive (+), CD117 positive (+), CD34 negative (-), CD48 negative (-), CD49b low expression (low), CD135 negative (-), and CD150 positive (+). Markers of intermediate hematopoietic stem cells (IT-HSCs) are Sca-1 positive (+), CD117 positive (+), CD34 negative (-), CD49 high expression (high), CD135 negative (-), and CD150 positive (+). Markers of short-term hematopoietic stem cells (ST-HSCs) are Sca-1 positive (+), CD117 positive (+), CD34 positive (+), CD48 negative (-), CD135 negative (-), and CD150 negative (-). Mouse-derived hematopoietic progenitor cells are Sca-1 positive (+), CD117 positive (+), CD34 positive (+), CD48 negative (-), and CD135 positive (+).

[0098] (Cell culture method) The cell culture method of the present invention is a method for culturing at least one of hematopoietic stem cells and hematopoietic progenitor cells using the above-described cell culture device of the present invention. The cell culture method preferably includes a seeding step and a culture step, and may further include a harvesting step, a recovery step, a subculture step, etc., and may further include other steps as necessary.

[0099] <Seeding process> The seeding step is a step of seeding at least one of hematopoietic stem cells and hematopoietic progenitor cells into the cell culture device. The seeding step is carried out to introduce at least one of hematopoietic stem cells and hematopoietic progenitor cells into the micropores in the cell culture device.

[0100] In the seeding step, at least one of hematopoietic stem cells and hematopoietic progenitor cells does not need to be introduced into all of the multiple micropores, but it is sufficient that they can be introduced into at least one micropore. However, from the standpoint of cell proliferation efficiency, the greater the number of micropores into which at least one of hematopoietic stem cells and hematopoietic progenitor cells have been introduced, the better.

[0101] In the seeding step, the number of at least one of hematopoietic stem cells and hematopoietic progenitor cells introduced into each micropore is not particularly limited, and can be appropriately selected depending on the average length (La), average depth (H), aspect ratio (H / La), average pitch (P), etc. of the opening of the micropore. The number of at least one of hematopoietic stem cells and hematopoietic progenitor cells introduced into each micropore can be adjusted by the number of at least one of hematopoietic stem cells and hematopoietic progenitor cells initially seeded in the seeding step.

[0102] At least one of the hematopoietic stem cells and hematopoietic progenitor cells used in the seeding step can be the same as those described in the section "<<Hematopoietic stem cells and hematopoietic progenitor cells>>" above.

[0103] The method for seeding at least one of hematopoietic stem cells and hematopoietic progenitor cells into the cell culture device is not particularly limited and can be appropriately selected from known cell seeding methods depending on the purpose, but it is preferable to seed at least one of hematopoietic stem cells and hematopoietic progenitor cells in a single-cell state after the cells have been seeded. Specifically, one example is a method in which at least one of hematopoietic stem cells and hematopoietic progenitor cells in a single-cell state is suspended in a solution such as a culture medium, and the suspension is dropped into the cell culture device using a pipette or the like.

[0104] The method for converting at least one of hematopoietic stem cells and hematopoietic progenitor cells into a single-cell state is not particularly limited and can be appropriately selected from known methods, such as a method involving physical treatment such as pipetting.

[0105] The number of at least one of hematopoietic stem cells and hematopoietic progenitor cells to be seeded into the cell culture device is not particularly limited, and can be selected appropriately depending on the size of the culture section in the cell culture device, the size and number of the micropores in the culture section, the number of desired at least one of hematopoietic stem cells and hematopoietic progenitor cells after culture, etc. For example, when the cell culture device is used as an insert for a known 96-well plate, and the shape and size of the culture section in the cell culture device are approximately the same as the shape and size of the bottom of the wells in the 96-well plate, at least one of hematopoietic stem cells and hematopoietic progenitor cells is preferably seeded at a cell count of 1,000 to 10,000 cells / well, more preferably 1,000 to 5,000 cells / well.

[0106] <Culture process> The culturing step is a step of culturing at least one of the hematopoietic stem cells and the hematopoietic progenitor cells seeded in the seeding step. The culture step is carried out to efficiently proliferate at least one of hematopoietic stem cells and hematopoietic progenitor cells in vitro while maintaining their self-renewal ability and pluripotency.

