Method for producing hematopoietic progenitor cells and method for producing leukocytes

Abstract

Provided is a method for producing hematopoietic progenitor cells with which it is possible to stably obtain hematopoietic progenitor cells from pluripotent stem cells and with which it is possible to improve the efficiency of inducing differentiation from pluripotent stem cells into hematopoietic progenitor cells. This method for producing hematopoietic progenitor cells is provided with: a cell mass formation step in which pluripotent stem cells are cultured using a cell culture substrate provided with a substrate and a cell scaffold material arranged in a dot shape on a first surface of the substrate, and cell mass is formed on the surface of the cell scaffold material; and a differentiation induction step for inducing the differentiation of the obtained cell mass into a cell population containing hematopoietic progenitor cells.

Description

Technical Field

[0001] This invention relates to a method for manufacturing hematopoietic progenitor cells. Additionally, this invention relates to a method for manufacturing leukocytes. Background Technology

[0002] In recent years, attempts have been made to induce hematopoietic progenitor cells (hematopoietic stem cells) or leukocytes from the differentiation of induced pluripotent stem cells (iPS cells). iPS cells typically differentiate into hematopoietic progenitor cells via mesoderm and hematopoietic endothelial cells. Additionally, hematopoietic progenitor cells differentiate into leukocytes.

[0003] For example, Non-Patent Document 1 describes the use of a plate with a layer of adhesion protein coated on the bottom surface, inducing the differentiation of monocytes, a type of white blood cell, by iPS cells.

[0004] However, Patent Document 1 discloses a method comprising: using a membrane with an area of ​​0.001 to 5 mm that has cell adhesion and cell proliferation properties. 2 The method describes a cell culture medium consisting of an island-like region (A) and a region (B) adjacent to region (A) that does not have cell adhesion or cell proliferation, and a method for inducing ectodermal cell differentiation from pluripotent stem cells. However, the method described in Patent Document 1 is a method for inducing pluripotent stem cells to differentiate into ectodermal cell-like cells. Patent Document 1 does not describe the differentiation of pluripotent stem cells into mesoderm, and therefore, it does not describe the differentiation of pluripotent stem cells into hematopoietic progenitor cells.

[0005] Furthermore, Patent Document 2 describes a cell culture scaffold material comprising a substrate and protrusions formed by patterning dots or lines on the substrate. The protrusions comprise a synthetic resin, which may be a polyvinyl alcohol derivative or poly(meth)acrylic acid resin. Examples of Patent Document 2 describe the use of a cell culture container incorporating the aforementioned cell culture scaffold material to culture iPS cells. However, Patent Document 2 does not describe any method for differentiating iPS cells.

[0006] Existing technical documents

[0007] Patent documents

[0008] Patent Document 1: Japanese Patent Application Publication No. 2021-158960

[0009] Patent Document 2: WO2021 / 024943A1

[0010] Non-patent literature

[0011] Non-patent document 1: High-Yield Human Induced Pluripotent Stem Cell-DerivedMonocytes and Macrophages Are Functionally Comparable With Primary Cells (Front Cell Dev Biol. 2021 Apr 13;9:656867. doi: 10.3389 / fcell.2021.656867) Summary of the Invention

[0012] The technical problem solved by the invention

[0013] As described in Non-Patent Document 1, previous attempts have involved using plates with a layer of adhesion protein coated on the entire bottom surface to differentiate iPS cells into blood-like cells such as hematopoietic progenitor cells and leukocytes. However, in these previous methods, the number of differentiated cells (hematopoietic progenitor cells or leukocytes) obtained can vary depending on the seeding conditions, type, and state of the pluripotent stem cells, or the expression ratio of differentiation markers can also vary, making it difficult to consistently obtain differentiated cells.

[0014] The purpose of this invention is to provide a method for manufacturing hematopoietic progenitor cells that can stably obtain hematopoietic progenitor cells from pluripotent stem cells and improve the differentiation induction efficiency from pluripotent stem cells to hematopoietic progenitor cells. The purpose of this invention is to provide a method for manufacturing leukocytes that can stably obtain leukocytes from pluripotent stem cells and improve the differentiation induction efficiency from pluripotent stem cells to leukocytes.

[0015] Problem Solving Methods

[0016] This specification discloses the following methods for manufacturing hematopoietic progenitor cells and leukocytes.

[0017] Item 1. A method for manufacturing hematopoietic progenitor cells, the method comprising:

[0018] The cell block formation process involves culturing pluripotent stem cells using a cell culture medium containing a substrate and a cell scaffold material disposed in dots on a first surface of the substrate, and forming cell blocks on the surface of the cell scaffold material; and

[0019] The differentiation induction process induces the resulting cell blocks to differentiate into a cell population containing hematopoietic progenitor cells.

[0020] Item 2. The method for manufacturing hematopoietic progenitor cells according to Item 1, wherein,

[0021] The average equivalent circle diameter of the upper surface of the above-mentioned cell scaffold material is above 200 μm and below 3000 μm.

[0022] Item 3. The method for manufacturing hematopoietic progenitor cells according to Item 1 or 2, wherein,

[0023] The average distance between the centroids of the upper surface of the aforementioned cell scaffold material is greater than 0.5 mm and less than 10 mm.

[0024] Item 4. A method for manufacturing hematopoietic progenitor cells according to any one of items 1 to 3, wherein,

[0025] The aforementioned cell scaffold material comprises a peptide-containing resin having a synthetic resin portion and a peptide portion.

[0026] Item 5. The method for manufacturing hematopoietic progenitor cells according to Item 4, wherein,

[0027] The aforementioned peptide-containing resin has a polyvinyl acetal resin portion and a peptide portion.

[0028] Item 6. A method for manufacturing hematopoietic progenitor cells according to any one of items 1 to 5, wherein,

[0029] The area on the first surface of the substrate where the cell scaffold material is not disposed has an area treated with low cell adhesion.

[0030] Item 7. A method for manufacturing hematopoietic progenitor cells according to any one of items 1 to 6, wherein,

[0031] The aforementioned pluripotent stem cells are iPS cells.

[0032] Item 8. A method for manufacturing leukocytes, the method comprising:

[0033] The process of differentiating and inducing a cell population comprising hematopoietic progenitor cells obtained by the manufacturing method of any one of items 1 to 7 into a cell population comprising monocytes, lymphocytes or granulocytes.

[0034] Item 9. A method for manufacturing leukocytes, the method comprising:

[0035] The cell block formation process involves culturing pluripotent stem cells using a cell culture medium containing a substrate and a cell scaffold material disposed in dots on a first surface of the substrate, and forming cell blocks on the surface of the cell scaffold material; and

[0036] The differentiation induction process induces the resulting cell blocks to differentiate into a cell population containing monocytes, lymphocytes, or granulocytes.

[0037] Item 10. The method for manufacturing leukocytes according to Item 9, wherein,

[0038] The average equivalent circle diameter of the upper surface of the above-mentioned cell scaffold material is above 200 μm and below 3000 μm.

[0039] Item 11. The method for manufacturing leukocytes according to Item 9 or 10, wherein,

[0040] The average distance between the centroids of the upper surface of the aforementioned cell scaffold material is greater than 0.5 mm and less than 10 mm.

[0041] Item 12. A method for manufacturing leukocytes according to any one of items 9 to 11, wherein,

[0042] The aforementioned cell scaffold material comprises a peptide-containing resin having a synthetic resin portion and a peptide portion.

[0043] Item 13. The method for manufacturing leukocytes according to Item 12, wherein,

[0044] The aforementioned peptide-containing resin has a polyvinyl acetal resin portion and a peptide portion.

[0045] Item 14. A method for manufacturing leukocytes according to any one of items 9 to 13, wherein,

[0046] The area on the first surface of the substrate where the cell scaffold material is not disposed has an area treated with low cell adhesion.

[0047] Item 15. A method for manufacturing leukocytes according to any one of items 9 to 14, wherein,

[0048] The aforementioned pluripotent stem cells are iPS cells.

[0049] The effects of the invention

[0050] The method for manufacturing hematopoietic progenitor cells of the present invention comprises: a cell block formation step, wherein pluripotent stem cells are cultured on a cell culture medium having a substrate and a cell scaffold material disposed in dots on a first surface of the substrate, and cell blocks are formed on the surface of the cell scaffold material; and a differentiation induction step, wherein the obtained cell blocks are differentiated and induced into a cell population containing hematopoietic progenitor cells. With the above-described configuration, the method for manufacturing hematopoietic progenitor cells of the present invention allows for the stable generation of hematopoietic progenitor cells from pluripotent stem cells and improves the efficiency of differentiation induction from pluripotent stem cells to hematopoietic progenitor cells.

[0051] The method for manufacturing leukocytes according to the present invention comprises: a cell block formation step, in which pluripotent stem cells are cultured using a cell culture medium having a substrate and a cell scaffold material disposed in dots on a first surface of the substrate, and cell blocks are formed on the surface of the cell scaffold material; and a differentiation induction step, in which the obtained cell blocks are differentiated and induced into a cell population comprising monocytes, lymphocytes, or granulocytes. With the above-described configuration, the method for manufacturing leukocytes according to the present invention can stably obtain leukocytes from pluripotent stem cells and can improve the efficiency of differentiation induction from pluripotent stem cells to leukocytes. Attached Figure Description

[0052] [ Figure 1 ] Figure 1 (a) and (b) are top and cross-sectional views schematically illustrating examples of cell culture materials that can be used in methods for manufacturing hematopoietic progenitor cells and leukocytes.

[0053] [ Figure 2 ] Figure 2 This is an example of the process by which iPS cells differentiate into leukocytes (monocytes, lymphocytes, or granulocytes) via hematopoietic progenitor cells.

[0054] Symbol Explanation

[0055] 1. Cell culture materials

[0056] 2···Substrate

[0057] 2a···First Surface

[0058] 3. Cell scaffold materials

[0059] 3a··· Upper surface

[0060] D: Diameter (equivalent circle diameter)

[0061] H: Height

[0062] L: Distance between centers of gravity Detailed Implementation

[0063] The present invention will now be described in detail.

[0064] The method for manufacturing hematopoietic progenitor cells of the present invention comprises: a cell block formation step, wherein pluripotent stem cells are cultured on a cell culture medium having a substrate and a cell scaffold material disposed in dots on a first surface of the substrate, and a cell block is formed on the surface of the cell scaffold material; and a differentiation induction step, wherein the obtained cell block is differentiated and induced into a cell population containing hematopoietic progenitor cells.

[0065] In the method for manufacturing hematopoietic progenitor cells of the present invention, since the above-described structure is provided, hematopoietic progenitor cells can be stably obtained from pluripotent stem cells, and the differentiation induction efficiency from pluripotent stem cells to hematopoietic progenitor cells can be improved.

[0066] The inventors have discovered that by using the aforementioned cell culture medium and controlling the size of the cell blocks, hematopoietic progenitor cells can be stably obtained from pluripotent stem cells, thereby improving the differentiation induction efficiency from pluripotent stem cells to hematopoietic progenitor cells. In the method for manufacturing hematopoietic progenitor cells according to the present invention, compared to conventional cell culture medium where the bottom surface is entirely coated with cell scaffold material, hematopoietic progenitor cells can be stably obtained from pluripotent stem cells. For example, in the method for manufacturing hematopoietic progenitor cells according to the present invention, compared to conventional cell culture medium where the bottom surface is entirely coated with cell scaffold material, hematopoietic progenitor cells can be stably obtained even under conditions of high pluripotent stem cell seeding density.

[0067] The method for manufacturing leukocytes according to the present invention comprises: a cell block forming step, wherein pluripotent stem cells are cultured using a cell culture medium having a substrate and a cell scaffold material disposed in dots on a first surface of the substrate, and cell blocks are formed on the surface of the cell scaffold material; and a differentiation induction step, wherein the obtained cell blocks are differentiated and induced into a cell population comprising monocytes, lymphocytes or granulocytes.

[0068] In the method for manufacturing leukocytes according to the present invention, since the above-described structure is provided, leukocytes can be stably obtained from pluripotent stem cells, and the differentiation induction efficiency from pluripotent stem cells to leukocytes can be improved.

[0069] The inventors have discovered that by using the aforementioned cell culture medium and controlling the size of the cell blocks, leukocytes can be stably obtained from pluripotent stem cells, and the differentiation induction efficiency from pluripotent stem cells to leukocytes can be improved. In the leukocyte manufacturing method of the present invention, compared to conventional cell culture medium where a cell scaffold material is integrally coated on the bottom surface, leukocytes can be stably obtained from pluripotent stem cells, and the differentiation induction efficiency from pluripotent stem cells to leukocytes can be improved. In the leukocyte manufacturing method of the present invention, compared to conventional cell culture medium where a cell scaffold material is integrally coated on the bottom surface, the number of leukocytes obtained from one pluripotent stem cell can be increased.

