Cell scaffold material, cell culture substrate, and method for introducing molecule into cell

Abstract

Provided is a cell scaffold material that improves the efficiency of introducing molecules into cells.　A cell scaffold material according to the present invention is for introducing molecules into cells, and contains a peptide-containing resin (A) having a synthetic resin part and a peptide part. The peptide part has a first peptide part and a second peptide part. The first peptide part and the second peptide part have different amino acid sequences. The first peptide part can bind to a first integrin, and the second peptide part can bind to a second integrin.

Description

Cell scaffold material, cell culture substrate, and method for introducing molecules into cells

[0001] The present invention relates to a cell scaffold material. The present invention also relates to a cell culture substrate comprising the above cell scaffold material. Further, the present invention relates to a method for introducing molecules into cells using the above cell scaffold material.

[0002] Techniques for introducing molecules such as DNA, RNA, and proteins into cells are known. As methods for introducing molecules into cells, various methods such as methods using transfection reagents, methods performing plasma treatment, and methods using viruses are known.

[0003] For example, Patent Document 1 below discloses a method for producing a cell population into which a desired gene is introduced, comprising the following steps (1) and (2). (1) A step of culturing a cell population containing T cells and / or precursor cells of T cells in a container containing fibronectin or a fragment thereof and a CD3 ligand. (2) A step of adding a vector carrying a desired gene to the container of step (1).

[0004] WO2009 / 119793A1

[0005] As a method for introducing molecules into cells, a method of culturing cells on the surface of a cell scaffold material and introducing molecules into the cells adhered to the cell scaffold material is known. Conventionally, cell adhesion proteins such as fibronectin as described in Patent Document 1 have been widely used as cell scaffold materials. Also, cell scaffold materials containing synthetic resins are known as cell scaffold materials.

[0006] However, when using a conventional cell scaffold material containing a synthetic resin, it may not be possible to enhance the efficiency of introducing molecules into cells.

[0007] An object of the present invention is to provide a cell scaffold material capable of enhancing the efficiency of introducing molecules into cells. The present invention also aims to provide a cell culture substrate comprising the above cell scaffold material. Further, the present invention also aims to provide a method for introducing molecules into cells using the above cell scaffold material.

[0008] This specification discloses the following cell scaffold materials, cell culture substrates, and methods for introducing molecules into cells.

[0009] Item 1. A cell scaffold material for introducing molecules into cells, comprising a peptide-containing resin (A) having a synthetic resin portion and a peptide portion, wherein the peptide portion comprises a first peptide portion and a second peptide portion, the first peptide portion and the second peptide portion have different amino acid sequences, the first peptide portion is capable of binding to a first integrin, and the second peptide portion is capable of binding to a second integrin.

[0010] Item 2. The cell scaffold material according to Item 1, wherein the first integrin is VLA-5 and the second integrin is VLA-4.

[0011] Item 3. The cell scaffold material according to item 1 or 2, wherein the first peptide portion has an RGD sequence and the second peptide portion has an LDV sequence.

[0012] Item 4. The cell scaffold material according to any one of items 1 to 3, wherein the first peptide portion has a c-RGDfK sequence.

[0013] Item 5. The cell scaffold material according to any one of items 1 to 4, wherein the second peptide portion has an EILDV sequence.

[0014] Item 6. The cell scaffold material according to any one of items 1 to 5, wherein the peptide-containing resin (A) has a total content P1 of the first peptide portion and a total content P2 of the second peptide portion of 0.05 mol% or more and 3.5 mol% or less.

[0015] Item 7. The cell scaffold material according to any one of items 1 to 6, wherein the peptide-containing resin (A) has a content P1 of the first peptide portion of 0.02 mol% or more and a content P2 of the second peptide portion of 0.03 mol% or more and a content P2 of 0.8 mol% or less.

[0016] Item 8. The cell scaffold material according to any one of items 1 to 7, wherein the synthetic resin portion is a polyvinyl acetal resin portion or a (meth)acrylic polymer portion.

[0017] Item 9. The cell scaffold material according to any one of items 1 to 8, wherein the synthetic resin portion is a polyvinyl acetal resin portion.

[0018] Item 10. The cell scaffold material according to any one of items 1 to 9, wherein the peptide-containing resin (A) has a linker portion, and the synthetic resin portion and the peptide portion are bonded via the linker portion.

[0019] Item 11. The cell scaffold material according to item 10, wherein the linker portion has structural units derived from polymerizable monomers.

[0020] Item 12. The cell scaffold material according to item 11, wherein the polymerizable monomer is a (meth)acrylate compound.

[0021] Item 13. The cell scaffold material according to item 11 or 12, wherein the polymerizable monomer comprises a compound having a carboxyl group.

[0022] Item 14. A cell scaffold material for introducing molecules into cells, comprising a peptide-containing resin (1) having a synthetic resin portion and a first peptide portion, and a peptide-containing resin (2) having a synthetic resin portion and a second peptide portion, wherein the first peptide portion and the second peptide portion have different amino acid sequences, the first peptide portion is capable of binding to a first integrin, and the second peptide portion is capable of binding to a second integrin.

[0023] Item 15. The cell scaffold material according to item 14, wherein the first integrin is VLA-5 and the second integrin is VLA-4.

[0024] Item 16. The cell scaffold material according to item 14 or 15, wherein the first peptide portion has an RGD sequence and the second peptide portion has an LDV sequence.