[0107] The medium (culture solution) used for culturing at least one of hematopoietic stem cells and hematopoietic progenitor cells is not particularly limited and can be appropriately selected from known media. Typically, for the purpose of maintaining the undifferentiated state of at least one of hematopoietic stem cells and hematopoietic progenitor cells, they are cultured in a medium containing cytokines such as stem cell factor (SCF), thrombopoietin (TPO), Flt-3 ligand (FL), interleukin (IL)-3, IL-6, and IL-11, and serum albumin such as bovine serum albumin (BSA), but the culture medium is not limited to these.

[0108] The culture conditions (temperature, carbon dioxide (CO2) concentration, oxygen concentration, etc.) for at least one of hematopoietic stem cells and hematopoietic progenitor cells are not particularly limited and can be appropriately selected from known conditions. The culture temperature for at least one of hematopoietic stem cells and hematopoietic progenitor cells is generally 30°C to 40°C, and about 37°C is preferred. The CO2 concentration when culturing at least one of hematopoietic stem cells and hematopoietic progenitor cells is generally 1% to 10% by volume, and preferably 2% to 5% by volume. The culture conditions for at least one of hematopoietic stem cells and hematopoietic progenitor cells can be adjusted using a commercially available cell culture device such as an incubator.

[0109] The culture period for at least one of hematopoietic stem cells and hematopoietic progenitor cells is not particularly limited and can be appropriately selected depending on the number of at least one of hematopoietic stem cells and hematopoietic progenitor cells desired after culture.

[0110] In the culture step, when the culture period is long, the medium may be appropriately replaced with a new one or circulated, or a subculture step described below may be carried out.

[0111] The cells cultured in the culturing step can be confirmed to be at least one of hematopoietic stem cells and hematopoietic progenitor cells by confirming the expression of a cell surface marker specific to at least one of hematopoietic stem cells and hematopoietic progenitor cells, as described in the section above under <<Hematopoietic Stem Cells and Hematopoietic Progenitor Cells>>. Methods for confirming the expression of the cell surface markers include the aforementioned methods using a cell sorter, magnetic beads, etc., as well as immunostaining, enzyme activity measurement, real-time RT-PCR, etc.

[0112] <Collection process> The collecting step is a step of collecting at least one of the hematopoietic stem cells and the hematopoietic progenitor cells cultured in the culturing step. When the cell culture method includes the recovery step, the collection step can be performed on the cell culture device recovered in the recovery step.

[0113] The method for collecting at least one of the hematopoietic stem cells and hematopoietic progenitor cells cultured in the culture step is not particularly limited and can be appropriately selected from known cell collection methods, such as a method using pipetting, or a method in which the cell culture device (when the cell culture device is used as the insert, the culture vessel) is vibrated by shaking or tapping with a hand. The cells suspended or detached from the cell culture device by the above method can be collected together with the culture medium.

[0114] <Recovery process> The recovery step is a step of recovering the cell culture device together with at least one of the cultured hematopoietic stem cells and hematopoietic progenitor cells from the culture vessel after the culture step, when the cell culture device is used as the insert, or recovering the cell culture device after the collection step.

[0115] The method for recovering the cell culture device is not particularly limited and can be appropriately selected depending on the purpose. For example, the cell culture device can be recovered using tweezers.

[0116] In the recovery step, the removal section or the connection section of the cell culture device is preferably used.

[0117] <Subculture process> The subculture step is a step of further subculturing at least one of the hematopoietic stem cells and the hematopoietic progenitor cells cultured in the culture step.

[0118] The method for the subculture is not particularly limited and can be appropriately selected depending on the purpose, but is preferably performed by repeating the collection step, the seeding step, the culture step, and, if necessary, the recovery step. The subculture step may be performed only once or multiple times.

[0119] When the cell culture method is performed only once, the number of cells that can be obtained is limited, and when the culture period is extended over a long period of time, there is a risk of cell aging or cell death due to an increase in cell density, etc., and the self-renewal ability and pluripotency of at least one of hematopoietic stem cells and hematopoietic progenitor cells may not be maintained. Therefore, by including the subculture step, the cell culture method is advantageous in that it allows the proliferation of at least one of hematopoietic stem cells and hematopoietic progenitor cells in a larger number while maintaining the self-renewal ability and pluripotency of at least one of hematopoietic stem cells and hematopoietic progenitor cells.