[0070] In the methods for manufacturing hematopoietic progenitor cells and leukocytes of the present invention, the following possibilities can be considered as reasons for achieving the aforementioned effects. In conventional methods using cell culture media with a cell scaffold material integrally coated on the bottom surface, the size of the cell blocks and the distance between them are typically controlled by reducing the seeding density of pluripotent stem cells to prevent the formed cell blocks from adhering to each other. However, in this method, the arrangement of cell blocks is random, so some or a large number of cell blocks will adhere to each other, making it difficult to control the size of the cell blocks and the distance between them. The inventors have found that in conventional methods, the reason why differentiated cells (hematopoietic progenitor cells or leukocytes) cannot be stably obtained and the differentiation induction efficiency cannot be improved is that there are large deviations in the size of the cell blocks and the distance between them. In contrast, in the manufacturing method of the present invention, since a cell culture media having a cell scaffold material arranged in a dotted pattern is used, the size of the cell blocks, the number of cell blocks, and the distance between them can be controlled. Therefore, differentiation-inducing factors are easily and uniformly distributed throughout the cells. Furthermore, the cytokines secreted by the cell blocks become more uniform, and the differentiation induction promotion among the cell blocks becomes more uniform (paracrine effect). Therefore, differentiated cells (hematopoietic progenitor cells or leukocytes) can be stably obtained from pluripotent stem cells, and the differentiation induction efficiency from pluripotent stem cells to differentiated cells (hematopoietic progenitor cells or leukocytes) can be improved. In the manufacturing method of the present invention, for example, deviations in the number of differentiated cells (hematopoietic progenitor cells or leukocytes) and the expression ratio of differentiation markers can be effectively suppressed, and differentiated cells can be stably obtained. In addition, the number of differentiated cells (hematopoietic progenitor cells or leukocytes) obtained from one pluripotent stem cell can be increased, thereby improving the differentiation induction efficiency. However, the above is presumptive, and the reasons for the above effects in the manufacturing method of the present invention are not limited to this.

[0071] First, the cell culture materials that can be used in the methods for manufacturing hematopoietic progenitor cells and leukocytes of the present invention will be described.

[0072] Cell culture materials

[0073] The aforementioned cell culture medium comprises a substrate and a cell scaffold material disposed in a dotted pattern on a first surface of the substrate. The first surface of the substrate has: a region disposed of the cell scaffold material and a region not disposed of the cell scaffold material.

[0074] The following description, with reference to the accompanying drawings, illustrates examples of cell culture materials.

[0075] Figure 1(a) and (b) are top and cross-sectional views schematically illustrating examples of cell culture materials that can be used in methods for manufacturing hematopoietic progenitor cells and leukocytes. Figure 1 (a) is a top view. Figure 1 (b) is along Figure 1 (a) Cross-sectional view of line II.

[0076] Figure 1 The cell culture medium 1 shown includes a substrate 2 and a cell scaffold material 3. The cell scaffold material 3 is disposed in a dotted pattern on a first surface 2a of the substrate 2. The first surface 2a of the substrate 2 is in direct contact with the cell scaffold material 3. The cell scaffold material 3 is periodically disposed on the first surface 2a of the substrate 2 and is disposed at equal intervals. The cell scaffold material 3 has an upper surface 3a. The upper surface 3a of the cell scaffold material 3 is the surface of the cell scaffold material 3 that is opposite to the side of the first surface 2a of the substrate 2.

[0077] In cell culture medium 1, the upper surface 3a of cell scaffold material 3 is circular in shape. Figure 1 In (a), the symbol D represents the diameter of the upper surface 3a of the cell scaffold material 3. In the cell culture medium 1, since the upper surface 3a of the cell scaffold material 3 is circular, the diameter D of the upper surface 3a is equivalent to the diameter of the circle of the upper surface 3a of the cell scaffold material 3. Figure 1 In (a), the symbol L represents the distance between the centroids of the upper surfaces 3a of the cell scaffold material 3. In the cell culture medium 1, since the upper surfaces 3a of the cell scaffold material 3 are circular, the distance L between the centroids of the upper surfaces 3a of the cell scaffold material 3 is the distance between the centers of the circles that define the shape of the upper surfaces 3a of the cell scaffold material 3. Figure 1 In (b), the symbol H represents the height of the cell scaffold material 3. The height H of the cell scaffold material 3 is the distance from the first surface 2a of the substrate 2 to the upper surface 3a of the cell scaffold material 3.

[0078] (Substrate)

[0079] The aforementioned cell culture medium includes a substrate (the main body of the cell culture medium). There are no particular limitations on the material, size, or shape of the substrate. Conventionally known substrates can be used as the substrate.

[0080] Examples of materials that can be used as the base material include synthetic resins, metals, and glass. Examples of synthetic resins include polystyrene, polyethylene, polypropylene, polyethersulfone, polycarbonate, polyester, polyisoprene, cyclic olefin polymers, polyimide, polyamide, polyamide-imide, (meth)acrylic resins, epoxy resins, and silicone.

[0081] Examples of the shapes of the aforementioned substrates include plate-like, film-like, porous film-like, and bag-like forms. Furthermore, the aforementioned substrate can be a container.

[0082] The aforementioned substrate is preferably a container. The aforementioned cell culture material is preferably a cell culture container. The aforementioned cell culture material is preferably a cell culture container comprising a container (cell culture container body) and cell scaffold material arranged in a dotted pattern on the first surface of the container. The shape and size of the aforementioned container (cell culture container body) are not particularly limited. Examples of the aforementioned container (cell culture container body) include 2-384 well plates, monolayer culture flasks, multilayer culture flasks, multifaceted culture flasks, culture dishes, roller bottles, bags, insert cups, and microfluidic chips. The aforementioned first surface of the aforementioned substrate is preferably the bottom surface of the aforementioned container.

[0083] (Cell scaffold materials)

[0084] The aforementioned cell culture medium includes a cell scaffold material. The cell scaffold material is disposed in a dotted pattern on a first surface of the substrate. The cell scaffold material has an upper surface. The upper surface of the cell scaffold material is the surface of the cell scaffold material opposite to the first surface of the substrate.

[0085] The shape of the upper surface of the aforementioned cell scaffold material is not particularly limited. The upper surface of the aforementioned cell scaffold material can be circular, elliptical, polygonal, or any other shape.

[0086] The number (dots) of the cell scaffold material disposed on the first surface of the substrate can be appropriately varied depending on the type, shape, and size of the substrate. For example, the number (dots) of the cell scaffold material disposed on the first surface of the substrate may be per 1 cm of the first surface of the substrate. 2 The area can contain more than 1, more than 3, more than 5, more than 7, less than 150, less than 120, less than 90, or less than 60.

[0087] The cell scaffold material described above can be arranged at equal intervals or at unequal intervals on the first surface of the substrate. The cell culture medium may have regions on the first surface of the substrate where the cell scaffold material is arranged at equal intervals and regions where it is not arranged at equal intervals. The cell scaffold material may be arranged periodically on the first surface of the substrate or not periodically. The cell culture medium may have regions on the first surface of the substrate where the cell scaffold material is periodically arranged and regions where it is not periodically arranged.

[0088] The average aspect ratio of the upper surface of the above-mentioned cell scaffold material is preferably 6 or less, more preferably 5 or less, even more preferably 4 or less, even more preferably 3 or less, even more preferably 2 or less, particularly preferably 1.5 or less, and most preferably 1.25 or less. When the average aspect ratio is below the above-mentioned upper limit, cell blocks can be formed more effectively. It should be noted that the average aspect ratio of the upper surface of the above-mentioned cell scaffold material can be 1 or more, or even exceed 1.

[0089] The aforementioned aspect ratio is the ratio of the major diameter of the upper surface of the cell scaffold material to the minor diameter of the upper surface of the cell scaffold material (major diameter / minor diameter). This aspect ratio can be calculated, for example, by photographing the surface of the cell culture medium under a microscope and analyzing the microscope image using commercially available image analysis software. The average aspect ratio can be obtained by averaging the aspect ratios of the upper surfaces of multiple cell scaffold materials. Preferably, the average aspect ratio is obtained by averaging the aspect ratios of the upper surfaces of at least 10 cell scaffold materials arbitrarily selected from the entire first surface of the substrate. If there are fewer than 10 cell scaffold materials on the first surface of the substrate, it is preferable to obtain the average aspect ratio by averaging the aspect ratios of the upper surfaces of all the cell scaffold materials.

[0090] The average value of the equivalent circular diameter of the upper surface of the above-mentioned cell scaffold material is preferably 200 μm or more, more preferably 300 μm or more, even more preferably 350 μm or more, even more preferably 400 μm or more, particularly preferably 500 μm or more, preferably 3000 μm or less, more preferably 2500 μm or less, even more preferably 2000 μm or less, even more preferably 1500 μm or less, and particularly preferably 1000 μm or less. When the average value of the above-mentioned equivalent circular diameter is above or below the lower limit and below the upper limit, it is easy to control the size of the cell block within a suitable range, and the effects of the present invention can be further and more effectively exerted.

[0091] The aforementioned equivalent circle diameter refers to the diameter of a circle having an area equal to the area of ​​the upper surface of the aforementioned cell scaffold material. The equivalent circle diameter can be calculated using the following formula. For example, the equivalent circle diameter can be calculated by photographing the surface of the cell culture medium under a microscope and analyzing the microscope image using commercially available image analysis software. The average value of the equivalent circle diameter can be obtained by averaging the equivalent circle diameters of the upper surfaces of multiple cell scaffold materials. Preferably, the average value of the equivalent circle diameter is obtained by averaging the equivalent circle diameters of the upper surfaces of 10 or more cell scaffold materials arbitrarily selected from the entire first surface of the substrate. If there are fewer than 10 cell scaffold materials on the first surface of the substrate, it is preferable to obtain the average value by averaging the equivalent circle diameters of the upper surfaces of all the aforementioned cell scaffold materials.

[0092] [Mathematical Expression 1]

[0093]

[0094] D: Equivalent circle diameter of the upper surface of the cell scaffold material

[0095] S: Area of ​​the upper surface of the cell scaffold material

[0096] The average distance between the centers of gravity on the upper surface of the aforementioned cell scaffold material is preferably 0.5 mm or more, more preferably 0.7 mm or more, even more preferably 1 mm or more, even more preferably 1.5 mm or more, particularly preferably 2 mm or more, preferably 10 mm or less, more preferably 9 mm or less, even more preferably 8 mm or less, even more preferably 7 mm or less, even more preferably 6 mm or less, and particularly preferably 5 mm or less. When the average distance between the centers of gravity is above or below the aforementioned lower limit and below the aforementioned upper limit, it is easy to control the distance between cell blocks within an appropriate range, thereby further and effectively exerting the effects of the present invention.

[0097] The aforementioned centroid distance is the distance from the centroid of the upper surface of the cell scaffold material to the centroid of the upper surface of the cell scaffold material closest to its own (the distance from one centroid to the other centroid closest to it). This centroid distance can be calculated, for example, by taking a microscopic photograph of the cell culture medium surface and analyzing the microscopic image using commercially available image analysis software. The average centroid distance can be obtained by averaging the centroid distances of the upper surfaces of multiple cell scaffold materials. Preferably, the average centroid distance is obtained by averaging the centroid distances of the upper surfaces of at least 10 cell scaffold materials arbitrarily selected from the entire first surface of the substrate. If there are fewer than 10 cell scaffold materials on the first surface of the substrate, it is preferable to obtain the average centroid distance by averaging the centroid distances of the upper surfaces of all the cell scaffold materials.

[0098] The average height of the aforementioned cell scaffold material can be greater than 1 nm, greater than 5 nm, greater than 10 nm, greater than 15 nm, greater than 20 nm, less than 10 μm, less than 5 μm, less than 2 μm, or less than 1 μm. When the average height of the aforementioned cell scaffold material is greater than or equal to the lower limit mentioned above, the adhesion of cells to the cell scaffold material can be improved, and cell proliferation can be enhanced. When the average height of the aforementioned cell scaffold material is less than or equal to the upper limit mentioned above, the manufacturing cost of the aforementioned cell culture material can be kept relatively low.

[0099] The height of the aforementioned cell scaffold material is the distance from the first surface of the aforementioned substrate to the upper surface of the aforementioned cell scaffold material. Preferably, the height of the aforementioned cell scaffold material is the distance from the first surface of the aforementioned substrate to the center of gravity of the upper surface of the aforementioned cell scaffold material. The average height can be obtained by averaging the heights of multiple cell scaffold materials. Preferably, the average height is obtained by averaging the heights of 10 or more cell scaffold materials arbitrarily selected from the entire first surface of the aforementioned substrate. If there are fewer than 10 cell scaffold materials on the first surface of the aforementioned substrate, it is preferable to obtain the average height by averaging the heights of all the aforementioned cell scaffold materials.

[0100] It should be noted that, for example, the microscope used to measure the aspect ratio, equivalent circle diameter, and distance between centroids of the upper surface of the cell scaffold material, as well as the height of the cell scaffold material, can be the "BZ-X800" manufactured by KEYENCE, and the analytical application program can be the "BZ-H4M / Measurement Application" manufactured by KEYENCE.

[0101] Examples of components found in the aforementioned cell scaffold materials include cell adhesion proteins and synthetic resins. Only one of these components may be used, or two or more may be used in combination.

[0102] Examples of cell adhesion proteins include laminin, vitronectin, and collagen. The components of the aforementioned cell scaffold material may include cell adhesion proteins such as matrix gel. Examples of matrix gels include matrix gel derived from mouse sarcoma.

[0103] As described below, the aforementioned synthetic resin may include a synthetic resin having a synthetic resin portion and a peptide portion (a synthetic resin with bonded peptides). The aforementioned synthetic resin may also include a synthetic resin without a peptide portion (a synthetic resin without bonded peptides). Examples of the aforementioned synthetic resin include polyvinyl alcohol derivatives, peptide-containing polyvinyl alcohol derivatives having a polyvinyl alcohol derivative portion and a peptide portion, (meth)acrylic acid copolymers, and peptide-containing (meth)acrylic acid copolymers having a poly(meth)acrylate portion and a peptide portion. Examples of the aforementioned polyvinyl alcohol derivatives include polyvinyl alcohol acetal resins.

[0104] Hereinafter, in this specification, the term "synthetic resin having a synthetic resin portion and a peptide portion" will sometimes be referred to as "peptide-containing resin".