[0025] Item 17. The cell scaffold material according to any one of items 14 to 16, wherein the first peptide portion has a c-RGDfK sequence.

[0026] Item 18. The cell scaffold material according to any one of items 14 to 17, wherein the second peptide portion has an EILDV sequence.

[0027] Item 19. The cell scaffold material according to any one of items 14 to 18, wherein the sum of the content P1 of the first peptide portion in the peptide-containing resin (1) and the content P2 of the second peptide portion in the peptide-containing resin (2) is 0.05 mol% or more and 3.5 mol% or less.

[0028] Item 20. The cell scaffold material according to any one of items 14 to 19, wherein the content P1 of the first peptide portion in the peptide-containing resin (1) is 0.02 mol% or more and 1.7 mol% or less, and the content P2 of the second peptide portion in the peptide-containing resin (2) is 0.03 mol% or more and 1.8 mol% or less.

[0029] Item 21. The cell scaffold material according to any one of items 14 to 20, wherein the synthetic resin portion in the peptide-containing resin (1) is a polyvinyl acetal resin portion or a (meth)acrylic polymer portion, and the synthetic resin portion in the peptide-containing resin (2) is a polyvinyl acetal resin portion or a (meth)acrylic polymer portion.

[0030] Item 22. The cell scaffold material according to any one of items 14 to 21, wherein the synthetic resin portion in the peptide-containing resin (1) is a polyvinyl acetal resin portion, and the synthetic resin portion in the peptide-containing resin (2) is a polyvinyl acetal resin portion.

[0031] Item 23. The cell scaffold material according to any one of items 14 to 22, wherein the peptide-containing resin (1) has a linker portion, and in the peptide-containing resin (1), the synthetic resin portion and the first peptide portion are linked via the linker portion, and the peptide-containing resin (2) has a linker portion, and in the peptide-containing resin (2), the synthetic resin portion and the second peptide portion are linked via the linker portion.

[0032] Item 24. The cell scaffold material according to item 23, wherein the linker portion in the peptide-containing resin (1) has structural units derived from polymerizable monomers, and the linker portion in the peptide-containing resin (2) has structural units derived from polymerizable monomers.

[0033] Item 25. The cell scaffold material according to item 23 or 24, wherein the linker portion in the peptide-containing resin (1) has structural units derived from a (meth)acrylate compound, and the linker portion in the peptide-containing resin (2) has structural units derived from a (meth)acrylate compound.

[0034] Item 26. The cell scaffold material according to any one of items 23 to 25, wherein the linker portion in the peptide-containing resin (1) has structural units derived from a compound having a carboxyl group, and the linker portion in the peptide-containing resin (2) has structural units derived from a compound having a carboxyl group.

[0035] Section 27. Dispersion component γ of the surface free energy of the cell scaffold material. ｄ However, 24.5 mJ / m ２ 45.0mJ / m or more ２ The dipole component γ of the surface free energy of the cell scaffold material is as follows: ｐ However, 1.0 mJ / m ２ 20.0mJ / m or more ２ The cell scaffold material described in any one of items 1 to 26 below.

[0036] Item 28. A cell scaffold material for introducing molecules into cells using endocytosis, as described in any one of items 1 to 27.

[0037] Item 29. A cell scaffold material for introducing molecules into cells using a nonviral introduction method, as described in any one of items 1 to 28.

[0038] Item 30. A cell scaffold material for introducing molecules into cells using a virus introduction method, as described in any one of items 1 to 28.

[0039] Item 31. A cell scaffold material for introducing DNA or RNA into cells, as described in any one of items 1 to 30.

[0040] Item 32. A cell culture substrate comprising a substrate body and a cell scaffold material disposed on a first surface of the substrate body, wherein the cell scaffold material is the cell scaffold material described in any one of items 1 to 31.

[0041] Item 33. The cell culture substrate according to item 32, wherein the substrate body is a container.

[0042] Item 34. A method for introducing molecules into cells, comprising a culture step of culturing cells on the surface of a cell scaffold material described in any one of items 1 to 31, and a molecule introduction step of introducing molecules into cells adhered to the surface of the cell scaffold material.

[0043] Item 35. The method for introducing molecules into cells according to Item 34, wherein in the molecular introduction step, molecules are introduced into cells using endocytosis.

[0044] Item 36. The method for introducing a molecule into a cell according to item 34 or 35, wherein the molecule is DNA or RNA.

[0045] The cell scaffold material according to the present invention is a cell scaffold material for introducing molecules into cells. The cell scaffold material according to the present invention comprises a peptide-containing resin (A) having a synthetic resin portion and a peptide portion, wherein the peptide portion has a first peptide portion and a second peptide portion. The first peptide portion and the second peptide portion have different amino acid sequences. In the cell scaffold material according to the present invention, the first peptide portion is capable of binding to a first integrin, and the second peptide portion is capable of binding to a second integrin. Since the cell scaffold material according to the present invention has the above configuration, the efficiency of introducing molecules into cells can be increased.

[0046] The cell scaffold material according to the present invention is a cell scaffold material for introducing molecules into cells. The cell scaffold material according to the present invention comprises a peptide-containing resin (1) having a synthetic resin portion and a first peptide portion, and a peptide-containing resin (2) having a synthetic resin portion and a second peptide portion, wherein the first peptide portion and the second peptide portion have different amino acid sequences. In the cell scaffold material according to the present invention, the first peptide portion is capable of binding to a first integrin, and the second peptide portion is capable of binding to a second integrin. Since the cell scaffold material according to the present invention is provided with the above configuration, the efficiency of introducing molecules into cells can be increased.