[0120] <Application> The cell culture method can efficiently proliferate at least one of hematopoietic stem cells and hematopoietic progenitor cells in vitro while maintaining the self-renewal ability and pluripotency of the cells, and therefore can be suitably used for the in vitro culture of at least one of hematopoietic stem cells and hematopoietic progenitor cells.

[0121] At least one of hematopoietic stem cells and hematopoietic progenitor cells obtained by the cell culture method is preferably used for the treatment of blood cancers such as leukemia, malignant lymphoma, and multiple myeloma, and for research on the maintenance, proliferation, differentiation, etc. of hematopoietic stem cells. At least one of hematopoietic stem cells and hematopoietic progenitor cells obtained by the cell culture method may be cryopreserved by a known method. [Example]

[0122] The present invention will be specifically explained below with reference to examples, comparative examples, and test examples, but the present invention is not limited to these examples and test examples.

[0123] Example 1 <Mold making> Martensitic stainless steel (SUS420J2, width 70 mm, length 70 mm, thickness 8 mm) was used as the mold material. The machined surface of the mold material was treated with electroless Ni-P plating to form a 150 μm-thick plating layer. Next, using an ultra-precision machining center, the plated surface was cut and smoothed with a single-crystal diamond bit with a curvature radius of 5 mm. Next, a fine convex structure (lattice-like) was machined within a 20 mm wide and 20 mm long area of ​​the plated surface using a substantially rectangular single-crystal diamond bit with a tip width of 40.3 μm and a tip angle of 8° (one side). Specifically, the fine convex structure was formed by forming a group of rectangular pillars with a width of 47 μm, a length of 47 μm, a height of 50 μm, a widthwise pitch (center-to-center distance between convex portions in the width direction in the convex structure) of 87.3 μm, and a pitch (center-to-center distance between convex portions in the length direction in the convex structure) of 87.3 μm. This fine convex structure is an inverted structure of the structure to be formed on the surface of the culture portion of the final cell culture device.

[0124] <Forming the substrate for cell culture devices> The substrate used in the cell culture device was molded by heat compression molding using the above mold. Specifically, a 0.1 mm-thick unstretched polystyrene film (hereinafter sometimes simply referred to as "polystyrene film") was used as the substrate for the cell culture device. The polystyrene film was placed on the surface of the mold with the fine convex structure, and a SUS plate (SUS304, width 70 mm, length 70 mm, thickness 1 mm) was placed on the polystyrene film. Each was heated to 130°C without pressure, and once it reached 130°C, a pressure of 0.3 MPa was applied for 300 seconds. Thereafter, to solidify the polystyrene film, the temperature was cooled to 40°C while maintaining the pressure. Next, the pressure was released, and once the load was removed, the polystyrene film was peeled off from the mold to obtain the substrate for the cell culture device.

[0125] <Punching> A carbon dioxide laser was used to cut out a circular culture area with a diameter of 6 mm and a rectangular extraction area with a length of 8 mm and a width of 2 mm from the substrate used in the obtained cell culture device (see Figure 6).

[0126] Example 2 The cell culture device of Example 2 was obtained in the same manner as in Example 1, except that in the <Preparation of mold> of Example 1, the fine convex structure of the mold was changed to a group of rectangular pillars with a width of 15 μm, a length of 15 μm, a height of 15 μm, a pitch (the center-to-center distance between the convex portions in the width direction of the convex structure) of 30.0 μm, and a pitch (the center-to-center distance between the convex portions in the length direction of the convex structure) of 30.0 μm.

[0127] Example 3 The cell culture device of Example 3 was obtained in the same manner as in Example 1, except that in <Preparation of mold> of Example 1, the fine convex structure of the mold was changed to a group of rectangular pillars with a width of 100 μm, a length of 100 μm, a height of 90 μm, and a pitch (center-to-center distance between convex portions in the width direction of the convex structure) of 200.0 μm, and a pitch (center-to-center distance between convex portions in the length direction of the convex structure) of 200.0 μm, and the base material used in the cell culture device was changed to an unstretched polystyrene film with a thickness of 0.2 mm.

[0128] Example 4 The cell culture device of Example 4 was obtained in the same manner as in Example 1, except that in the <Preparation of mold> of Example 1, the fine convex structure of the mold was changed to a group of rectangular pillars with a width of 30 μm, a length of 30 μm, a height of 30 μm, a pitch (center-to-center distance between convex portions in the width direction of the convex structure) of 64.1 μm, and a pitch (center-to-center distance between convex portions in the length direction of the convex structure) of 64.1 μm.