[0105] The aforementioned cell scaffold material preferably comprises cell adhesion proteins or synthetic resins, more preferably laminin, fibronectin, or peptide-containing resins, and even more preferably peptide-containing resins. In this case, the effects of the present invention can be more effectively achieved.

[0106] <peptide-conjugated resin>

[0107] From the viewpoint of further effectively realizing the effects of the present invention and improving the manufacturing efficiency of cell culture materials, the aforementioned cell scaffold material preferably comprises a peptide-containing resin having a synthetic resin portion and a peptide portion. The aforementioned peptide-containing resin is a synthetic resin with peptides bonded to it. The aforementioned peptide-containing resin has a synthetic resin portion and a peptide portion. Only one type of peptide-containing resin may be used, or two or more types may be used in combination.

[0108] The aforementioned synthetic resin portion preferably comprises a polyvinyl alcohol derivative portion or a poly(meth)acrylate portion, more preferably a polyvinyl alcohol derivative portion or a poly(meth)acrylate portion. The aforementioned peptide-containing resin preferably comprises both a polyvinyl alcohol derivative portion or a poly(meth)acrylate portion and a peptide portion. In this case, the effects of the present invention can be achieved more effectively. The aforementioned peptide-containing resin may comprise both a polyvinyl alcohol derivative portion and a poly(meth)acrylate portion.

[0109] In the peptide-containing resin having the aforementioned polyvinyl alcohol derivative portion, it is preferable that the polyvinyl alcohol derivative portion and the peptide portion are bonded together via a connector portion. Therefore, the peptide-containing resin having the aforementioned polyvinyl alcohol derivative portion preferably has a polyvinyl alcohol derivative portion, a peptide portion, and a connector portion.

[0110] In the peptide-containing resin having the aforementioned poly(meth)acrylate portion, the poly(meth)acrylate portion and the peptide portion can be bonded together via a connector portion, or they can be directly bonded without a connector portion. The peptide-containing resin having the aforementioned poly(meth)acrylate portion can have a poly(meth)acrylate portion, a peptide portion, and a connector portion.

[0111] It should be noted that in this specification, "(meth)acrylic acid" refers to one or both of "acrylic acid" and "methacrylic acid", and "(meth)acrylate" refers to one or both of "acrylate" and "methacrylate".

[0112] <<Polyvinyl Alcohol Derivatives Section>>

[0113] The aforementioned polyvinyl alcohol derivative portion (polyvinyl alcohol derivative skeleton) is a structural portion derived from a polyvinyl alcohol derivative. The aforementioned polyvinyl alcohol derivative is a compound derived from polyvinyl alcohol.

[0114] The aforementioned peptide-containing resin is preferably a peptide-containing polyvinyl alcohol derivative having a polyvinyl alcohol derivative portion and a peptide portion. The aforementioned cell scaffold material preferably comprises a peptide-containing polyvinyl alcohol derivative having a polyvinyl alcohol derivative portion and a peptide portion. By using the aforementioned peptide-containing polyvinyl alcohol derivative, the effects of the present invention can be further effectively achieved, and cell proliferation can be further improved.

[0115] From the viewpoint of further effectively realizing the effects of the present invention and further improving cell proliferation, the aforementioned polyvinyl alcohol derivative is preferably a polyvinyl alcohol acetal resin, and the aforementioned polyvinyl alcohol derivative portion is preferably a polyvinyl alcohol acetal resin portion. That is, the aforementioned peptide-containing resin preferably has a polyvinyl alcohol acetal resin portion and a peptide portion. The aforementioned cell scaffold material preferably comprises a peptide-containing polyvinyl alcohol acetal resin having a polyvinyl alcohol acetal resin portion and a peptide portion. The aforementioned polyvinyl alcohol derivative and the aforementioned polyvinyl alcohol acetal resin may each be used individually, or two or more may be used in combination.

[0116] The polyvinyl alcohol derivative portion and the polyvinyl alcohol acetal resin portion preferably have acetal groups, hydroxyl groups, and acetyl groups in their side chains. However, the polyvinyl alcohol derivative portion and the polyvinyl alcohol acetal resin portion may also be without acetyl groups. For example, by bonding all the acetyl groups of the polyvinyl alcohol acetal resin portion to the linker, the polyvinyl alcohol derivative portion and the polyvinyl alcohol acetal resin portion may also be without acetyl groups.

[0117] The above-mentioned polyvinyl alcohol acetal resin can be synthesized by acetalizing polyvinyl alcohol with aldehyde.

[0118] The aldehyde used in the acetalization of polyvinyl alcohol is not particularly limited. Examples of such aldehydes include those with 1 to 10 carbon atoms. The aldehyde may have chain aliphatic groups, cyclic aliphatic groups, or aromatic groups, or may not have them. The aldehyde may be chain-like or cyclic. Only one of the aldehydes may be used, or two or more may be used in combination.

[0119] From the viewpoint of further improving the adhesion between the cell scaffold material and cells and from the viewpoint of further effectively utilizing the effects of the present invention, the aldehyde is preferably formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, or valeraldehyde, and more preferably butyraldehyde. Therefore, the polyvinyl acetal resin is more preferably polyvinyl butyral resin. From the viewpoint of further improving the adhesion between the cell scaffold material and cells and from the viewpoint of further effectively utilizing the effects of the present invention, the polyvinyl acetal resin portion is preferably a polyvinyl butyral resin portion. The peptide-containing resin preferably has a polyvinyl butyral resin portion and a peptide portion. The cell scaffold material preferably comprises a peptide-containing polyvinyl butyral resin having a polyvinyl butyral resin portion and a peptide portion.

[0120] In the above-mentioned peptide-containing polyvinyl alcohol acetal resin, the degree of acetalization of the polyvinyl alcohol acetal resin portion (or the degree of butyralization in the case of the polyvinyl alcohol butyral resin portion) is preferably 40 mol% or more, more preferably 50 mol% or more, more preferably 90 mol% or less, and more preferably 85 mol% or less. When the degree of acetalization is above or below the aforementioned lower limit, the peptide-containing polyvinyl alcohol acetal resin does not easily swell in the culture medium. When the degree of acetalization is below or below the aforementioned upper limit, it can be made to have good solubility in solvents.

[0121] In the above-mentioned peptide-containing polyvinyl acetal resin, the content (hydroxyl content) of hydroxyl groups in the polyvinyl acetal resin portion is preferably 15 mol% or more, more preferably 20 mol% or more, more preferably 45 mol% or less, more preferably 30 mol% or less, and even more preferably 25 mol% or less.

[0122] In the above-mentioned peptide-containing polyvinyl acetal resin, the degree of acetylation (acetyl group content) of the polyvinyl acetal resin portion is preferably 1 mol% or more, more preferably 2 mol% or more, more preferably 5 mol% or less, and more preferably 4 mol% or less. When the degree of acetylation is above or below the lower limit and below the upper limit, the reaction efficiency between the polyvinyl acetal resin and the connector can be improved.

[0123] The degree of acetalization, degree of acetylation, and amount of hydroxyl groups in the above-mentioned polyvinyl acetal resin portion can be determined by... 1 The determination was performed using H-NMR (nuclear magnetic resonance spectroscopy). The degree of acetalization, degree of acetylation, and amount of hydroxyl groups in the above-mentioned polyvinyl acetal resin portion are typically expressed in moles relative to the total amount (100 mol%) of each structural unit constituting the above-mentioned peptide-containing polyvinyl acetal resin.

[0124] <<Poly(meth)acrylate section>>

[0125] The aforementioned poly(meth)acrylate portion (poly(meth)acrylate backbone) is a structural part derived from poly(meth)acrylate.

[0126] The aforementioned peptide-containing resin preferably has a poly(meth)acrylate portion, more preferably a peptide-containing (meth)acrylate copolymer having both a poly(meth)acrylate portion and a peptide portion. The aforementioned cell scaffold material preferably comprises a peptide-containing (meth)acrylate copolymer having both a poly(meth)acrylate portion and a peptide portion. By using the aforementioned peptide-containing (meth)acrylate copolymer, the effects of the present invention can be further effectively achieved, and cell proliferation can be further improved.

[0127] The aforementioned poly(meth)acrylate portion has a backbone derived from (meth)acrylate. The aforementioned poly(meth)acrylate can be obtained by polymerizing (meth)acrylate. The aforementioned poly(meth)acrylate can be used alone, or in combination of two or more.

[0128] Examples of the aforementioned (meth)acrylates include alkyl (meth)acrylates, cyclic (meth)acrylates, aryl (meth)acrylates, polyethylene glycol (meth)acrylates, and phosphocholine (meth)acrylates. Only one of these (meth)acrylates may be used, or two or more may be used in combination.

[0129] Examples of the aforementioned alkyl methacrylates include methyl methacrylate, ethyl methacrylate, n-propyl methacrylate, isopropyl methacrylate, n-butyl methacrylate, isobutyl methacrylate, tert-butyl methacrylate, n-octyl methacrylate, isooctyl methacrylate, 2-ethylhexyl methacrylate, nonyl methacrylate, isononyl methacrylate, decyl methacrylate, isodecyl methacrylate, lauryl methacrylate, stearyl methacrylate, and isotetradecyl methacrylate.

[0130] The aforementioned alkyl (meth)acrylates can be substituted with substituents such as alkoxy groups having 1 to 3 carbon atoms and tetrahydrofurfuryl groups. Examples of such alkyl (meth)acrylates include methoxyethyl acrylate and tetrahydrofurfuryl acrylate.

[0131] Examples of the above-mentioned cyclic alkyl esters of (meth)acrylate include cyclohexyl (meth)acrylate and isobornyl (meth)acrylate.

[0132] Examples of aryl esters of (meth)acrylate include phenyl (meth)acrylate and benzyl (meth)acrylate.

[0133] Examples of the aforementioned polyethylene glycol (meth)acrylates include methoxy-polyethylene glycol (meth)acrylate, ethoxy-polyethylene glycol (meth)acrylate, hydroxy-polyethylene glycol (meth)acrylate, methoxy-diethylene glycol (meth)acrylate, ethoxy-diethylene glycol (meth)acrylate, hydroxy-diethylene glycol (meth)acrylate, methoxy-triethylene glycol (meth)acrylate, ethoxy-triethylene glycol (meth)acrylate, and hydroxy-triethylene glycol (meth)acrylate.

[0134] Examples of the aforementioned (meth)acrylic acid phosphocholine include 2-(meth)acryloyloxyethyl phosphocholine.

[0135] The peptide-containing resin having the above-mentioned poly(meth)acrylate portion may have a backbone derived from monomers other than (meth)acrylate.

[0136] Other monomers besides the aforementioned (meth)acrylates include (meth)acrylamides and vinyl compounds. Only one of these monomers may be used, or two or more may be used in combination.

[0137] Examples of the aforementioned (meth)acrylamides include (meth)acrylamide, N-isopropyl (meth)acrylamide, N-tert-butyl (meth)acrylamide, N,N'-dimethyl (meth)acrylamide, (3-(meth)acrylamidopropyl)trimethylammonium chloride, 4-(meth)acryloylmorpholine, and 3-(meth)acryloyl-2- Zolpidemone, N-[3-(dimethylamino)propyl](methyl)acrylamide, N-(2-hydroxyethyl)(methyl)acrylamide, N-hydroxymethyl(methyl)acrylamide, and 6-(methyl)acrylamide hexanoic acid, etc.

[0138] Examples of the aforementioned vinyl compounds include ethylene, allylamine, vinylpyrrolidone, maleic anhydride, maleimide, itaconic acid, (meth)acrylic acid, and ethyleneamine.

[0139] <<Peptide Section>>

[0140] The aforementioned peptide portion is a structural part derived from a peptide. The aforementioned peptide portion has an amino acid sequence. The peptides constituting the aforementioned peptide portion can be oligopeptides or polypeptides. Only one type of peptide can be used, or two or more can be used in combination.

[0141] The number of amino acid residues in the peptide portion is preferably 3 or more, more preferably 4 or more, even more preferably 5 or more, preferably 10 or less, more preferably 8 or less, and even more preferably 6 or less. When the number of amino acid residues is above the lower limit and below the upper limit, the adhesion between the cell scaffold material and the seeded cells can be further improved, and the cell proliferation rate can be further improved. The number of amino acid residues in the peptide portion can exceed 10 or exceed 15.

[0142] The aforementioned peptide preferably possesses an amino acid sequence with cell adhesion properties. It should be noted that the amino acid sequence with cell adhesion properties refers to an amino acid sequence whose cell adhesion activity has been confirmed by phage display, agarose bead method, or plate coating method. For example, the phage display method described in "The Journal of Cell Biology, Volume 130, Number 5, September 1995 1189-1196" can be used. For example, the agarose bead method described in "Protein Nuclease Vol. 45 No. 15 (2000) 2477" can be used. For example, the plate coating method described in "Protein Nuclease Vol. 45 No. 15 (2000) 2477" can be used.

[0143] Examples of amino acid sequences that contribute to cell adhesion include, for instance, the RGD sequence (Arg-Gly-Asp), YIGSR sequence (Tyr-Ile-Gly-Ser-Arg), PDSGR sequence (Pro-Asp-Ser-Gly-Arg), HAV sequence (His-Ala-Val), ADT sequence (Ala-Asp-Thr), QAV sequence (Gln-Ala-Val), LDV sequence (Leu-Asp-Val), IDS sequence (Ile-Asp-Ser), REDV sequence (Arg-Glu-Asp-Val), IDAPS sequence (Ile-Asp-Ala-Pro-Ser), KQAGDV sequence (Lys-Gln-Ala-Gly-Asp-Val), and TDE sequence (Thr-Asp-Glu). Furthermore, examples of the aforementioned amino acid sequences contributing to cell adhesion include those described in "Pathological Physiology, Vol. 9, No. 7, pp. 527-535, 1990" and "Osaka Prefectural Maternal and Child Health Center Journal, Vol. 8, No. 1, pp. 58-66, 1992". The aforementioned peptide may contain only one of these cell-adhesive amino acid sequences, or it may contain two or more.