[0047] Figure 1 is a schematic diagram showing an example of a peptide-containing resin (A) that can be used in the present invention. Figure 2(a) is a schematic diagram showing an example of a peptide-containing resin (1) that can be used in the present invention, and Figure 2(b) is a schematic diagram showing an example of a peptide-containing resin (2) that can be used in the present invention. Figure 3 is a schematic cross-sectional view showing a cell culture substrate according to one embodiment of the present invention.

[0048] The details of the present invention will be described below.

[0049] [Cell scaffold material] The cell scaffold material (A) according to the present invention is a cell scaffold material for introducing molecules into cells. The cell scaffold material (A) according to the present invention comprises a peptide-containing resin (A) having a synthetic resin portion and a peptide portion, wherein the peptide portion has a first peptide portion and a second peptide portion. The first peptide portion and the second peptide portion have different amino acid sequences. In the cell scaffold material (A) according to the present invention, the first peptide portion is capable of binding to a first integrin, and the second peptide portion is capable of binding to a second integrin.

[0050] The cell scaffold material (B) according to the present invention is a cell scaffold material for introducing molecules into cells. The cell scaffold material (B) according to the present invention comprises a peptide-containing resin (1) having a synthetic resin portion and a first peptide portion, and a peptide-containing resin (2) having a synthetic resin portion and a second peptide portion, wherein the first peptide portion and the second peptide portion have different amino acid sequences. In the cell scaffold material (B) according to the present invention, the first peptide portion is capable of binding to a first integrin, and the second peptide portion is capable of binding to a second integrin.

[0051] Hereinafter, in this specification, cell scaffold material (A) and cell scaffold material (B) may be collectively referred to as cell scaffold material.

[0052] The cell scaffold material according to the present invention has the above-described configuration, and therefore can improve the efficiency of molecular introduction into cells. The cell scaffold material according to the present invention can improve the efficiency of molecular introduction into cells even though it uses components (resins) that are not cell adhesion proteins.

[0053] The inventors of the present invention have found that the efficiency of molecular introduction into cells changes depending on the types of components contained in the cell scaffold material. And the inventors have found that the efficiency of molecular introduction into cells can be enhanced by using a cell scaffold material containing a peptide-containing resin having a specific amino acid sequence. By using the cell scaffold material of the present invention, it is presumed that the reason why the efficiency of molecular introduction into cells can be enhanced is that endocytosis of the cells adhering to the cell scaffold material is promoted, but it is not limited thereto.

[0054] The dispersive component γ of the surface free energy of the above cell scaffold material ｄ is preferably 24.5 mJ / m ２ or more, more preferably 28.0 mJ / m ２ or more, still more preferably 30.0 mJ / m ２ or more, particularly preferably 32.5 mJ / m ２ or more, preferably 45.0 mJ / m ２ or less, more preferably 38.0 mJ / m ２ or less, still more preferably 36.0 mJ / m ２ or less. When the above dispersive component γ ｄ is within the above lower limit and the above upper limit, the efficiency of molecular introduction into cells can be further enhanced. Also, when the above dispersive component γ ｄ is within the above lower limit and the above upper limit, the cell adhesion to the cell scaffold material can be enhanced, and the cell growth efficiency can be enhanced.

[0055] The dipole component γ of the surface free energy of the above cell scaffold material ｐ is preferably 1.0 mJ / m ２ or more, more preferably 2.5 mJ / m ２ or more, preferably 20.0 mJ / m ２ or less, more preferably 10.0 mJ / m ２ or less, still more preferably 5.0 mJ / m ２ or less. When the above dipole component γ ｐ is within the above lower limit and the above upper limit, the efficiency of molecular introduction into cells can be further enhanced. Also, when the above dipole component γ ｐWhen the values ​​are above the lower limit and below the upper limit, the ability of cells to adhere to the cell scaffold material can be improved, and the efficiency of cell proliferation can be increased.

[0056] The dispersion component γ of the above surface free energy ｄ and dipole component γ ｐ This is calculated using the Kaelble-Uy theoretical formula. The Kaelble-Uy theoretical formula is as shown in equation (1) below, where the total surface free energy γ is equal to the dispersion component γ ｄ and dipole component γ ｐ This is a theoretical formula based on the assumption that it will be the sum of [two factors].

[0057]

[0058] Furthermore, in the Kaelble-Uy theoretical formula, the surface free energy of the liquid is γ ｌ (mJ / m ２ ) and the surface free energy of the solid is γ ｓ (mJ / m ２ If we assume that the contact angle is θ (°), then the following equation (2) holds true.

[0059]

[0060] Therefore, the surface free energy γ of the liquid ｌ Using two known liquids, the contact angle θ with respect to a resin film (membrane cell scaffold material) formed using cell scaffold material was measured, and γ ｓ ｄ and γ ｓ ｐ By solving the simultaneous equations, the dispersion component γ of the surface free energy of the cell scaffold material can be obtained. ｄ and dipole component γ ｐ It is possible to find this.

[0061] In this invention, the above surface free energy γ ｌ As two known types of liquids, pure water and diiodomethane are used.

[0062] The above contact angle θ is measured using a contact angle meter (for example, "DMo-701" manufactured by Kyowa Interface Chemical Co., Ltd.) as follows.