[0129] Example 5 The cell culture device of Example 5 was obtained in the same manner as in Example 1, except that in the <Preparation of mold> of Example 1, the fine convex structure of the mold was changed to a group of rectangular pillars with a width of 80 μm, a length of 80 μm, a height of 80 μm, and a pitch (center-to-center distance between convex portions in the width direction of the convex structure) of 160 μm, and a pitch (center-to-center distance between convex portions in the length direction of the convex structure) of 160 μm.

[0130] Example 6 A cell culture device of Example 6 was obtained in the same manner as in Example 1, except that the base material used in the cell culture device in Example 1 was changed to polycarbonate having a thickness of 0.1 mm.

[0131] (Comparative Example 1) A cell culture device of Comparative Example 1 was obtained in the same manner as in Example 1, except that the base material used in the cell culture device in Example 1 was changed to low-density polyethylene with a thickness of 0.1 mm.

[0132] The cell culture devices of Examples 1 to 6 and Comparative Example 1 are summarized in Table 1 below.

[0133] [Table 1]

[0134] The Young's modulus, average length [La] of the openings of the micropores, average depth [H], and average pitch [P] shown in Table 1 were measured as follows.

[0135] -Young's modulus- The Young's modulus of the substrate (polystyrene) used in Examples 1 to 5, the substrate (polycarbonate) used in Example 6, and the substrate (low-density polyethylene) used in Comparative Example 1 was measured in accordance with JIS K 7161-1 and JIS K 7161-2.

[0136] -Average length of opening [La]- Ten micropores were randomly selected from the surface of the culture area, and the length of each side of the shape formed by the outer edge of the opening was measured using a field emission scanning electron microscope S-4700 (Hitachi High-Technologies Corporation). The average values ​​(Sa1) to (Sa 10 ) was calculated. Next, the average values ​​(Sa1) to (Sa 10 ) was calculated and used as the average length [La] of the opening of the micropores.

[0137] -Average depth [H]- Ten micropores were randomly selected from the surface of the culture section, and the depth (h) of each micropore in the thickness direction of the substrate of the culture section from the reference plane of the substrate surface to the bottom of the micropore (the length in the thickness direction of the cross section of the micropore) was measured using a field emission scanning electron microscope S-4700 (manufactured by Hitachi High-Technologies Corporation).The average depth (h) of the 10 micropores was calculated, and this was used as the average depth [H] of the micropores.

[0138] -Average pitch [P]- The shortest center-to-center distance (pitch (p)) between the center of the shape formed by the outer edge of the opening of one arbitrarily selected micropore on the surface of the culture area and the center of the shape formed by the outer edges of the openings of other micropores adjacent to the arbitrarily selected micropore was measured using a field emission scanning electron microscope S-4700 (Hitachi High-Technologies Corporation). This shortest center-to-center distance was measured for 10 arbitrarily selected micropores, and the average value of the shortest center-to-center distances at 10 locations was taken as the average pitch [P].

[0139] (Preparation Example 1: Preparation of CD34-positive cells) CD34-positive cells used in the following test examples were prepared by the following method.

[0140] Ficoll (Nacalai Tesque, Inc.) was dispensed into 50 mL tubes, 12.5 mL each. 250 mL of human umbilical cord blood was placed in a sterilized 500 mL culture bottle, and 250 mL of PBS was added and mixed to obtain a diluted umbilical cord blood solution. Next, 37.5 mL of the diluted umbilical cord blood solution was layered on top of the Ficoll in each tube. The tubes were centrifuged at 1,500 rpm and 25°C for 25 minutes. The separated top layer (containing plasma) was removed, and the layer containing mononuclear cells and CD34+ cells (liquid phase) was collected and transferred to a new 50 mL tube. 25 mL of Iscove's Modified Dulbecco's Medium (IMDM, GE Healthcare) was added to the resulting 25 mL of solution containing mononuclear cells and CD34+ cells, followed by centrifugation at 1,500 rpm and 4°C for 10 minutes, and the supernatant was removed. The resulting precipitate was resuspended by adding 2 mL of staining buffer (SM; PBS containing 2% by volume of fetal bovine serum (FBS, manufactured by Sigma-Aldrich)), and all the tubes were combined into one tube. Here, the number of cells obtained was measured using a Burker-Türk counting chamber, and was found to be 1.10 × 10 9 It was a cell.