[0144] The peptide portion preferably has at least one of the amino acid sequences described above that promote cell adhesion, more preferably at least an RGD sequence, a YIGSR sequence, or a PDSGR sequence, even more preferably an RGD sequence, and particularly preferably at least an RGD sequence as shown in formula (1) below. In this case, the adhesion between the cell scaffold material and the seeded cells can be further improved, and the cell proliferation rate can be further increased.

[0145] Arg-Gly-Asp-X ···Equation (1)

[0146] In the above formula (1), X represents Gly, Ala, Val, Ser, Thr, Phe, Met, Pro or Asn.

[0147] The aforementioned peptide may be linear or have a cyclic peptide backbone. The aforementioned cyclic peptide backbone refers to a cyclic backbone composed of multiple amino acids. From the viewpoint of more effectively utilizing the effects of the present invention, the aforementioned cyclic peptide backbone is preferably composed of 4 or more amino acids, more preferably 5 or more amino acids, and most preferably 10 or fewer amino acids.

[0148] In the aforementioned peptide-containing resin, the content of the peptide portion is preferably 0.05 mol% or more, more preferably 0.1 mol% or more, even more preferably 0.15 mol% or more, particularly preferably 0.2 mol% or more, preferably 25 mol% or less, more preferably 20 mol% or less, even more preferably 15 mol% or less, and particularly preferably 10 mol% or less. When the content of the peptide portion is at or above the aforementioned lower limit, the adhesion between the cell scaffold material and the seeded cells can be further improved, and the cell proliferation rate can be further improved. Furthermore, if the content of the peptide portion is below or above the aforementioned upper limit, manufacturing costs can be suppressed.

[0149] The content (molar percentage) of the peptide portion is the mass of the peptide portion relative to the sum of the mass of each structural unit constituting the peptide-containing resin.

[0150] The content of the aforementioned peptides can be determined, for example, by NMR (nuclear magnetic resonance).

[0151] <<Joint Section>>

[0152] The aforementioned connector portion is a structural part derived from a connector. This connector portion is typically located between the aforementioned polyvinyl alcohol derivative portion or the aforementioned poly(meth)acrylate portion and the aforementioned peptide portion. The aforementioned polyvinyl alcohol derivative portion or the aforementioned poly(meth)acrylate portion and the aforementioned peptide portion are bonded together via the aforementioned connector portion. The connector portion is formed by a connector (crosslinking agent). Only one type of connector may be used, or two or more types may be used in combination.

[0153] The aforementioned connector is preferably a compound having a functional group capable of bonding with the aforementioned peptide, and more preferably a compound having a functional group capable of condensing with the carboxyl or amino group of the aforementioned peptide.

[0154] Examples of functional groups capable of condensing with the carboxyl or amino groups of the aforementioned peptides include carboxyl, thiol, amino, hydroxyl, and cyano groups.

[0155] From the viewpoint of good reaction with peptides, the above-mentioned linker is preferably a compound having a carboxyl or amino group, and more preferably a compound having a carboxyl group.

[0156] In the case of obtaining a peptide-containing resin having a polyvinyl alcohol derivative portion, examples of carboxyl-containing linkers include (meth)acrylic acid and carboxyl-containing acrylamide. By using a carboxylic acid (carboxylic acid monomer) with a polymerizable unsaturated group as the carboxyl-containing linker, and by polymerizing the carboxylic acid monomer using graft polymerization during linker introduction, the number of carboxyl groups capable of reacting with the peptide can be increased.

[0157] From the viewpoint of enabling good bonding between polyvinyl alcohol derivatives and peptides, the aforementioned linker is preferably (meth)acrylic acid, and more preferably acrylic acid.

[0158] When obtaining a peptide-containing resin having a poly(meth)acrylate portion, the aforementioned connector preferably has a functional group capable of bonding with (meth)acrylate. Examples of such functional groups capable of bonding with (meth)acrylate include vinyl, (meth)acryloyl, and allyl groups. More preferably, the connector has a (meth)acryloyl group. As the aforementioned functional group capable of bonding with (meth)acrylate, compounds having both a carboxyl or amino group and a (meth)acryloyl group are preferred.

[0159] Examples of the above-mentioned connectors obtained when a peptide-containing resin having a poly(meth)acrylate portion is obtained include (meth)acrylic acid, itaconic acid, and acrylamide.

[0160] From the viewpoint of enabling good bonding between poly(meth)acrylate and peptide, the aforementioned connector is preferably (meth)acrylate or itaconic acid, and more preferably (meth)acrylate.

[0161] <Other details about cell scaffold materials>

[0162] The number-average molecular weight of the aforementioned peptide-containing resin is preferably 10,000 or more, more preferably 50,000 or more, even more preferably 100,000 or more, preferably 5,000,000 or less, more preferably 2,500,000 or less, and even more preferably 1,000,000 or less. When the number-average molecular weight is above or below the aforementioned lower limit, the cell scaffold material is less likely to be eluted into the liquid culture medium during cell culture, and the cell scaffold material is less likely to be peeled off from the substrate or the like during cell culture. When the number-average molecular weight is below or below the aforementioned upper limit, the solubility in alcohol solvents can be improved.

[0163] It should be noted that the number-average molecular weight of the above-mentioned peptide-containing resin can be determined, for example, by the following method: The above-mentioned peptide-containing resin is dissolved in tetrahydrofuran (THF) to prepare a 0.2% by weight solution of the peptide-containing resin. Next, the solution is evaluated using a gel permeation chromatography (GPC) apparatus (APC system, Waters Corporation) under the following determination conditions.

[0164] Column: HSPgel HR MB-M 6.0×150mm

[0165] Flow rate: 0.5 mL / min

[0166] Column temperature: 40℃

[0167] Injection volume: 10 μL

[0168] Detectors: RI, PDA

[0169] Standard sample: Polystyrene

[0170] The aforementioned cell scaffold material may comprise a synthetic resin without a peptide portion (a synthetic resin without bonded peptides) and a peptide-containing resin. The aforementioned cell scaffold material may comprise: a synthetic resin having a polyvinyl alcohol derivative portion or a poly(meth)acrylate portion but without a peptide portion, and a peptide-containing resin. The aforementioned cell scaffold material may comprise other components such as resins without both the aforementioned polyvinyl alcohol derivative portion and poly(meth)acrylate portion. Examples of these other components include polyolefin resins, polyether resins, polyesters, epoxy resins, polyamide resins, polyimide resins, polyurethane resins, polycarbonate resins, cellulose, and polypeptides. Only one of these other components may be used, or two or more may be used in combination.

[0171] The cell scaffold material described above preferably comprises the aforementioned peptide-containing resin and is a synthetic resin that contains or does not contain unbonded peptides.

[0172] The cell scaffold material described above preferably has a layer comprising the aforementioned peptide-containing resin. The cell scaffold material may have only the layer comprising the aforementioned peptide-containing resin, or it may have a layer comprising the aforementioned peptide-containing resin and a layer comprising a synthetic resin containing unbonded peptides. From the viewpoint of further effectively realizing the effects of the present invention and further improving cell proliferation, it is preferable that the first surface comprising the layer comprising the aforementioned peptide-containing resin is the upper surface of the cell scaffold material. Preferably, the aforementioned peptide-containing resin is present at least on the upper surface of the cell scaffold material.

[0173] In the above-mentioned cell scaffold material (100% by weight), the content of the peptide-containing resin is preferably 90% by weight or more, more preferably 95% by weight or more, further preferably 97.5% by weight or more, particularly preferably 99% by weight or more, and most preferably 100% by weight (total amount). When the content of the peptide-containing resin is at or above the aforementioned lower limit, the effects of the present invention can be more effectively achieved. In the above-mentioned cell scaffold material (100% by weight), the content of the peptide-containing resin may be less than 100% by weight or less.

[0174] In the aforementioned 100% by weight of cell scaffold material, the total content of the peptide-containing resin and the synthetic resin containing unbonded peptides is preferably 90% by weight or more, more preferably 95% by weight or more, further preferably 97.5% by weight or more, particularly preferably 99% by weight or more, and most preferably 100% by weight (total amount). When the total content is at or above the aforementioned lower limit, the effects of the present invention can be more effectively achieved. In the aforementioned 100% by weight of cell scaffold material, the total content of the peptide-containing resin and the synthetic resin containing unbonded peptides may be 100% by weight or less, or may be less than 100% by weight.

[0175] Preferably, the cell scaffold material has a layer containing the aforementioned cell adhesion proteins. The cell scaffold material may have only a layer containing the aforementioned cell adhesion proteins, or it may have a layer containing the aforementioned cell adhesion proteins and a layer not containing cell adhesion proteins. From the viewpoint of further effectively realizing the effects of the present invention and further improving cell proliferation, the first surface containing the aforementioned cell adhesion proteins is preferably the upper surface of the cell scaffold material. Preferably, the aforementioned cell adhesion proteins are present at least on the upper surface of the cell scaffold material.

[0176] In the aforementioned 100% by weight of the cell scaffold material, the content of the aforementioned cell adhesion protein is preferably 90% by weight or more, more preferably 95% by weight or more, further preferably 97.5% by weight or more, particularly preferably 99% by weight or more, and most preferably 100% by weight (total amount). When the content of the aforementioned cell adhesion protein is at or above the aforementioned lower limit, the effects of the present invention can be more effectively achieved. In the aforementioned 100% by weight of the cell scaffold material, the content of the aforementioned cell adhesion protein may be less than 100% by weight, or even less than 100% by weight.

[0177] (Other details about cell culture materials)

[0178] In the aforementioned cell culture medium, the cell scaffold material is disposed in a dotted pattern on the first surface of the substrate. Preferably, the regions of the first surface of the substrate not containing the cell scaffold material have undergone a low-adhesion treatment. In this case, the differentiation induction efficiency from pluripotent stem cells to hematopoietic progenitor cells can be further improved. The reason for this is not yet clear, but the following possibilities are considered: By effectively inhibiting the extension of endothelial cells to the regions treated with low-adhesion, these endothelial cells are locally present on the cell scaffold material, thereby promoting the possibility of differentiation induction into hematopoietic progenitor cells. By effectively inhibiting the adsorption of supplements in the liquid culture medium to the substrate, the possibility of differentiation induction into hematopoietic progenitor cells can be promoted. However, the above description is presumptive, and the reason why the differentiation induction efficiency can be further improved by the presence of regions treated with low-adhesion on the substrate is not limited to these.

[0179] Examples of cell-low adhesion treatments include coatings with fluoropolymers, amphoteric polymers, MPC polymers, or hydrogels. Commercially available products using substrates that have undergone such cell-low adhesion treatments include, for example, low-adhesion treated containers and ultra-low-adhesion treated containers.

[0180] From the perspective of further improving differentiation induction efficiency, the regions treated with the above-mentioned low cell adhesion are preferably regions that have been coated with MPC polymer or hydrogel.

[0181] It should be noted that the area on the first surface of the substrate where the cell scaffold material is disposed may or may not undergo cell low adhesion treatment.

[0182] Cell culture media containing a cell scaffold material comprising peptide resin can be prepared, for example, by the following method (A) or (B). (A) is an example of a method for preparing cell culture media using a liquid-phase synthesis of peptide resin, and (B) is an example of a method for preparing cell culture media using a solid-phase synthesis of peptide resin.

[0183] (A) Prepare a solution containing a synthetic resin (hereinafter sometimes referred to as "synthetic resin X") having a functional group capable of reacting with an amino group or a functional group capable of reacting with a carboxyl group and a peptide. React the synthetic resin X with the peptide to obtain a peptide-containing resin. Apply the solution containing the peptide-containing resin and the solvent in dots onto a first surface of a substrate, and then allow the solvent to evaporate.

[0184] (B) Prepare a solution containing a synthetic resin (synthetic resin X) having a functional group capable of reacting with an amino group or a functional group capable of reacting with a carboxyl group. Apply the solution containing synthetic resin X in dots onto a first surface of a substrate. Add a liquid containing a peptide to the first surface of the substrate, allowing the synthetic resin X to react with the peptide.

[0185] It should be noted that, as the aforementioned synthetic resin X, examples include (meth)acrylate polymers and polyvinyl acetal derivatives with bonded joints. Furthermore, as a method for coating the above solution onto the first surface of the substrate in dots, examples include methods using a dispenser, methods using an inkjet device, methods using an impression device, and methods using a pipette.

[0186] For cell culture media containing cell scaffold materials that include cell adhesion proteins, for example, it can be prepared by coating a solution containing cell adhesion proteins in dots on a first surface of the substrate and then allowing the solvent to evaporate.

[0187] [Methods for manufacturing hematopoietic progenitor cells and white blood cells]

[0188] (Cell block formation process)

[0189] The method for manufacturing hematopoietic progenitor cells and the method for manufacturing leukocytes of the present invention include a step of culturing pluripotent stem cells using the above-mentioned cell culture medium and forming cell blocks on the surface of the above-mentioned cell scaffold material (cell block formation step).

[0190] Examples of pluripotent stem cells include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), and embryonic germline stem cells (EG cells).

[0191] From the perspective of regenerative medicine and drug discovery, the aforementioned pluripotent stem cells are preferably iPS cells.

[0192] The cell block formation process described above preferably includes the following steps (a), (b), and (c) in sequence.

[0193] Step (a): The step of seeding pluripotent stem cells onto the above-mentioned cell culture medium.

[0194] Process (b): The process of culturing pluripotent stem cells.