[0063] 1 μL of pure water or diiodomethane is dropped onto the surface of a resin film formed using a cell scaffold material. The angle between the pure water and the resin film 10 seconds after dropping is defined as the contact angle θ with respect to the pure water. Similarly, the angle between the diiodomethane and the resin film 10 seconds after dropping is defined as the contact angle θ with respect to the diiodomethane.

[0064] The above variance component γ ｄ Methods to increase the amount of nonpolar functional groups in the peptide-containing resin include increasing the amount of nonpolar functional groups and introducing functional groups having a cyclic structure in the peptide-containing resin. ｄ Methods to reduce the dipole component γ include reducing the amount of butyl groups, which are nonpolar functional groups, in the peptide-containing resin. ｐ Methods to increase the dipole component γ include increasing the amount of polar functional groups in the peptide-containing resin, and introducing functional groups containing ether structures into the peptide-containing resin. ｐ Methods to reduce this include increasing the amount of butyl groups, which are nonpolar functional groups, in the peptide-containing resin.

[0065] (Peptide-conjugated resin) The cell scaffold material (A) includes a peptide-conjugated resin (A) having a synthetic resin portion and a peptide portion. The peptide-conjugated resin (A) is a synthetic resin to which peptides are bound. The peptide-conjugated resin (A) has a synthetic resin portion and a peptide portion. The peptide portion has a first peptide portion and a second peptide portion. Therefore, the peptide-conjugated resin (A) has a synthetic resin portion, a first peptide portion and a second peptide portion. The peptide-conjugated resin (A) preferably has a linker portion. In the peptide-conjugated resin (A), it is preferable that the synthetic resin portion and the peptide portion are bound via the linker portion. Only one type of peptide-conjugated resin (A) may be used, or two or more types may be used in combination.

[0066] The cell scaffold material (B) above includes a peptide-containing resin (1) having a synthetic resin portion and a first peptide portion. The cell scaffold material (B) above includes a peptide-containing resin (2) having a synthetic resin portion and a second peptide portion. The peptide-containing resins (1) and (2) above are synthetic resins to which peptides are bound. The peptide-containing resin (1) above has a synthetic resin portion and a first peptide portion. The peptide-containing resin (2) above has a synthetic resin portion and a second peptide portion. The peptide-containing resin (1) above preferably has a linker portion. In the peptide-containing resin (1) above, it is preferable that the synthetic resin portion and the first peptide portion are bound via the linker portion. The peptide-containing resin (2) above preferably has a linker portion. In the peptide-containing resin (2) above, it is preferable that the synthetic resin portion and the second peptide portion are bound via the linker portion. The peptide-containing resin (1) above may be used by one type only, or two or more types may be used in combination. The peptide-containing resin (2) described above may be used by one type only, or two or more types may be used in combination.

[0067] The preferred configuration of the synthetic resin portion in the peptide-containing resins (A), (1), and (2) described above will be explained below. Note that the common description of the synthetic resin portion in peptide-containing resin (A), the synthetic resin portion in peptide-containing resin (1), and the synthetic resin portion in peptide-containing resin (2) may simply be referred to as the "synthetic resin portion."

[0068] <Synthetic Resin Part> The above-mentioned synthetic resin part is a structural part derived from the synthetic resin in the peptide-containing resin (peptide-containing resin (A), (1), (2)). The type of the above-mentioned synthetic resin part is not particularly limited.

[0069] In the peptide-containing resin (A) described above, the synthetic resin portion is preferably a polyvinyl acetal resin portion or a (meth)acrylic polymer portion, and more preferably a polyvinyl acetal resin portion. In the peptide-containing resin (1) described above, the synthetic resin portion is preferably a polyvinyl acetal resin portion or a (meth)acrylic polymer portion, and more preferably a polyvinyl acetal resin portion. In the peptide-containing resin (2) described above, the synthetic resin portion is preferably a polyvinyl acetal resin portion or a (meth)acrylic polymer portion, and more preferably a polyvinyl acetal resin portion. In this case, the efficiency of molecular introduction into cells can be further increased. Also, the dispersion component γ of the surface free energy ｄ and dipole component γ ｐ This makes it easier to adjust the values ​​to the preferred range described above. Furthermore, it can improve the adhesion of cells to the cell scaffold material and increase the efficiency of cell proliferation. The synthetic resin portion may also consist of both a polyvinyl acetal resin portion and a (meth)acrylic polymer portion.

[0070] In the peptide-containing resin (A) having the polyvinyl acetal resin portion described above, it is preferable that the polyvinyl acetal resin portion and the peptide portion are bonded via the linker portion. Therefore, it is preferable that the peptide-containing resin (A) having the polyvinyl acetal resin portion comprises a polyvinyl acetal resin portion, a linker portion, and a peptide portion.

[0071] In the peptide-containing resin (1) having the polyvinyl acetal resin portion described above, it is preferable that the polyvinyl acetal resin portion and the first peptide portion are bonded via the linker portion. Therefore, it is preferable that the peptide-containing resin (1) having the polyvinyl acetal resin portion has a polyvinyl acetal resin portion, a linker portion, and a first peptide portion.

[0072] In the peptide-containing resin (2) having the polyvinyl acetal resin portion described above, it is preferable that the polyvinyl acetal resin portion and the second peptide portion are bonded via the linker portion. Therefore, it is preferable that the peptide-containing resin (2) having the polyvinyl acetal resin portion has a polyvinyl acetal resin portion, a linker portion, and a second peptide portion.