[0141] The cells suspended in the SM were centrifuged at 1,500 rpm and 4°C for 10 minutes, and the supernatant was removed. Staining medium (SM) was added to the resulting precipitate, and the cell count was adjusted to 1 x 10 8 The volume was adjusted to 50 μL, and 50 μL of FcR blocking reagent (IMMUNOSTEP) and 50 μL of CD34 microbeads (CD34 MicroBead Kit UltraPure, human, Miltenyi Biotec) were added. After incubating at 4°C for 30 minutes, the volume was adjusted to 50 mL with 4°C staining medium (SM), mixed by inversion, and centrifuged at 1,500 rpm at 4°C for 10 minutes, after which the supernatant was removed. The resulting precipitate was diluted with autoMACS® Running Buffer (Miltenyi Biotec) until the mononuclear cell count reached a maximum of 5 × 10 81 mL of this solution was added to the cells, and CD34-positive cells were separated from this solution using a magnetic bead cell separator (autoMACS (registered trademark), manufactured by Miltenyi Biotec) with an LS column (manufactured by Miltenyi Biotec). The number of isolated CD34-positive cells was 1.16 × 10 7 The number of CD34-negative cells (mononuclear cells) was 2.31 × 10 8 It was a cell.

[0142] (Test Example 1-1: Analysis of surface markers 1) The relationship between Young's modulus of the culture area and proliferation of hematopoietic stem cells was confirmed by the following method.

[0143] <Arrangement of cell culture devices> The cell culture devices of Example 1 and Comparative Example 1 were placed in the wells of a 96-well plate (made of polystyrene, flat bottom, made of TPP). A well without a cell culture device served as a control. Each experiment was carried out in triplicate (n=3).

[0144] <Surface coating treatment for cell culture devices> A fibronectin solution was prepared by diluting fibronectin (manufactured by SouthernBiotech) with PBS(-) (manufactured by Sigma-Aldrich) to 25 μg / mL. 50 μL of the fibronectin solution was dropped onto the culture area of ​​the cell culture device in each well and onto the bottom of the control well, and then the wells were left to stand at 37°C for 1 hour, thereby coating the surface of the culture area or the bottom of the control well with fibronectin. The fibronectin solution was then removed from the wells, and the wells were washed once with PBS.

[0145] <Culture of CD34-positive cells derived from cord blood> A culture medium was prepared by adding 2 mL of 10% (volume) bovine serum albumin (BSA, manufactured by Nacalai Tesque, Inc.), 20 μL of 100 ng / μL stem cell factor (Human, manufactured by SHENANDOAH), 200 μL of 10 ng / μL thrombopoietin (Human, manufactured by SHENANDOAH), and 20 μL of 100 ng / μL Flt-3 ligand (Human, manufactured by SHENANDOAH) to 18 mL of serum-free medium (X-Vivo10, manufactured by Lonza). The cord blood-derived CD34-positive cells obtained in Preparation Example 1 were seeded onto the surface-coated cell culture device and into control wells at 1,000 cells / well, and 200 μL of the culture medium was added to each well. The cells were then statically cultured for 7 days under conditions of 5% CO2 by volume and 37°C.

[0146] <Analysis of surface markers> -Preparation of antibody solution- An antibody solution was prepared by adding 0.7 μL of CD34-APC / Cy7 (BioLegend), 0.7 μL of CD38-PE / Cy7 (BioLegend), 0.7 μL of CD45RA-Brilliant Violet 421 (BioLegend), and 1.1 μL of CD90-APC (BioLegend) to 46.8 μL of PBS containing 2% by volume of fetal calf serum (FCS, Sigma-Aldrich).