[0195] Process (c): The process of forming cell blocks on the surface of the above-mentioned cell scaffold material.

[0196] <Process (a)>

[0197] In step (a), it is preferable to seed pluripotent stem cells that have been maintained in culture onto the cell culture medium described above.

[0198] The area of ​​the first surface of the substrate is 1 cm². 2 In step (a), the seeding density of pluripotent stem cells can be 5000 cells or more, 10000 cells or more, 15000 cells or more, less than 80000 cells, less than 65000 cells, or less than 50000 cells. In this invention, even with the above-mentioned high or low seeding densities, hematopoietic progenitor cells or leukocytes can be stably obtained.

[0199] <Process (b)>

[0200] In step (b), the pluripotent stem cells seeded in step (a) are cultured. The culture medium used for culturing the pluripotent stem cells in step (b) can be any conventionally known culture medium used for culturing pluripotent stem cells. The culture medium is preferably a liquid culture medium. Commercially available culture media or homemade culture media can be used. Examples of commercially available culture media include "Stem Fit AK03N" and "Stem Fit AK02N" manufactured by AJINOMOTO, "TeSR-E8," "mTeSR1," and "mTeSR Plus" manufactured by STEMCELL Technologies, and "StemFlex" and "Essential 8" manufactured by Gibco. The culture medium can contain a ROCK (Rho-binding kinase) specific inhibitor, or it can not contain a ROCK specific inhibitor. Alternatively, depending on the culture time, culture media containing and without ROCK specific inhibitors can be used separately.

[0201] The culture temperature for step (b) is preferably 35°C or higher, and preferably 38°C or lower. The CO2 concentration during the culture of step (b) is preferably 4% or higher, and preferably 6% or lower. For example, the culture conditions for step (b) are 37°C and a CO2 concentration of 5%. The culture time for step (b) can be, for example, 2 to 8 days (preferably 4 days). During the culture of step (b), the culture medium may or may not be changed. If the culture medium is changed, for example, it may be changed to fresh culture medium once a day.

[0202] <Process (c)>

[0203] In step (c), cell blocks can be obtained from pluripotent stem cells. By performing step (b), cell blocks are formed on the surface of the cell scaffold material. More specifically, by performing step (b), cell blocks can be formed on the upper surface of the cell scaffold material. These cell blocks are preferably cell blocks containing pluripotent stem cells. In step (c), cell blocks corresponding to the size of the upper surface of the cell scaffold material are typically formed.

[0204] (Differentiation Induction Process)

[0205] The method for manufacturing hematopoietic progenitor cells of the present invention includes a step of differentiating and inducing the obtained cell block into a cell population containing hematopoietic progenitor cells (a cell population containing hematopoietic progenitor cells) (differentiation induction step).

[0206] The method for manufacturing leukocytes according to the present invention includes a step of differentiating and inducing a obtained cell mass into a cell population (a cell population containing monocytes, lymphocytes, or granulocytes) (a differentiation induction step). The differentiation induction step in the method for manufacturing leukocytes according to the present invention can be a step of differentiating into a cell population containing monocytes, a step of differentiating into a cell population containing lymphocytes, or a step of differentiating into a cell population containing granulocytes.

[0207] In this specification, the common components of the differentiation induction process in the above-mentioned method for manufacturing hematopoietic progenitor cells and the differentiation induction process in the above-mentioned method for manufacturing leukocytes are described only as the "differentiation induction process".

[0208] In the above differentiation induction process, cell blocks obtained through the cell block formation process are used for differentiation induction.

[0209] The differentiation induction and culture conditions in the above-mentioned differentiation induction process can be based on conventionally known differentiation induction and culture conditions for the differentiation of pluripotent stem cells into hematopoietic progenitor cells or leukocytes. In the above-mentioned differentiation induction process, the same cell culture medium used in the cell block formation process can be used, or a different cell culture medium can be used. From the viewpoint of performing differentiation induction without separating the obtained cell blocks from the cell culture medium, it is preferable to use the same cell culture medium used in the cell block formation process at least at the beginning of the above-mentioned differentiation induction process. From the viewpoint of performing differentiation induction without separating cells from the cell culture medium before or during differentiation, it is preferable to use the same cell culture medium used in the cell block formation process at all points in the above-mentioned differentiation induction process.

[0210] Figure 2This document illustrates an example of a process for differentiating and inducing leukocytes (monocytes, lymphocytes, or granulocytes) from iPS cells via hematopoietic progenitor cells. Tables 1-3 show examples of liquid culture media that can be used in each step of the differentiation induction process, examples of differentiation-inducing factors that can be used, and standards during culture. Table 4 lists the formal names of the differentiation-inducing factors. It should be noted that... Figure 2 The names of each process (processes (1) to (5B)) in Tables 1-3 correspond to the names of the processes described later.

[0211]

[0212]

[0213]

[0214]

[0215] In the above-mentioned method for manufacturing hematopoietic progenitor cells, the differentiation induction process preferably includes the following steps (1), (2) and (3) in sequence.

[0216] Step (1): The process of culturing the obtained cell blocks in the presence of a culture medium containing differentiation-inducing factors to obtain a cell population containing mesoderm.

[0217] Step (2): The obtained cell population containing mesoderm is cultured in the presence of a culture medium containing differentiation inducing factors to obtain a cell population containing hematopoietic endothelial cells.

[0218] Step (3): The obtained cell population containing hematopoietic endothelial cells is cultured in the presence of a culture medium containing differentiation inducing factors to obtain a cell population containing hematopoietic progenitor cells.

[0219] <Process (1)>

[0220] In step (1), the cell block obtained in the cell block formation step is cultured. Step (1) is preferably a step in which the obtained cell block is cultured in the presence of a culture medium containing differentiation inducing factors in a state of adhering to the surface of the cell scaffold material to obtain a cell population containing mesoderm. Step (1) is preferably a step in which the pluripotent stem cells contained in the cell block are differentiated into a cell population containing mesoderm.

[0221] <Process (2)>

[0222] In step (2), the cell population containing mesoderm obtained in step (1) is cultured. Step (2) is preferably a step in which the obtained cell population containing mesoderm is cultured in the presence of a culture medium containing differentiation-inducing factors in a state of adhesion to the surface of the cell scaffold material to obtain a cell population containing hematopoietic endothelial cells. Step (2) is preferably a step in which mesoderm is induced to differentiate into a cell population containing hematopoietic endothelial cells.

[0223] <Process (3)>

[0224] In step (3), the cell population containing hematopoietic endothelial cells obtained in step (2) is cultured. Step (3) is preferably a step in which the obtained cell population containing hematopoietic endothelial cells is cultured in the presence of a culture medium containing differentiation-inducing factors in a state of adhesion to the surface of the cell scaffold material to obtain a cell population containing hematopoietic progenitor cells. Step (3) is preferably a step in which hematopoietic endothelial cells are induced to differentiate into a cell population containing hematopoietic progenitor cells.

[0225] Whether a cell has been induced to become a hematopoietic progenitor cell can be confirmed, for example, by observing cell morphology or by analyzing the expression of differentiation markers in the cells using flow cytometry. Known differentiation markers for hematopoietic progenitor cells include CD34 and CD43. For example, the simultaneous expression of CD34 and CD43 can confirm induction into hematopoietic progenitor cells.

[0226] The culture temperatures for steps (1), (2), and (3) are preferably 35°C or higher, and preferably 38°C or lower. The CO2 concentrations for steps (1), (2), and (3) are preferably 4% or higher, and preferably 6% or lower. For example, the culture conditions for steps (1), (2), and (3) are 37°C and a CO2 concentration of 5%. The culture time for steps (1), (2), and (3) can be set to a total of 4 to 13 days. During the culture of steps (1), (2), and (3), the culture medium may be changed or not. If the culture medium is changed, for example, it may be changed to fresh culture medium every two days.

[0227] Starting from the culture in step (1), for example, hematopoietic progenitor cells can be induced for about 7 days to obtain a cell population containing hematopoietic progenitor cells. The obtained cell population containing hematopoietic progenitor cells can be differentiated and induced into a cell population containing various blood cells using previously known differentiation induction and culture conditions. For example, white blood cells can be produced by differentiating and inducing the obtained cell population containing hematopoietic progenitor cells into a cell population containing monocytes, lymphocytes, or granulocytes.

[0228] In the above-described method for manufacturing leukocytes, it is preferable that the differentiation induction step, in addition to steps (1), (2), and (3), also includes the following step (4A) or step (4B). In the above-described method for manufacturing leukocytes, the differentiation induction step may sequentially include steps (1), (2), (3), and step (4A), or it may sequentially include steps (1), (2), (3), and step (4B).

[0229] Step (4A): The cell population containing hematopoietic progenitor cells is cultured in the presence of a culture medium containing differentiation-inducing factors to obtain a cell population containing bone marrow progenitor cells.

[0230] Step (4B): The cell population containing hematopoietic progenitor cells is cultured in the presence of a culture medium containing differentiation inducing factors to obtain a cell population containing lymphocyte progenitor cells.

[0231] <Process (4A)>

[0232] In step (4A), the cell population containing hematopoietic progenitor cells obtained in step (3) is cultured. Step (4A) can be a step in which the obtained cell population containing hematopoietic progenitor cells is cultured in the presence of a culture medium containing differentiation-inducing factors in a state of adhesion to the surface of the cell scaffold material to obtain a cell population containing bone marrow progenitor cells. Step (4A) is preferably a step in which hematopoietic progenitor cells are induced to differentiate into a cell population containing bone marrow progenitor cells.

[0233] <Process (4B)>

[0234] In step (4B), the cell population containing hematopoietic progenitor cells obtained in step (3) is cultured. Step (4B) preferably includes: a step of recovering suspended cells from the obtained cell population containing hematopoietic progenitor cells, and a step of culturing the recovered cells in the presence of a culture medium containing differentiation-inducing factors to obtain a cell population containing lymphocyte-like progenitor cells. In this case, the efficiency of differentiation induction into lymphocytes can be further improved, and in particular, the efficiency of differentiation induction into T cells can be further improved. Step (4B) is preferably a step of inducing hematopoietic progenitor cells to differentiate into a cell population containing lymphocyte-like progenitor cells.

[0235] The above-mentioned method for manufacturing leukocytes preferably includes the step of differentiating and inducing a cell population containing hematopoietic progenitor cells obtained by the above-mentioned method for manufacturing hematopoietic progenitor cells into a cell population containing monocytes, lymphocytes, or granulocytes. In the above-mentioned method for manufacturing leukocytes, it is preferable to manufacture leukocytes containing at least one of monocytes, lymphocytes, and granulocytes. The above-mentioned method for manufacturing leukocytes is preferably any one of the following methods: monocyte manufacturing, granulocyte manufacturing, and lymphocyte manufacturing. The above-mentioned method for manufacturing leukocytes can be a method for manufacturing monocytes, a method for manufacturing granulocytes, or a method for manufacturing lymphocytes.

[0236] When the above-mentioned method for manufacturing white blood cells is a method for manufacturing monocytes, the above-mentioned differentiation induction step preferably includes the above-mentioned step (4A) and the following step (5Aa). That is, the above-mentioned differentiation induction step preferably includes the following steps in sequence: the above-mentioned step (1), the above-mentioned step (2), the above-mentioned step (3), the above-mentioned step (4A) and the following step (5Aa).

[0237] Step (5Aa): The cell population containing bone marrow progenitor cells is cultured in the presence of a culture medium containing differentiation-inducing factors to obtain a cell population containing monocytes.

[0238] <Process (5Aa)>

[0239] In step (5Aa), it is preferable to culture the cell population containing bone marrow progenitor cells obtained in step (4A). Step (5Aa) can be a step in which the obtained cell population containing bone marrow progenitor cells is cultured in the presence of a culture medium containing differentiation-inducing factors, in a state of adhesion to the surface of the cell scaffold material, to obtain a cell population containing monocytes. Step (5Aa) is preferably a step in which bone marrow progenitor cells are induced to differentiate into a cell population containing monocytes.

[0240] Whether a cell has been induced into a monocyte can be confirmed, for example, by observing cell morphology or by analyzing the expression of differentiation markers in the cells using flow cytometry. Known differentiation markers for monocytes include CD14.

[0241] Alternatively, the obtained monocytes can be differentiated into a cell population containing dendritic cells and macrophages. The above-mentioned method for producing leukocytes can be further enhanced by a step of culturing the obtained cell population containing monocytes in the presence of a culture medium containing differentiation-inducing factors to obtain a cell population containing phagocytes.

[0242] When the above-mentioned method for manufacturing leukocytes is a method for manufacturing granulocytes, the above-mentioned differentiation induction step preferably includes the above-mentioned step (4A) and the following step (5Ab). That is, the above-mentioned differentiation induction step preferably includes the following steps in sequence: the above-mentioned step (1), the above-mentioned step (2), the above-mentioned step (3), the above-mentioned step (4A) and the following step (5Ab).

[0243] Step (5Ab): The cell population containing bone marrow progenitor cells is cultured in the presence of a culture medium containing differentiation-inducing factors to obtain a cell population containing granulocytes.

[0244] <Process (5Ab)>

[0245] In step (5Ab), it is preferable to culture the cell population containing bone marrow progenitor cells obtained in step (4A). Step (5Ab) can be a step of culturing the obtained cell population containing bone marrow progenitor cells in the presence of a culture medium containing differentiation-inducing factors, in a state of adhesion to the surface of the aforementioned cell scaffold material, to obtain a cell population containing granulocytes. Step (5Ab) is preferably a step of inducing the differentiation of bone marrow progenitor cells into a cell population containing granulocytes. Granulocytes are preferably at least one of neutrophils, eosinophils, and basophils.