[0073] In the peptide-containing resin (A) having the (meth)acrylic polymer portion described above, the (meth)acrylic polymer portion and the peptide portion may be bonded via a linker portion, or they may be directly bonded without a linker portion. The peptide-containing resin (A) having the (meth)acrylic polymer portion described above may also have a (meth)acrylic polymer portion, a linker portion, and a peptide portion.

[0074] In the peptide-containing resin (1) having the (meth)acrylic polymer portion described above, the (meth)acrylic polymer portion and the first peptide portion may be bonded via a linker portion, or they may be directly bonded without a linker portion. The peptide-containing resin (1) having the (meth)acrylic polymer portion may also have a (meth)acrylic polymer portion, a linker portion, and a first peptide portion.

[0075] In the peptide-containing resin (2) having the (meth)acrylic polymer portion described above, the (meth)acrylic polymer portion and the second peptide portion may be bonded via a linker portion, or they may be directly bonded without a linker portion. The peptide-containing resin (2) having the (meth)acrylic polymer portion may also have a (meth)acrylic polymer portion, a linker portion, and a second peptide portion.

[0076] Hereinafter, the polyvinyl acetal resin portion in peptide-containing resin (A), the polyvinyl acetal resin portion in peptide-containing resin (1), and the polyvinyl acetal resin portion in peptide-containing resin (2) will be described simply as "polyvinyl acetal resin portion." Similarly, the (meth)acrylic polymer portion in peptide-containing resin (A), the (meth)acrylic polymer portion in peptide-containing resin (1), and the (meth)acrylic polymer portion in peptide-containing resin (2) will be described simply as "(meth)acrylic polymer portion." In this specification, "(meth)acrylic" means either or both "acrylic" and "methacrylic," and "(meth)acrylate" means either or both "acrylate" and "methacrylate."

[0077] <<Polyvinyl acetal resin portion>> The above polyvinyl acetal resin portion (polyvinyl acetal resin skeleton) is a structural portion derived from polyvinyl acetal resin. The above synthetic resin portion is preferably a polyvinyl acetal resin portion. The above peptide-containing resin (A) preferably has a polyvinyl acetal resin portion and a peptide portion. The above peptide-containing resin (1) preferably has a polyvinyl acetal resin portion and a first peptide portion. The above peptide-containing resin (2) preferably has a polyvinyl acetal resin portion and a second peptide portion. The above cell scaffold material preferably contains a peptide-conjugated polyvinyl acetal resin having a polyvinyl acetal resin portion and a peptide portion. The above peptide-containing resin (A) preferably is a peptide-containing polyvinyl acetal resin. The above peptide-containing resin (1) preferably is a peptide-containing polyvinyl acetal resin. The above peptide-containing resin (2) preferably is a peptide-containing polyvinyl acetal resin. The above polyvinyl acetal resins may be used individually or in combination of two or more types.

[0078] The polyvinyl acetal resin portion described above preferably has structural units having acetal groups and structural units having hydroxyl groups, and more preferably has structural units having acetal groups, structural units having hydroxyl groups, and structural units having acetyl groups.

[0079] Polyvinyl acetal resin can be synthesized by acetalizing polyvinyl alcohol with an aldehyde.

[0080] The aldehyde used in the acetalization of polyvinyl alcohol is not particularly limited. Examples of the aldehyde include aldehydes having 1 to 10 carbon atoms. The aldehyde may or may not have a linear aliphatic group, a cyclic aliphatic group, or an aromatic group. The aldehyde may be a linear aldehyde or a cyclic aldehyde. Only one aldehyde may be used, or two or more may be used in combination.

[0081] The above-mentioned aldehyde is preferably formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, or pentanal, and more preferably butyraldehyde.

[0082] Therefore, the polyvinyl acetal resin portion is more preferably a polyvinyl butyral resin portion. The synthetic resin portion is more preferably a polyvinyl butyral resin portion. The peptide-containing resin (A) preferably has a polyvinyl butyral resin portion and a peptide portion. The peptide-containing resin (1) preferably has a polyvinyl butyral resin portion and a first peptide portion. The peptide-containing resin (2) preferably has a polyvinyl butyral resin portion and a second peptide portion. The cell scaffold material preferably contains a peptide-conjugated polyvinyl butyral resin having a polyvinyl butyral resin portion and a peptide portion. The peptide-containing resin (A) preferably is a peptide-containing polyvinyl butyral resin. The peptide-containing resin (1) preferably is a peptide-containing polyvinyl butyral resin. The peptide-containing resin (2) preferably is a peptide-containing polyvinyl butyral resin. In this case, the efficiency of molecular introduction into cells can be further increased. Furthermore, it can improve the adhesion of cells to the cell scaffold material, thereby increasing the efficiency of cell proliferation.

[0083] In the peptide-containing polyvinyl acetal resin (peptide-containing resin (A), (1), (2)) described above, the degree of acetalization A of the polyvinyl acetal resin portion (or the degree of butyralization in the case of the polyvinyl butyral resin portion) is preferably 40 mol% or more, more preferably 50 mol% or more, preferably 90 mol% or less, and more preferably 85 mol% or less. If the degree of acetalization A is above the lower limit, the peptide-containing polyvinyl acetal resin will not swell easily in the culture medium. If the degree of acetalization A is below the upper limit, good solubility in solvents can be achieved. Furthermore, if the degree of acetalization A is above the lower limit and below the upper limit, the dispersion component γ of the surface free energy ｄ and dipole component γ ｐ This makes it easier to adjust the range to the preferred range mentioned above.