[0147] -Confirmation of [CD34+, CD90+, CD45RA- cells]- Using a surface marker for hematopoietic stem cells, the number of hematopoietic stem cells among the cord blood-derived CD34-positive cells before and after culture was determined by the following method. 200 μL of the culture medium containing the cord blood-derived CD34-positive cells (cells before culture) obtained in Preparation Example 1 was transferred to a tube (n=3). After culturing the cord blood-derived CD34-positive cells for 7 days, the cells on the cell culture device were suspended in the culture medium by thoroughly pipetting the cells in the wells. The entire cell suspension obtained from each well was transferred to each tube. 10 μL of the antibody solution was added to each of the tubes containing the cells before and after culturing, and then cultured for 30 minutes at 4° C. in the dark. Next, the tubes were centrifuged at 600 g for 10 minutes, and the supernatant was removed. 250 μL of PBS containing 2% by volume of FCS and 10 μL of cell counting beads for flow cytometry (CountBright TM 5 μL of Absolute Counting Beads (manufactured by Thermo Fisher Scientific) was added, and the number of cells expressing each antibody was counted using a flow cytometer (FACSCanto II, manufactured by Becton Dickinson). Cells that are CD34 positive, CD90 positive, and CD45RA negative [CD34+, CD90+, CD45RA- cells] are hematopoietic stem cells. Figure 7 shows the results of analyzing the surface markers of the cells after culture. The vertical axis shows the average number of cells per well in the hematopoietic stem cell fraction [CD34+, CD90+, CD45RA- cells]. Although not shown in the figure, the average number of cells per well in the hematopoietic stem cell fraction [CD34+, CD90+, CD45RA- cells] before culture was 7 cells.

[0148] 7, the number of hematopoietic stem cells was significantly increased when the cell culture device of Example 1, in which the Young's modulus of the culture section was 3 GPa, was used compared to the control, in which no cell culture device was used. On the other hand, the number of hematopoietic stem cells was decreased compared to the control, in which the cell culture device of Comparative Example 1, in which the Young's modulus of the culture section was less than 3 GPa, was used. It is speculated that this result may be due in part to the fact that the cell culture device of Example 1, in which the Young's modulus of the culture area is 3 GPa, is close to the hard environment of cancellous bone within the bone marrow.

[0149] (Test Example 1-2: Analysis of surface markers 2) In Test Example 1-1, except that the <positioning of the cell culture device> and <culture of umbilical cord blood-derived CD34-positive cells> were changed to the following methods, the <surface coating treatment of the cell culture device> and <analysis of surface markers> were performed in the same manner as in Test Example 1-1, and the relationship between the average length (La) of the micropore openings and the proliferation of hematopoietic stem cells was confirmed.

[0150] <Arrangement of cell culture devices> The cell culture devices of Examples 1, 2, and 3 were placed in the wells of a 96-well plate (made of polystyrene, flat bottom, manufactured by TPP). A well without a cell culture device served as a control. Each experiment was carried out in triplicate (n=3).

[0151] <Culture of CD34-positive cells derived from cord blood> The cord blood-derived CD34-positive cells obtained in Preparation Example 1 were seeded onto the surface-coated cell culture device and into control wells at 5,000 cells / well, and 200 μL / well of the culture medium prepared in Test Example 1-1 was added. After that, the cells were statically cultured for 7 days under conditions of 5% CO2 by volume and 37°C. After 7 days of culture, the cells cultured in the cell culture devices of Examples 1, 2, and 3 were observed under a phase-contrast microscope, and then <surface marker analysis> was performed in the same manner as in Test Example 1-1.

[0152] The results of the surface marker analysis are shown in Figure 8. The vertical axis shows the average number of cells per well in the hematopoietic stem cell fraction [CD34+, CD90+, CD45RA- cells]. Although not shown in the figure, the average number of cells per well in the hematopoietic stem cell fraction [CD34+, CD90+, CD45RA- cells] before culture was 37 cells.

[0153] 9A to 9C show phase-contrast microscope photographs of the vicinity of the openings of the micropores after 7 days of culture. In Figures 9A to 9C, the areas surrounded by squares are the micropores on the surface of the cell culture device, and it was confirmed that cells had entered the micropores. Since the size of hematopoietic stem cells is approximately 10 μm to 15 μm, in micropores with an average opening length (La) of 15 μm (Example 2), only about one cell was observed per micropore. Furthermore, in micropores with an average opening length (La) of 100 μm (Example 3), multiple cells were observed per micropore, but the gaps between the cells were relatively wide. On the other hand, in micropores with an average opening length (La) of 47 μm (Example 1), when observing the vicinity of the opening, so many cells were observed that there were no gaps between them.

[0154] (Test Example 1-3: Analysis of surface markers 3) In Test Example 1-1, except that the <positioning of the cell culture device> and <culture of umbilical cord blood-derived CD34-positive cells> were changed to the following methods, the <surface coating treatment of the cell culture device> and <analysis of surface markers> were performed in the same manner as in Test Example 1-1, and the relationship between the Young's modulus of the culture area and the proliferation of hematopoietic stem cells was further confirmed.