[0246] Whether a cell has been induced into a granulocyte can be confirmed, for example, by observing cell morphology or by analyzing the expression of differentiation markers in the cells using flow cytometry. Known differentiation markers for granulocytes include CD15 and CD62L.

[0247] When the above-mentioned method for manufacturing leukocytes is a method for manufacturing lymphocytes, the above-mentioned differentiation induction step preferably includes the above-mentioned step (4B) and the following step (5B). That is, the above-mentioned differentiation induction step preferably includes the following steps in sequence: the above-mentioned step (1), the above-mentioned step (2), the above-mentioned step (3), the above-mentioned step (4B) and the following step (5B).

[0248] Step (5B): The cell population containing lymphocyte progenitor cells is cultured in the presence of a culture medium containing differentiation inducing factors to obtain a cell population containing lymphocytes.

[0249] <Process (5B)>

[0250] In step (5B), it is preferable to culture the cell population containing lymphocyte progenitor cells obtained in step (4B). Step (5B) is preferably a step of inducing the differentiation of lymphocyte progenitor cells into a cell population containing lymphocytes. The lymphocytes are preferably at least one of B cells, T cells, and NK cells. When obtaining a cell population containing T cells, step (5B) is preferably performed in the presence of Notch ligand.

[0251] Whether a cell has been induced into a lymphocyte can be confirmed, for example, by observing cell morphology or by analyzing the expression of differentiation markers in the cells using flow cytometry. Known differentiation markers for lymphocytes include CD3, CD4, CD8 (T cell differentiation markers), and CD56 (NK cell differentiation markers).

[0252] The present invention will be specifically described below with reference to specific embodiments. The present invention is not limited to the following embodiments.

[0253] (Example 1)

[0254] (1) Preparation of cell culture materials

[0255] As the substrate (main body of cell culture medium), 12-well plates (manufactured by Corning, surface treatment: TC (Tissue Culture treated), bottom area of ​​each well: 3.8 cm²) were prepared. 2 (This is recorded as 12wP in the table). Additionally, as a polyvinyl acetal resin, a polyvinyl butyral resin with a degree of acetalization (degree of butyralization) of 65 mol%, hydroxyl content of 32 mol%, and acetyl content of 3 mol% was prepared. A polymer mixture solution was obtained by dissolving 30 parts by weight of acrylic acid and 70 parts by weight of polyvinyl acetal resin in 255 parts by weight of tetrahydrofuran. 0.015 parts by weight of PERBUTYL O (manufactured by Nippon Oil Co., Ltd.) was dissolved in the obtained polymer mixture solution, and the reaction was carried out at 90°C for 6 hours. Next, the reacted solution was mixed with 30,000 parts by weight of water. The resulting precipitate was vacuum dried at 80°C for 3 hours to prepare a polyvinyl acetal resin with a joint (acrylic acid) (a polyvinyl butyral resin with a joint). Hereinafter, "a polyvinyl acetal resin with a joint" will sometimes be referred to as "polyvinyl acetal resin (X)".

[0256] The obtained polyvinyl acetal resin (X) was mixed with ethanol. The resulting mixture was applied in dots onto the first surface of the substrate using a dispenser, and then dried to allow the solvent to evaporate.

[0257] A cyclic peptide (5 amino acid residues, forming a cyclic backbone through Arg-Gly-Asp-Phe-Lys bonding, Phe being D-type, c-RGDfK) with the amino acid sequence Arg-Gly-Asp-Phe-Lys was prepared. Ten parts by weight of this peptide and ten parts by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (condensing agent) were added to 80 parts by weight of methanol to prepare a peptide-containing liquid. This peptide-containing liquid was added to the first surface of a substrate and reacted at 30°C, causing the carboxyl groups in the acrylic structural units derived from polyvinyl acetal resin (X) to undergo dehydration condensation with the amino groups of the Lys group of the peptide.

[0258] Thus, a cell culture medium having a substrate and a cell scaffold material disposed in a dotted pattern on a first surface of the substrate is obtained. The cell scaffold material comprises a peptide-containing polyvinyl acetal resin (peptide-containing resin).

[0259] Details of the obtained cell culture materials are shown in the table below. It should be noted that the cell scaffold material prepared as described above is listed as "PVB-AA (Composition 1)" in the table below.

[0260] (2) Cell culture

[0261] The differentiation induction media A1~E1 shown in the table below were prepared.

[0262]

[0263] (2-1) Preservation culture of iPS cells

[0264] As iPS cells, the iPS cell line established from human fibroblasts through the Kyoto University iPS Cell Research Foundation (strain 053018A, referred to as strain 1 in the table below) was used. iPS cells were seeded into 6-well plates with laminin coated on the bottom surface and cultured in StemFit AK03N medium (manufactured by AJINOMOTO) at 37°C and 5% CO2 concentration.

[0265] (2-2) iPS cell culture (cell block formation process)

[0266] iPS cells that had undergone maintenance culture were detached from 6-well plates using a detachment agent (TrypLE Select, Thermo Fisher Scientific) at 37°C for 10 minutes. The detached iPS cells were then diluted with StemFit AK03N medium containing 10 μM of the ROCK inhibitor (Y-27632) and seeded at a density of 8.0 × 10⁶ cells / well. 4Cells / well were seeded with 1.5 mL of the prepared cell culture medium. They were then cultured at 37°C and 5% CO2 in the presence of StemFit AK03N medium containing 10 μM of the ROCK inhibitor (Y-27632). The day after seeding, the medium was replaced with StemFit AK03N medium without the ROCK inhibitor (Y-27632). They were then cultured for another 3 days at 37°C and 5% CO2, resulting in cell blocks (containing iPS cells) forming on the upper surface of the cell scaffold material of the cell culture medium.

[0267] (2-3) Differentiation induction process (process (1), Day 0~Day 2)

[0268] The obtained cell blocks containing iPS cells were washed with 1.6 mL of Essential-8 medium. Then, 1.6 mL of medium A1 was added to the cell culture medium, and the cells were cultured for 2 days at 37°C and 5% CO2, with the cell blocks adhering to the surface of the cell scaffold material. This yielded a cell population containing mesoderm.

[0269] (2-4) Differentiation induction process (process (2), Day 2~Day 4)

[0270] Culture medium A1 was removed from the cell culture medium, and 1.6 mL of culture medium B1 was added to the cell culture medium. The cells, including those containing mesoderm, were cultured at 37°C and 5% CO2 for 2 days, with the cell population adhering to the surface of the cell scaffold material. This yielded a cell population containing hematopoietic endothelial cells.

[0271] (2-5) Differentiation induction process (process (3), Day 4~Day 7)

[0272] Culture medium B1 was removed from the cell culture medium, and 3.2 mL of culture medium C1 was added to the cell culture medium. The cell population containing hematopoietic endothelial cells was cultured at 37°C and 5% CO2 for 3 days, so that the cell population was adhered to the surface of the cell scaffold material. Thus, a cell population containing hematopoietic progenitor cells was obtained.

[0273] (2-6) Differentiation induction process (process (4A), Day 7~Day 10)

[0274] Culture medium C1 was removed from the cell culture medium, and 2 mL of culture medium D1 was added to the cell culture medium. The cell population containing hematopoietic progenitor cells was cultured for 3 days at 37°C and 5% CO2 concentration, with the cell population adhering to the surface of the cell scaffold material. Thus, a cell population containing bone marrow progenitor cells was obtained.

[0275] (2-7) Differentiation induction process (process (5Aa), Day 10~Day 15)

[0276] Culture medium D1 was removed from the cell culture medium, and 1.6 mL of culture medium E1 was added to the cell culture medium. Cells containing bone marrow progenitor cells were cultured at 37°C and 5% CO2, with the cell population adhered to the surface of the cell scaffold material. During culture, half the volume of liquid in the cell culture medium was replaced with fresh culture medium E1 every two days. It should be noted that the suspended cells recovered during medium replacement were cultured in non-TC culture plates (Corning Electron). On day 5 from the start of culture in culture medium E1, the supernatant containing the suspended cells was recovered. Next, using a stripping agent (TrypLE Select, Thermo Fisher Scientific), pipetting and stripping were performed to recover all cells from the cell culture medium.

[0277] (Examples 2-5)

[0278] The composition of the cell scaffold material in the cell culture medium was modified according to the table below. Otherwise, the cell culture medium was obtained in the same manner as in Example 1. Furthermore, the cell culture medium obtained was used to perform “(2) cell culture” in the same manner as in Example 1.

[0279] (Example 6)

[0280] As the substrate (main body) of the cell culture medium, a 24-well plate (manufactured by Corning, surface treatment: ultra-low adhesion treatment, bottom area of ​​each well: 1.9 cm²) was used. 2 (24wP is recorded in the table). Furthermore, the composition of the cell scaffold material was modified according to the table below. Otherwise, the cell culture medium was obtained in the same manner as in Example 1. Additionally, using the obtained cell culture medium, the seeding density of iPS cells in the cell culture medium was set to 4.0 × 10⁻⁶. 4 The cells / well were cultured, and the culture medium volume was set to 1 / 2. Otherwise, the same procedure as in Example 1 was performed for "(2) cell culture".

[0281] (Comparative Example 1)

[0282] Cell scaffold material was disposed on the entire first surface of the substrate (cell culture medium body), and the cell culture medium was obtained in the same manner as in Example 1. In addition, the cell culture medium obtained was used to perform “(2) cell culture” in the same manner as in Example 1.

[0283] (Example 7)

[0284] (1) Preparation of cell culture materials

[0285] As the substrate (main body of cell culture medium), 12-well plates (manufactured by Corning, surface treatment: TC (Tissue Culture treated), bottom area of ​​each well: 3.8 cm²) were prepared. 2 Additionally, polyvinyl acetal resin (X) was prepared in the same manner as in Example 1.

[0286] Dimethylformamide (DMF) was prepared as the first solvent. Dimethylformamide (DMF) was prepared as the second solvent. As a peptide, a cyclic peptide (5 amino acid residues, forming a cyclic backbone through Arg-Gly-Asp-Phe-Lys bonds, Phe being D-type, c-RGDfK) with the amino acid sequence Arg-Gly-Asp-Phe-Lys was prepared. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was prepared as a condensing agent. A first solution was prepared by mixing 50 parts by weight of polyvinyl acetal resin (X) and 2 parts by weight of the peptide in 1000 parts by weight of the first solvent. A second solution was prepared by mixing 1 part by weight of the condensing agent in 1000 parts by weight of the second solvent. The first and second solutions were mixed to prepare a solution containing polyvinyl acetal resin (X), the peptide, and the condensing agent.

[0287] The resulting solution was reacted at 40°C for 2 hours to allow the carboxyl group in the acrylic structural unit derived from polyvinyl acetal resin (X) to undergo dehydration condensation with the amino group of the peptide Lys, resulting in a solution containing peptide-containing polyvinyl acetal resin (peptide-containing resin).

[0288] The obtained solution containing peptide-containing polyvinyl acetal resin was diluted 100 times with DMF and washed by adding it dropwise at a rate of 0.3 mL / min into a column packed with ion exchange resin (Organo). The washed solution was then vacuum dried at 60°C for 3 hours to obtain a dried solid, which was then dissolved in butanol (alcohol solvent). Finally, 5 parts by weight of acetic acid (pH adjuster) were added relative to 100 parts by weight of butanol (alcohol solvent) to obtain a solution containing peptide-containing polyvinyl acetal resin and butanol (alcohol solvent).

[0289] Using an inkjet printer, the obtained solution containing peptide-containing polyvinyl acetal resin and butanol (alcohol solvent) is coated in dots on the first surface of the substrate, and then dried to allow the solvent to evaporate.

[0290] Thus, a cell culture medium having a substrate and a cell scaffold material disposed in a dotted pattern on a first surface of the substrate is obtained. The cell scaffold material comprises a peptide-containing polyvinyl acetal resin (peptide-containing resin).

[0291] Details of the obtained cell culture materials are shown in the table below. It should be noted that the cell scaffold material prepared as described above is referred to as "PVB-AA (Composition 2)" in the table below.

[0292] (2) Cell culture

[0293] The obtained cell culture medium was used, and the following items were changed. Except for this, “(2) cell culture” was performed in the same manner as in Example 1.

[0294] (a) strain 201B7 (referred to as strain 2 in the table below) was used as iPS cells.

[0295] (b) The culture media A2 to E2 shown in the table below were used instead of culture media A1 to E1.

[0296]

[0297] (Example 8)

[0298] The composition of the cell scaffold material in the cell culture medium was modified according to the table below. Otherwise, the cell culture medium was obtained in the same manner as in Example 7. Using the obtained cell culture medium, “(2) Cell culture” was performed in the same manner as in Example 7.

[0299] (Example 9)

[0300] (1) Preparation of cell culture materials

[0301] Using a pipette, a mixture of laminin (iMatrix-511, manufactured by NIPPI) and phosphate-buffered saline (PBS) was applied as dots on the first surface of a substrate, followed by drying to allow the solvent to evaporate. This yielded a cell culture medium comprising a substrate and a cell scaffold material with dots applied to the first surface of that substrate. The aforementioned cell scaffold material contains laminin.

[0302] (2) Cell culture

[0303] Using the obtained cell culture medium, “(2) cell culture” was performed in the same manner as in Example 7.

[0304] (Comparative Example 2)

[0305] Cell scaffold material was disposed on the entire first surface of the substrate (cell culture medium body), and a cell culture medium was obtained in the same manner as in Example 7. Using the obtained cell culture medium, “(2) cell culture” was performed in the same manner as in Example 7.