[0084] In the peptide-containing polyvinyl acetal resins (peptide-containing resins (A), (1), (2)), the hydroxyl group content (hydroxyl group amount B) of the polyvinyl acetal resin portion is preferably 15 mol% or more, more preferably 20 mol% or more, preferably 45 mol% or less, more preferably 35 mol% or less, and even more preferably 30 mol% or less. When the hydroxyl group amount B is above the lower limit and below the upper limit, the dispersion component γ of the surface free energy ｄ and dipole component γ ｐ This makes it easier to adjust the range to the preferred range mentioned above.

[0085] In the above peptide-containing polyvinyl acetal resin (peptide-containing resin (A), (1), (2)), the degree of acetylation C (amount of acetyl groups) of the polyvinyl acetal resin portion is preferably 0.1 mol% or more, more preferably 0.5 mol% or more, preferably 5 mol% or less, and more preferably 4 mol% or less. When the degree of acetylation C is above the lower limit and below the upper limit, the reaction efficiency between the polyvinyl acetal resin and the linker can be increased. Furthermore, when the degree of acetylation C is above the lower limit and below the upper limit, the dispersion component γ of the surface free energy ｄ and dipole component γ ｐ This makes it easier to adjust the range to the preferred range mentioned above.

[0086] In the above-mentioned peptide-containing polyvinyl acetal resin, the degree of acetalization A of the polyvinyl acetal resin portion is usually the content (mol%) of structural units having acetal groups relative to the total content (100 mol%) of structural units constituting the peptide-containing polyvinyl acetal resin.

[0087] In the above-mentioned peptide-containing polyvinyl acetal resin, the amount of hydroxyl groups B in the polyvinyl acetal resin portion is usually the content (mol%) of structural units having hydroxyl groups relative to the total content (100 mol%) of structural units constituting the peptide-containing polyvinyl acetal resin.

[0088] In the above-mentioned peptide-containing polyvinyl acetal resin, the degree of acetylation C of the polyvinyl acetal resin portion is usually the content (mol%) of the structural units having the acetyl group relative to the total content (100 mol%) of the structural units constituting the peptide-containing polyvinyl acetal resin.

[0089] In the peptide-containing polyvinyl acetal resin described above, the content of structural units in the linker portion (for example, the content of structural units derived from polymerizable monomers) is defined as content L (mol%). In the peptide-containing polyvinyl acetal resin described above, the content of the peptide portion is defined as content P (mol%). The sum of the content of structural units constituting the peptide-containing polyvinyl acetal resin usually refers to the sum of the degree of acetalization A, the amount of hydroxyl groups B, the degree of acetylation C, content L, and content P.

[0090] The degree of acetalization A, the amount of hydroxyl groups B, and the degree of acetylation C are: １ It can be measured using 1H-NMR (nuclear magnetic resonance spectroscopy).

[0091] <<(meth)acrylic polymer portion>> The above (meth)acrylic polymer portion ((meth)acrylic polymer skeleton) is a structural portion derived from the (meth)acrylic polymer. The above synthetic resin portion is preferably the (meth)acrylic polymer portion. The above peptide-containing resin (A) preferably has a (meth)acrylic polymer portion and a peptide portion. The above peptide-containing resin (1) preferably has a (meth)acrylic polymer portion and a first peptide portion. The above peptide-containing resin (2) preferably has a (meth)acrylic polymer portion and a second peptide portion. The above cell scaffold material preferably contains a peptide-containing (meth)acrylic polymer having a (meth)acrylic polymer portion and a peptide portion. The above peptide-containing resin (A) is preferably a peptide-containing (meth)acrylic polymer. The above peptide-containing resin (1) is preferably a peptide-containing (meth)acrylic polymer. The above peptide-containing resin (2) is preferably a peptide-containing (meth)acrylic polymer. Only one type of (meth)acrylic polymer may be used, or two or more types may be used in combination.

[0092] The (meth)acrylic polymer portion described above has structural units derived from a (meth)acrylate compound. The (meth)acrylate compound may be used alone or in combination of two or more types.

[0093] The above (meth)acrylic polymer portion preferably has structural units derived from a (meth)acrylate compound (A) represented by the following formula (A). In this case, the efficiency of molecular introduction into cells can be further increased. Also, the dispersion component γ of the above surface free energy ｄ and dipole component γ ｐ This makes it easier to adjust the value to the preferred range described above. Furthermore, it can improve the adhesion of cells to the cell scaffold material and increase the efficiency of cell proliferation. The (meth)acrylate compound (A) represented by the following formula (A) may be used alone or in combination of two or more types.

[0094]

[0095] In the above formula (A), R １ R represents a hydrogen atom or a methyl group. ２ This represents a hydrocarbon group with 2 to 18 carbon atoms.

[0096] In the above formula (A), R １ This may be a hydrogen atom or a methyl group.

[0097] In the above formula (A), R ２ R in formula (A) above may be an aliphatic hydrocarbon group or an aromatic hydrocarbon group. ２ R is preferably an aliphatic hydrocarbon group. The aliphatic hydrocarbon group may be linear, branched, have a double bond, or not have a double bond. ２ This may be an alkyl group or an alkylene group.