[0155] <Arrangement of cell culture devices> The cell culture devices of Example 1 and Example 6 were placed in the wells of a 96-well plate (made of polystyrene, flat bottom, manufactured by TPP), respectively. Each experiment was carried out in duplicate (n=2).

[0156] <Culture of CD34-positive cells derived from cord blood> The cord blood-derived CD34-positive cells obtained in Preparation Example 1 were seeded onto the surface-coated cell culture device and into control wells at 5,000 cells / well, and 200 μL / well of the culture medium prepared in Test Example 1-1 was added. After that, the cells were statically cultured for 7 days under conditions of 5% CO2 by volume and 37°C. After 7 days of culture, the cells cultured in the cell culture devices of Examples 1 and 6 were subjected to <surface marker analysis> in the same manner as in Test Example 1-1.

[0157] The results of the analysis of cell surface markers after culture are shown in Figure 10. The vertical axis shows the average number of cells in the hematopoietic stem cell fraction [CD34+, CD90+, CD45RA- cells] per well.

[0158] (Test Example 1-4: Analysis of surface markers 4) In Test Example 1-1, except that the <positioning of the cell culture device> and <culture of umbilical cord blood-derived CD34-positive cells> were changed to the following methods, the <surface coating treatment of the cell culture device> and <analysis of surface markers> were performed in the same manner as in Test Example 1-1, and the relationship between the average length [La] and average depth [H] of the openings of the micropores and the proliferation of hematopoietic stem cells was confirmed when the aspect ratio [H / La] of the micropores was kept approximately the same.

[0159] <Arrangement of cell culture devices> The cell culture devices of Examples 1, 2, 4, and 5 were placed in the wells of a 96-well plate (made of polystyrene, flat bottom, manufactured by TPP). Each experiment was carried out in duplicate (n=2).

[0160] <Culture of CD34-positive cells derived from cord blood> The cord blood-derived CD34-positive cells obtained in Preparation Example 1 were seeded onto the surface-coated cell culture device and into control wells at 5,000 cells / well, and 200 μL / well of the culture medium prepared in Test Example 1-1 was added. After that, the cells were statically cultured for 7 days under conditions of 5% CO2 by volume and 37°C. After 7 days of culture, the cells cultured in the cell culture devices of Examples 1, 2, 4, and 5 were subjected to <surface marker analysis> in the same manner as in Test Example 1-1.

[0161] The results of analyzing the surface markers of the cells after culture are shown in Figure 11. The vertical axis shows the average number of cells in the hematopoietic stem cell fraction [CD34+, CD90+, CD45RA- cells] per well.

[0162] (Test Example 1-5: Analysis of surface markers 5) In Test Example 1-1, except that the <positioning of the cell culture device> and <culture of umbilical cord blood-derived CD34-positive cells> were changed to the following methods, the <surface coating treatment of the cell culture device> and the <analysis of surface markers> were performed in the same manner as in Test Example 1-1, and it was confirmed that the cells grown on the cell culture device contained hematopoietic progenitor cells in addition to hematopoietic stem cells. The method for <surface marker analysis> was the same as in Test Example 1-1, but the human-derived hematopoietic stem cells and hematopoietic progenitor cells were CD34-positive cells [CD34+ cells].

[0163] <Arrangement of cell culture devices> The cell culture devices of Examples 1, 2, 4, 5, and 6 were placed in the wells of a 96-well plate (made of polystyrene, flat bottom, manufactured by TPP). Each experiment was carried out in duplicate (n=2).

[0164] <Culture of CD34-positive cells derived from cord blood> The cord blood-derived CD34-positive cells obtained in Preparation Example 1 were seeded onto the surface-coated cell culture device and into control wells at 5,000 cells / well, and 200 μL / well of the culture medium prepared in Test Example 1-1 was added. After that, the cells were statically cultured for 7 days under conditions of 5% CO2 by volume and 37°C. After 7 days of culture, the cells cultured in the cell culture devices of Examples 1, 2, 4, 5, and 6 were subjected to <surface marker analysis> in the same manner as in Test Example 1-1.

[0165] The results of analyzing the surface markers of the cells after culture are shown in Figure 12. The vertical axis shows the average number of hematopoietic stem cells and hematopoietic progenitor cell fractions [CD34+ cells] per well.