[0306] (Evaluation: Examples 1-9 and Comparative Examples 1 and 2)

[0307] (1) Presence of suspended cells

[0308] On day 15 (the fifth day after culturing in either culture medium E1 or E2), the cell culture medium was examined under a microscope to confirm the presence of suspended cells. The presence of suspended cells presumably indicates the formation of mononuclear cells.

[0309] <Evaluation Criteria for the Presence of Suspended Cells>

[0310] ○: Suspended cells can be confirmed

[0311] ×: Unable to confirm suspended cells

[0312] (2) Number of recovered cells

[0313] The number of recovered cells was determined using a cell counter (M&S TechnoSystems "NC3000").

[0314] (3) Every 10 mm of the upper surface of the cell scaffold material 2 Number of cells recovered per area

[0315] The area of ​​the upper surface of the cell scaffold material was calculated by dividing the number of recovered cells by the area of ​​the upper surface of the cell scaffold material. 2 The number of cells recovered per area.

[0316] (4) Proportion of CD14 positive cells

[0317] After the recovered cells were labeled with an anti-CD14 antibody (manufactured by BD Pharmingen), they were measured and analyzed using a flow cytometer (Sony SA3800 Cell Analyzer).

[0318] The proportion of CD14-positive cells was calculated using the following formula.

[0319] The percentage of CD14-positive cells (%) = A / B × 100

[0320] A: The number of cells containing the CD14 marker, detected by flow cytometry.

[0321] B: Cell count as determined by flow cytometry.

[0322] (5) Induction of CD14-positive cells into macrophage-like cells

[0323] The CD14-positive cells obtained in Examples 1-6 were evaluated as follows. TexMACS medium (Miltenyi Biotec) supplemented with 50 ng / mL M-CSF (granulocyte-macrophage colony-stimulating factor, Miltenyi Biotec) was prepared. 4 mL of this medium was added to the cell culture medium, and the CD14-positive cells were cultured for 7 days. During culture, half the volume of the medium in the cell culture medium was replaced with fresh medium every two days. The differentiation of the cultured cells into macrophage-like cells was confirmed by the following method.

[0324] (a) After the cultured cells were labeled with anti-CD11b antibody, flow cytometry was used for assays. The results showed that cells expressing CD11b, a marker of macrophages, were identified in all examples.

[0325] (b) The cultured cells were further cultured for 24 hours in 4 mL of TexMACS medium supplemented with 20 ng / mL IL-4. Subsequently, the cultured cells were labeled with anti-CD163 and anti-CD206 antibodies and analyzed by flow cytometry. The results showed that cells expressing CD163 and CD206, markers of M2 macrophages, were identified in all examples. Furthermore, cytokines were confirmed by ELISA, and the production of IL-10 was confirmed in all examples.

[0326] The details and results are shown in the table below. It should be noted that in the "Culture Conditions" column of the table below, "Condition 1" refers to the conditions using culture media A1~E1, and "Condition 2" refers to the conditions using culture media A2~E2. Additionally, ".E+0N" in the table below refers to "×10"... N For example, "3.2.E+06" means 3.2 × 10⁻⁶. 6 It should be noted that, based on the results of (a) and (b) above, the evaluation result of "differentiation induction of CD14 positive cells into macrophage-like cells" in Examples 1-6 is determined to be "○".

[0327]

[0328]

[0329] (Examples 10 and 11)

[0330] (1) Preparation of cell culture materials

[0331] The composition of the cell scaffold material in the cell culture medium was modified according to the table below. Otherwise, the cell culture medium was obtained in the same manner as in Example 7.

[0332] (2) Cell culture

[0333] The obtained cell culture medium was used, and the following items were changed. Except for this, “(2) cell culture” was performed in the same manner as in Example 7.

[0334] (a) 253G1 (referred to as strain 3 in the table below) was used as iPS cells.

[0335] (b) at 2.0 × 10 4 cells / well, 4.0 × 10 4 cells / well, 8.0×10 4 cells / well, 1.6 × 10 5 The seeding density of iPS cells into the obtained cell culture medium was tested under four conditions in cells / well.

[0336] (c) At the seeding density of the above 4 conditions, the procedures from “(2-1) iPS cell maintenance culture” to “(2-5) differentiation induction process (step (3), Day 4 to Day 7)” were performed under the same conditions as in Example 7. The culture in medium C2 was then continued for 5 days. During the culture using medium C2, half the volume of liquid in the cell culture medium was replaced with fresh medium C2 every two days. On Day 9, the supernatant containing the suspended cells was recovered, and then, using a stripping agent (TrypLE Select, Thermo Fisher Scientific), pipetting and stripping were performed to recover all the cells in the cell culture medium.

[0337] (Comparative Example 3)

[0338] Cell scaffold material was disposed on the entire first surface of the substrate (cell culture medium body), and a cell culture medium was obtained in the same manner as in Example 10. Using the obtained cell culture medium, “(2) cell culture” was performed in the same manner as in Example 10.

[0339] (Example 12)

[0340] (1) Preparation of cell culture materials

[0341] As the substrate (main body) of the cell culture medium, a 24-well plate (manufactured by Corning, surface treatment: TC, bottom area of ​​each well: 1.9 cm²) was used.2 (24wP, as recorded in the table), and the composition of the cell scaffold material was changed according to the table below. Otherwise, the cell culture material was obtained in the same manner as in Example 7.

[0342] (2) Cell culture

[0343] The obtained cell culture medium was used, and except that “(2) cell culture” was performed in the same manner as in Example 10.

[0344] (Example 13)

[0345] (1) Preparation of cell culture materials

[0346] As the substrate (main body) of the cell culture medium, a 24-well plate (manufactured by Corning, surface treatment: ultra-low adhesion treatment, bottom area of ​​each well: 1.9 cm²) was used. 2 (24wP, as recorded in the table), and the composition of the cell scaffold material was changed according to the table below. Otherwise, the cell culture material was obtained in the same manner as in Example 7.

[0347] (2) Cell culture

[0348] Using the obtained cell culture medium, “(2) cell culture” was performed in the same manner as in Example 12.

[0349] (Evaluation: Examples 10-13 and Comparative Example 3)

[0350] (1) Presence of suspended cells

[0351] On day 5 of culturing in the initial C2 medium, the cell culture medium was examined under a microscope to confirm the presence of suspended cells. The presence of suspended cells presumably indicates the formation of hematopoietic progenitor cells.

[0352] <Evaluation Criteria for the Presence of Suspended Cells>

[0353] ○: Suspended cells can be confirmed

[0354] ×: Unable to confirm suspended cells

[0355] (2) Number of recovered cells

[0356] The number of recovered cells was determined using a cell counter (M&S TechnoSystems "NC3000").

[0357] (3) Number of CD34 / CD43 positive cells

[0358] The recovered cells were labeled with anti-CD34 antibody (manufactured by BD Pharmingen) and anti-CD43 antibody (manufactured by BD Pharmingen), and then measured and analyzed using a flow cytometer (Sony SA3800 Cell Analyzer). The number of cells that were positive for both CD34 and CD43 (CD34 / CD43 positive cell count) was calculated.

[0359] Details and results are shown in the table below. It should be noted that "Condition 2" in the culture medium conditions column of the table below refers to the conditions using culture media A2~E2. Additionally, "E+0N" in the table below means "×10 to the power of N", for example, "3.2E+06" means 3.2 × 10⁻⁶. 6 .

[0360]

[0361]

[0362] As shown in Table 9, in Examples 10 and 11, hematopoietic progenitor cells were stably produced even at high seeding densities, compared to Comparative Example 3. Furthermore, as shown in Table 10, in Example 13, which used a substrate treated with ultra-low adhesion, more hematopoietic progenitor cells were obtained compared to Example 12, under any seeding conditions.

[0363] (Example 14)

[0364] (1) Preparation of cell culture materials

[0365] The composition of the cell scaffold material was modified according to the table below, except that the cell culture material was obtained in the same manner as in Example 7.

[0366] (2) Cell culture

[0367] The obtained cell culture medium was used, and StemFit AK02N was used instead of StemFit AK03N. Otherwise, the process was carried out in the same manner as in Example 7, from “(2-1) iPS cell maintenance culture” to “(2-5) differentiation induction process (process (3), Day 4 to Day 7)”.

[0368] In addition, the differentiation induction media F1 and G1 shown below were prepared.

[0369] Culture medium F1: StemSpan Lymphoid Progenitor Expansion Medium (STEMCELL Technologies)

[0370] Culture medium G1: StemSpan NK Cell Differentiation Medium (STEMCELL Technologies)

[0371] (2-6) Differentiation induction process (process (4B), Day 7~Day 21)

[0372] Culture medium C2 was removed from the cell culture medium, and 1.6 mL of culture medium F1 was added to the cell culture medium. The cell population, containing hematopoietic progenitor cells, was cultured for 14 days at 37°C and 5% CO2, with the cell population adhered to the surface of the cell scaffold material. During culture, half the volume of liquid in the cell culture medium was replaced with fresh culture medium F1 every 3 days. This yielded a cell population containing lymphocyte-like progenitor cells.

[0373] (2-7) Differentiation induction process (process (5B), Day 21~Day 44)

[0374] Culture medium F1 was removed from the cell culture medium, and 1.6 mL of culture medium G1 was added to the cell culture medium. The cells, containing lymphocyte progenitor cells, were cultured at 37°C and 5% CO2, with the culture medium adhering to the surface of the cell scaffold material. During culture, half the volume of liquid in the cell culture medium was replaced with fresh culture medium G1 every 3 days. It should be noted that the suspended cells recovered during medium replacement were cultured in non-TC culture plates (Corning Electron). On Day 44, the supernatant containing the suspended cells and the cells detached by pipetting were recovered. Next, using a detaching agent (TrypLE Select, Thermo Fisher Scientific), pipetting and detachment were performed to recover all cells from the cell culture medium. This yielded a cell population containing lymphocytes (NK cells).

[0375] (Example 15)

[0376] The composition of the cell scaffold material in the cell culture medium was modified according to the table below. Otherwise, the cell culture medium was obtained in the same manner as in Example 14. Using the obtained cell culture medium, “(2) Cell culture” was performed in the same manner as in Example 14.

[0377] (Comparative Example 4)

[0378] Cell scaffold material was disposed on the entire first surface of the substrate (cell culture medium body), and the cell culture medium was otherwise obtained in the same manner as in Example 7. Using the obtained cell culture medium, “(2) cell culture” was performed in the same manner as in Example 14.

[0379] (Evaluation: Examples 14 and 15 and Comparative Example 4)

[0380] (1) Presence of suspended cells

[0381] On day 44 (the 23rd day of culture in the initial G1 medium), the cell culture medium was examined under a microscope to confirm the presence of suspended cells. The presence of suspended cells presumably indicates the production of lymphocytes (NK cells).

[0382] <Evaluation Criteria for the Presence of Suspended Cells>

[0383] ○: Suspended cells can be confirmed

[0384] ×: Unable to confirm suspended cells

[0385] (2) The proportion of cells detached by pipetting

[0386] Before using the stripping agent, the proportion of cells detached from the cell culture medium by pipetting was visually confirmed (number of cells detached by pipetting / number of cells adhering to the surface of the cell scaffold material before pipetting × 100 (%)).

[0387] (3) Number of iPS cells at Day 0 time point

[0388] The number of iPS cells (cells / well) at Day 0 was determined using a cell counter (M&S TechnoSystems "NC3000").

[0389] (4) Number of CD56 positive cells

[0390] After the recovered cells were labeled with an anti-CD56 antibody (manufactured by BD Pharmingen), they were measured and analyzed using a flow cytometer (Sony SA3800 Cell Analyzer).

[0391] The proportion of CD56-positive cells was calculated using the following formula, and the number of CD56-positive cells (cells / well) was calculated based on the number of cells recovered at the Day 44 time point.

[0392] The percentage of CD56-positive cells (%) = A / B × 100

[0393] A: The number of cells containing the CD56 marker, detected by flow cytometry.

[0394] B: Cell count as determined by flow cytometry.

[0395] Details and results are shown in the table below. It should be noted that "Condition 3" in the culture medium conditions column of the table below refers to the conditions using media A2~C2, F1, and G1. Additionally, ".E+0N" in the table below means "×10 to the power of N", for example, "3.2.E+06" means 3.2 × 10⁻⁶. 6 .

[0396]

[0397] As shown in Table 11, in Examples 14 and 15, compared with Comparative Example 4, the number of iPS cells per unit Day 0 time point and the cell scaffold material per 1 cm of upper surface area were significantly different. 2 The area yielded a higher number of CD56-positive cells.

[0398] (Example 16)

[0399] (1) Preparation of cell culture materials

[0400] The composition of the cell scaffold material was modified according to the table below. Otherwise, cell culture material A1 was obtained in the same manner as in Example 7.

[0401] In addition, the rhDLL4 / Fc chimeric protein solution (10 μg / mL, manufactured by Sino Biological) was diluted with an equal volume of RetroNectin solution (10 μg / mL, manufactured by TAKARABIO), and 1 mL of the diluted solution was added to each well of a 6-well plate (surface treated: TC), and incubated overnight at 4°C. This yielded cell culture medium B1. Similarly, the rhDLL4 / Fc chimeric protein solution (10 μg / mL, manufactured by Sino Biological) was diluted with an equal volume of RetroNectin solution (10 μg / mL, manufactured by TAKARABIO), and 0.4 mL of the diluted solution was added to each well of a 24-well plate (surface treated: TC), and incubated overnight at 4°C. This yielded cell culture medium B2.

[0402] (2) Cell culture

[0403] The culture media A3~C3 and F2 shown in the table below were prepared for differentiation induction.