[0098] In the above formula (A), R ２The number of carbon atoms is preferably 4 or more, and preferably 16 or less. When the number of carbon atoms is above the lower limit and below the upper limit, the efficiency of molecular introduction into cells can be further increased. Also, the dispersion component γ of the surface free energy ｄ and dipole component γ ｐ This makes it easier to adjust the values ​​to the preferred range described above. Furthermore, it can improve the adhesion of cells to the cell scaffold material and increase the efficiency of cell proliferation.

[0099] Of the total structural units of the (meth)acrylic polymer portion described above, the content of structural units derived from the (meth)acrylate compound (A) is preferably 25 mol% or more, more preferably 30 mol% or more, preferably 98 mol% or less, and more preferably 95 mol% or less. When the above content is above the lower limit and below the upper limit, the efficiency of molecular introduction into cells can be further increased. In addition, the dispersion component γ of the surface free energy described above. ｄ and dipole component γ ｐ This makes it easier to adjust the values ​​to the preferred range described above. Furthermore, it can improve the adhesion of cells to the cell scaffold material and increase the efficiency of cell proliferation.

[0100] The (meth)acrylic polymer portion preferably has structural units derived from a (meth)acrylate compound (B) different from the (meth)acrylate compound (A) mentioned above.

[0101] Examples of the above (meth)acrylate compound (B) include (meth)acrylic acid, 3-butenoic acid, 4-pentenoic acid, 5-hexenoic acid, 6-heptenoic acid, 7-octenoic acid, benzeneacrylic acid, (meth)acryloyloxyethyl succinic acid, (meth)acryloyloxyethyl phthalic acid, (meth)acryloyloxypropyl succinic acid, (meth)acryloyloxypropyl phthalic acid, (meth)acryloyloxyethyl hexahydrosuccinate, (meth)acryloyloxyethyl hexahydrophthalic acid, (meth)acryloyloxypropyl hexahydrosuccinate, and (meth)acryloyloxypropyl hexahydrophthalic acid.

[0102] The above (meth)acrylate compound (B) is preferably (meth)acrylic acid, (meth)acryloyloxyethyl succinic acid, (meth)acryloyloxypropyl succinic acid, (meth)acryloyloxyethyl hexahydrosuccinic acid, (meth)acryloyloxypropyl hexahydrosuccinic acid, or butenic acid, and more preferably (meth)acrylic acid. In this case, the effects of the present invention can be exhibited even more effectively.

[0103] Of the total structural units of the (meth)acrylic polymer portion described above, the content of structural units derived from the (meth)acrylate compound (B) is preferably 2 mol% or more, more preferably 5 mol% or more, more preferably 75 mol% or less, and more preferably 70 mol% or less. When the above content is above the lower limit and below the upper limit, the efficiency of molecular introduction into cells can be further increased. In addition, the dispersion component γ of the surface free energy described above. ｄ and dipole component γ ｐ This makes it easier to adjust the values ​​to the preferred range described above. Furthermore, it can improve the adhesion of cells to the cell scaffold material and increase the efficiency of cell proliferation.

[0104] In the total structural units of the (meth)acrylic polymer portion described above, the combined content of structural units derived from (meth)acrylate compound (A) and structural units derived from (meth)acrylate compound (B) is preferably 50 mol% or more, more preferably 65 mol% or more, even more preferably 80 mol% or more, still more preferably 90 mol% or more, particularly preferably 95 mol% or more, and most preferably 100 mol%. When the above combined content is above the lower limit, the efficiency of molecular introduction into cells can be further increased. Also, the dispersion component γ of the surface free energy described above. ｄ and dipole component γ ｐ This makes it easier to adjust the concentration to the preferred range described above. Furthermore, it can improve the adhesion of cells to the cell scaffold material and increase the efficiency of cell proliferation. The total concentration may be 100 mol% or less, or 90 mol% or less.

[0105] The preferred configurations of the peptide portions (first peptide portion, second peptide portion) in the peptide-containing resins (A), (1), and (2) described above will be explained below. Note that the first peptide portion in peptide-containing resin (A) and the first peptide portion in peptide-containing resin (1) may be simply referred to as the "first peptide portion." Similarly, the second peptide portion in peptide-containing resin (A) and the second peptide portion in peptide-containing resin (2) may be simply referred to as the "second peptide portion."

[0106] <Peptide portion> The peptide portions described above (first peptide portion, second peptide portion) are structural parts derived from peptides.

[0107] The peptide portion in the peptide-containing resin (A) has a first peptide portion and a second peptide portion. In the peptide-containing resin (A), the first peptide portion and the second peptide portion have different amino acid sequences. In the peptide-containing resin (A), the first peptide portion and the second peptide portion are not directly bonded. In the peptide-containing resin (A), one first peptide portion constitutes one structural unit. In the peptide-containing resin (A), one second peptide portion constitutes one structural unit. The first peptide portion and the second peptide portion have amino acid sequences. The peptide constituting the first peptide portion may be an oligopeptide or a polypeptide. The peptide constituting the second peptide portion may be an oligopeptide or a polypeptide. Only one type of peptide may be used, or two or more types may be used in combination.

[0108] The peptide-containing resin (1) has a first peptide portion. The peptide-containing resin (2) has a second peptide portion. The first peptide portion in the peptide-containing resin (1) and the second peptide portion in the peptide-containing resin (2) have different amino acid sequences. In the peptide-containing resin (1), one first peptide portion constitutes one structural unit. In the peptide-containing resin (2), one second peptide portion constitutes one structural unit. The first peptide portion and the second peptide portion have amino acid sequences. The peptide constituting the first peptide portion may be an oligopeptide or a polypeptide. The peptide constituting the second peptide portion may be an oligopeptide or a polypeptide. Only one type of peptide may be used, or two or more types may be used in combination.