[0166] (Test Example 2: Methylcellulose colony assay) The self-renewal ability and pluripotency of hematopoietic stem cells and hematopoietic progenitor cells grown on the cell culture device were confirmed by the following method.

[0167] Using the cell culture devices of Examples 1 to 3, the <arrangement of the cell culture device>, <surface coating treatment of the cell culture device>, and <culture of cord blood-derived CD34-positive cells> were carried out in the same manner as in Test Example 1-1.

[0168] After 7 days of culturing the cord blood-derived CD34+ cells, the cells on the cell culture device were suspended in the culture medium by thoroughly pipetting the cells in the wells. The entire cell suspension obtained from each well was transferred to a tube. The tubes were centrifuged at 600 g for 10 minutes, and the supernatant was removed. 150 μL of Iscove's Modified Dulbecco's Medium (IMDM, GE Healthcare) was added per tube to obtain a cell suspension. 1.35 mL of MethoCult medium for human hematopoietic progenitor cell colony measurement (STEMCELL Technology) was dispensed into a petri dish (polystyrene, flat-bottom, TPP) and 150 μL of the cell suspension was seeded. The cells were then cultured statically at 5% CO2 and 37°C for 14 days. After 14 days of culture, the formed colonies were observed under a phase-contrast microscope, and the number of granulocytic and monocytic progenitor cells, early erythroid progenitor cells, and mixed colonies containing multiple blood cell lineages was counted across the entire dish (STEMCELL Technology, TECHNICAL MANUAL, Human Colony-Forming Unit (CFU) Assays Using MethoCult™, DOCUMENT #28404, VERSION 4.6.0, MAR 2019, see pages 27 to 32).

[0169] The results of the methylcellulose colony assay are shown in Figure 13. The vertical axis shows the number of colonies. 13, proliferation of hematopoietic stem cells and hematopoietic progenitor cells was observed using the cell culture devices of Examples 1 to 3. Among these, when the cell culture device of Example 1 was used, particularly favorable proliferation of hematopoietic stem cells and hematopoietic progenitor cells was observed. [Industrial Applicability]

[0170] The cell culture device of the present invention can be easily produced at low cost and can efficiently grow at least one of hematopoietic stem cells and hematopoietic progenitor cells in vitro while maintaining their self-renewal ability and pluripotency, and therefore can be suitably used as a scaffold for culturing and growing at least one of hematopoietic stem cells and hematopoietic progenitor cells in vitro.Furthermore, it can be suitably used in research on the maintenance, proliferation, differentiation, etc. of hematopoietic stem cells. Furthermore, the cell culture method of the present invention can efficiently proliferate at least either hematopoietic stem cells or hematopoietic progenitor cells in vitro while maintaining the self-renewal ability and pluripotency of the cells, and therefore can be suitably used for the in vitro culture of at least either hematopoietic stem cells or hematopoietic progenitor cells. [Explanation of symbols]

[0171] 1 well 20 Cell culture device 21 Culture Department 21a Cell seeding surface 22 Removal section 23 Connection 30 micropores 31 Opening 32 Bottom 40 Master 41 Resin film T Angle T between cell seeding surface 21a and removal section 22 X: length of one side of the opening 31 Y: the length of the other side of the opening 31 p pitch h Depth of micropores

Claims

1. A cell culture device having a substrate with a culture section used for culturing at least one of hematopoietic stem cells and hematopoietic progenitor cells, The material of the culture part is either polystyrene or polycarbonate, The culture section has a plurality of micropores, the ratio [H / La] of the average depth (H) of the micropores to the average length (La) of the openings of the micropores is 1.1 or more; A cell culture device characterized in that the culture section has a Young's modulus of at least 3 GPa as measured in accordance with JIS K 7161-1 and JIS K 7161-2.

2. A take-out section, and Further provided is a connection part that connects the culture part and the removal part. The cell culture device according to claim 1 .

3. 3. The cell culture device according to claim 1, wherein the inside of the micropores is surface-treated with fibronectin.

4. 4. The cell culture device according to claim 1, wherein the openings of the micropores have an average length of 30 μm to 80 μm.

5. 5. The cell culture device according to claim 1, wherein the average depth of the micropores is 30 μm to 160 μm.

6. A cell culture method, comprising culturing at least one of hematopoietic stem cells and hematopoietic progenitor cells using the cell culture device according to any one of claims 1 to 5.

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