[0404]

[0405] The obtained cell culture medium A1 was used, and the cells were inoculated at a density of 1.6 × 10⁶ cells / year in cell culture medium A1. 5 Cells / wells were seeded in 6 wells. Apart from this, the procedures from “(2-1) iPS cell maintenance culture” to “(2-2) iPS cell culture (cell block formation process)” were performed in the same manner as in Example 10.

[0406] (2-3) Differentiation induction process (process (1), Day 0~Day 4)

[0407] 1 mL of culture medium A3 was added to cell culture medium A1, and the cells were cultured for 4 days at 37°C and 5% CO2, with the cell clumps adhering to the surface of the cell scaffold material. On Day 2, 6 μM of SB431542 (manufactured by Fujifilm and Koko Pure Chemical Industries, Ltd.) was added. This yielded a cell population containing mesoderm.

[0408] (2-4) Differentiation induction process (process (2), Day 4~Day 7)

[0409] Culture medium A3 was removed from cell culture medium A1, and 1 mL of culture medium B3 was added to cell culture medium A1. The cells, including those containing mesoderm, were cultured at 37°C and 5% CO2 for 3 days, with the cell population adhering to the surface of the cell scaffold material. This yielded a cell population containing hematopoietic endothelial cells.

[0410] (2-5) Differentiation induction process (process (3), Day 7~Day 15)

[0411] Culture medium B3 was removed from cell culture medium A1, and 1 mL of culture medium C3 was added to cell culture medium A1. The cells, containing hematopoietic endothelial cells, were cultured for 8 days at 37°C and 5% CO2, ensuring the cell population adhered to the surface of the cell scaffold material. During culture, the entire volume of liquid in cell culture medium A1 was replaced with fresh culture medium C3 every two days until Day 11. After Day 11, the culture medium was not replaced. This yielded a cell population containing hematopoietic progenitor cells.

[0412] (2-6) Differentiation induction process (processes (4B)~(5B), Day 15~Day 37)

[0413] Cells in cell culture medium A1 were peeled and recovered using a peeling agent (TrypLE Select, Thermo Fisher Scientific) at 37°C for 15 minutes. The recovered cells were then thinned by inserting and withdrawing a 2.5 mL 21 G syringe (TERUMO) with a syringe needle approximately 15 times, and then collected as single cells using a 40 μm cell analyzer (BD, Japan). CD34-positive cells were recovered from the collected cells using the magnetic bead method of the Easy Sep Human CD34 Positive Selection Kit II (Stemcell Technologies). The number of recovered CD34-positive cells was determined using a cell counter (BioRad TC20).

[0414] In the recovered CD34-positive cells, 2.0 × 10⁶ cells were added. 4 CD34-positive cells were seeded onto culture medium B1, and 2 mL of culture medium F2 was added to culture medium B1. The cells were cultured at 37°C and 5% CO2 for 8 days. On Day 17 and Day 23, the liquid in culture medium B1 was collected into tubes and centrifuged to remove culture medium F2. The culture medium F2 was then replaced with an equal volume of fresh culture medium F2 and returned to the original culture medium B1. On Day 30, the liquid in culture medium B1 was collected into tubes and centrifuged to remove culture medium F2. 400 μL of fresh culture medium F2 was added, and the cells were then seeded onto freshly prepared culture medium B2. On Day 36, the liquid in culture medium B2 was collected into tubes and centrifuged to remove culture medium F2. An equal volume of fresh culture medium F2 was then added. On Day 37, all cells in culture medium B2 were recovered by pipetting.

[0415] (Example 17)

[0416] (1) Preparation of cell culture materials

[0417] The composition of the cell scaffold material was modified according to the table below. Otherwise, cell culture material A2 was obtained in the same manner as in Example 7.

[0418] In addition, the rhDLL4 / Fc chimeric protein solution (10 μg / mL, manufactured by Sino Biological) was diluted with an equal volume of RetroNectin solution (10 μg / mL, manufactured by TAKARABIO), and 0.4 mL of the diluted solution was added to each well of a 48-well plate (surface treated: TC), and incubated overnight at 4°C. This yielded cell culture medium B3. Similarly, the rhDLL4 / Fc chimeric protein solution (10 μg / mL, manufactured by Sino Biological) was diluted with an equal volume of RetroNectin solution (10 μg / mL, manufactured by TAKARABIO), and 0.8 mL of the diluted solution was added to each well of a 12-well plate (surface treated: TC), and incubated overnight at 4°C. This yielded cell culture medium B4.

[0419] (2) Cell culture

[0420] The differentiation induction media A4~C4 and F3 shown in the table below were prepared.

[0421]

[0422] The obtained cell culture medium A2 was used, and one well in cell culture medium A2 was seeded at a density of 1.4 × 10⁻⁶. 5 Cells / wells were seeded, and the process from “(2-1) iPS cell maintenance culture” to “(2-2) iPS cell culture (cell block formation process)” was performed in the same manner as in Example 10.

[0423] (2-3) Differentiation induction process (process (1), Day 0~Day 2)

[0424] 1 mL of culture medium A4 was added to cell culture medium A2, and the cells were cultured for 2 days at 37°C and 5% CO2 concentration, with the cell blocks adhering to the surface of the cell scaffold material. This yielded a cell population containing mesoderm.

[0425] (2-4) Differentiation induction process (process (2), Day 2~Day 4)

[0426] Culture medium A4 was removed from cell culture medium A2, and 1 mL of culture medium B4 was added to cell culture medium A2. The cells, including those containing mesoderm, were cultured at 37°C and 5% CO2 for 2 days, with the cell population adhering to the surface of the cell scaffold material. This yielded a cell population containing hematopoietic endothelial cells.

[0427] (2-5) Differentiation induction process (process (3), Day 4~Day 14)

[0428] Culture medium B4 was removed from cell culture medium A2, and 1 mL of culture medium C4 was added to cell culture medium A2. The cells, containing hematopoietic endothelial cells, were cultured at 37°C and 5% CO2 for 10 days, ensuring the cell population adhered to the surface of the cell scaffold material. During culture, the entire volume of liquid in cell culture medium A2 was replaced with fresh culture medium C4 every two days until Day 11. After Day 11, the culture medium was not replaced. This yielded a cell population containing hematopoietic progenitor cells.

[0429] (2-6) Differentiation induction process (processes (4B)~(5B), Day 14~Day 33)

[0430] Cells suspended in cell culture medium A2 (cells not adhering to the cell scaffold material) were recovered using pipettes. CD34-positive cells were recovered from the recovered cells using the magnetic bead method of the Easy Sep Human CD34 Positive Selection Kit II (Stemcell Technologies). The number of recovered CD34-positive cells was determined using a cell counter (BioRad "TC20").

[0431] 1.28 × 10⁻⁶ of the recovered CD34-positive cells 4 CD34-positive cells were seeded onto culture medium B3, and 0.5 mL of culture medium F3 was added to B3. The cells were cultured at 37°C and 5% CO2 for 14 days. On Day 19, Day 21, Day 23, and Day 26, the liquid in culture medium B3 was collected into tubes and centrifuged to remove culture medium F3. The culture medium was then replaced with an equal volume of fresh culture medium F3 and returned to the original culture medium B3. On Day 28, the liquid in culture medium B3 was collected into tubes and centrifuged to remove culture medium F3. After replacing the culture medium with 1 mL of fresh culture medium F3, the cells were seeded onto a newly prepared culture medium B4. On Day 33, all cells in culture medium B4 were recovered by pipetting.

[0432] (Example 18)

[0433] The process up to Day 13 of “(2-5) Differentiation Induction Process (Process (3)” was performed in the same manner as in Example 17.

[0434] (2-6) Differentiation induction process (processes (4B)~(5B), Day 13~Day 34)

[0435] Cells suspended in cell culture medium A2 (cells not adhering to the cell scaffold material) were recovered using pipettes. The number of recovered cells was determined using a cell counter (BioRad "TC20"). A portion of the recovered cells was sampled and labeled with anti-CD34 antibody (BD Pharmingen), and then analyzed using flow cytometry (BD FACSLyric) to determine the CD34 positivity rate. The number of recovered cells was multiplied by the CD34 positivity rate to determine the number of recovered CD34-positive cells.

[0436] To achieve a CD34-positive cell count of 3.6 × 10⁻⁶. 2 Cell culture was initiated from cell culture medium A2 and seeded onto cell culture medium B4. 1 mL of culture medium F3 was added to cell culture medium B4, and the cells were cultured at 37°C and 5% CO2 for 21 days. On Day 18, the liquid in cell culture medium B4 was collected into a tube, centrifuged to remove culture medium F3, and replaced with an equal volume of fresh culture medium F3, returning the cells to the original cell culture medium B4. On Days 20 and 22, the liquid in cell culture medium B4 was collected into a tube, centrifuged to remove culture medium F3, and replaced with 1 mL of fresh culture medium F3 before being seeded onto newly prepared cell culture medium B4. On Days 25, 28, and 32, the liquid in cell culture medium B4 was collected into a tube, centrifuged to remove culture medium F3, and replaced with an equal volume of fresh culture medium F3, returning the cells to the original cell culture medium B4. On Day 35, all cells in cell culture medium B4 were recovered by pipetting.

[0437] (Evaluation: Examples 16-18)

[0438] (1) Number of recovered cells

[0439] The number of recovered cells was determined using a cell counter (BioRad "TC20").

[0440] (2) Number of CD3 positive cells

[0441] After the recovered cells were labeled with FITC-antiCD3 antibody (manufactured by BD Biosciences), they were measured and analyzed using a flow cytometer (manufactured by BD Biosciences, Japan, "Lyric") to calculate the number of CD3-positive cells.

[0442] (3) CD3 positive cell count / iPS cell count

[0443] The number of CD3-positive cells obtained from one iPS cell can be calculated using the following formula: (number of CD3-positive cells / number of iPS cells).

[0444] (CD3-positive cells / iPS cells) = A / B × C / D

[0445] A: Number of CD3-positive cells

[0446] B: Number of CD34-positive cells inoculated

[0447] C: Number of recovered CD34-positive cells

[0448] D: Number of iPS cells seeded

[0449] Details and results are shown in the table below. It should be noted that ".E+0N" in the table below refers to "×10" N For example, "3.2.E+06" means 3.2 × 10⁻⁶. 6 .

[0450]

[0451]

[0452] As shown in Tables 14 and 15, CD3-positive cells were obtained in Examples 16-18. Furthermore, compared to Example 16, in Examples 17 and 18, which included the steps of recovering cells suspended in a cell population containing hematopoietic progenitor cells and culturing the recovered cells in the presence of a culture medium containing differentiation-inducing factors to obtain a cell population containing lymphocyte progenitor cells, the number of CD3-positive cells obtained from one iPS cell increased.

Claims

1. A method for manufacturing hematopoietic progenitor cells, comprising: The cell block formation process involves culturing pluripotent stem cells using a cell culture medium containing a substrate and a cell scaffold material disposed in dots on a first surface of the substrate, and forming cell blocks on the surface of the cell scaffold material; and The differentiation induction process induces the resulting cell blocks to differentiate into a cell population containing hematopoietic progenitor cells.

2. The method for manufacturing hematopoietic progenitor cells according to claim 1, wherein, The average equivalent circle diameter of the upper surface of the cell scaffold material is greater than 200 μm and less than 3000 μm.

3. The method for manufacturing hematopoietic progenitor cells according to claim 1 or 2, wherein, The average distance between the centroids of the upper surface of the cell scaffold material is greater than 0.5 mm and less than 10 mm.

4. The method for manufacturing hematopoietic progenitor cells according to any one of claims 1 to 3, wherein, The cell scaffold material comprises a peptide-containing resin having a synthetic resin portion and a peptide portion.

5. The method for manufacturing hematopoietic progenitor cells according to claim 4, wherein, The peptide-containing resin has a polyvinyl acetal resin portion and a peptide portion.

6. The method for manufacturing hematopoietic progenitor cells according to any one of claims 1 to 5, wherein, The area on the first surface of the substrate where the cell scaffold material is not disposed has an area treated with low cell adhesion.

7. The method for manufacturing hematopoietic progenitor cells according to any one of claims 1 to 6, wherein, The pluripotent stem cells are iPS cells.

8. A method for manufacturing white blood cells, comprising: The process of differentiating and inducing a cell population comprising hematopoietic progenitor cells obtained by the manufacturing method of hematopoietic progenitor cells according to any one of claims 1 to 7 into a cell population comprising monocytes, lymphocytes or granulocytes.

9. A method for manufacturing white blood cells, comprising: The cell block formation process involves culturing pluripotent stem cells using a cell culture medium containing a substrate and a cell scaffold material disposed in dots on a first surface of the substrate, and forming cell blocks on the surface of the cell scaffold material; and The differentiation induction process induces the resulting cell blocks to differentiate into a cell population containing monocytes, lymphocytes, or granulocytes.

10. The method for manufacturing leukocytes according to claim 9, wherein, The average equivalent circle diameter of the upper surface of the cell scaffold material is greater than 200 μm and less than 3000 μm.

11. The method for manufacturing leukocytes according to claim 9 or 10, wherein, The average distance between the centroids of the upper surface of the cell scaffold material is greater than 0.5 mm and less than 10 mm.

12. The method for manufacturing leukocytes according to any one of claims 9 to 11, wherein, The cell scaffold material comprises a peptide-containing resin having a synthetic resin portion and a peptide portion.

13. The method for manufacturing leukocytes according to claim 12, wherein, The peptide-containing resin has a polyvinyl acetal resin portion and a peptide portion.

14. The method for manufacturing leukocytes according to any one of claims 9 to 13, wherein, The area on the first surface of the substrate where the cell scaffold material is not disposed has an area treated with low cell adhesion.

15. The method for manufacturing leukocytes according to any one of claims 9 to 14, wherein, The pluripotent stem cells are iPS cells.

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