[0109] The first peptide portion in the peptide-containing resin (A) is capable of binding to a first integrin, and the second peptide portion in the peptide-containing resin (A) is capable of binding to a second integrin. The first peptide portion in the peptide-containing resin (A) has an amino acid sequence capable of binding to a first integrin, and the second peptide portion in the peptide-containing resin (A) has an amino acid sequence capable of binding to a second integrin. It is preferable that the first integrin and the second integrin are different integrins.

[0110] The first peptide portion in the peptide-containing resin (1) is capable of binding to a first integrin, and the second peptide portion in the peptide-containing resin (2) is capable of binding to a second integrin. The first peptide portion in the peptide-containing resin (1) has an amino acid sequence capable of binding to a first integrin, and the second peptide portion in the peptide-containing resin (2) has an amino acid sequence capable of binding to a second integrin. It is preferable that the first integrin and the second integrin are different integrins.

[0111] Examples of integrins include integrin α5β1 (VLA-5), integrin αVβ1, integrin αVβ3, integrin αVβ5, integrin αVβ6, integrin αVβ8, integrin αIIbβ3, integrin α4β1 (VLA-4), integrin α4β7, integrin α9β1, integrin αDβ2, integrin αLβ2, integrin αMβ2, integrin αXβ2, and integrin αEβ7. For references on integrins, see Targeting integrin pathways: mechanisms and advances in therapy, Signal Transduction and Targeted Therapy volume 8, Article number: 1 (2023), URL: https: / / doi.org / 10.1038 / s41392-022-01259-6.

[0112] Preferably, the first integrin is integrin α5β1 (VLA-5) and the second integrin is integrin α4β1 (VLA-4). Preferably, the first peptide portion of the peptide-containing resin (A) is capable of binding to integrin α5β1 (VLA-5), and the second peptide portion of the peptide-containing resin (A) is capable of binding to integrin α4β1 (VLA-4). Preferably, the first peptide portion of the peptide-containing resin (1) is capable of binding to integrin α5β1 (VLA-5), and the second peptide portion of the peptide-containing resin (2) is capable of binding to integrin α4β1 (VLA-4). In this case, the efficiency of molecular introduction into cells can be further increased.

[0113] The first peptide portion described above preferably has an RGD sequence (Arg-Gly-Asp). In this case, the efficiency of molecular introduction into cells can be further increased. The peptide portion having the RGD sequence can be bound to VLA-5.

[0114] The second peptide portion described above preferably has an LDV sequence (Leu-Asp-Val). In this case, the efficiency of molecular introduction into cells can be further increased. The peptide portion having the LDV sequence can be bound to VLA-4.

[0115] The first peptide portion described above may or may not have an LDV sequence, but it is preferable that it does not have an LDV sequence.

[0116] The second peptide portion described above may or may not have an RGD sequence, but it is preferable that it does not have an RGD sequence.

[0117] In the peptide-containing resin (A) described above, it is preferable that the synthetic resin portion and the first peptide portion are bonded via the linker portion, and that the synthetic resin portion and the second peptide portion are bonded via the linker portion. In addition, in the peptide-containing resin (A), there may be a structural portion in which the first peptide portion and the second peptide portion are bonded to the same linker portion.

[0118] The number of amino acid residues in the first peptide portion is preferably three or more, more preferably four or more, even more preferably five or more, preferably ten or fewer, more preferably eight or fewer, and even more preferably six or fewer. When the number of amino acid residues in the first peptide portion is above the lower limit and below the upper limit, the efficiency of molecular introduction into cells can be further increased. In addition, the ability of cells to adhere to the cell scaffold material can be improved, and the efficiency of cell proliferation can be increased. The number of amino acid residues in the first peptide portion may exceed ten or exceed fifteen.

[0119] The number of amino acid residues in the second peptide portion is preferably three or more, more preferably four or more, even more preferably five or more, preferably ten or fewer, more preferably eight or fewer, and even more preferably six or fewer. When the number of amino acid residues in the second peptide portion is above the lower limit and below the upper limit, the efficiency of molecular introduction into cells can be further increased. In addition, the ability of cells to adhere to the cell scaffold material can be improved, and the efficiency of cell proliferation can be increased. The number of amino acid residues in the second peptide portion may exceed ten or exceed fifteen.

[0120] The first peptide portion described above may be linear or may have a cyclic peptide skeleton. The cyclic peptide skeleton is a cyclic skeleton composed of multiple amino acids. It is preferable that the first peptide portion has a cyclic peptide skeleton. In this case, the efficiency of molecular introduction into cells can be further increased. In addition, the ability of cells to adhere to the cell scaffold material can be improved, and the efficiency of cell proliferation can be increased.

[0121] When the first peptide portion has the cyclic peptide skeleton, the cyclic peptide skeleton is preferably composed of four or more amino acids, more preferably of five or more amino acids, and preferably of ten or fewer amino acids.

[0122] The second peptide portion may be linear or may have a cyclic peptide skeleton. It is preferable that the second peptide portion is linear. It is preferable that the second peptide portion does not have a cyclic peptide skeleton. In this case, the efficiency of molecular introduction into cells can be further increased. Furthermore, the ability of cells to adhere to the cell scaffold material can be improved, thereby increasing the efficiency of cell proliferation.

[0123] The first peptide portion described above preferably has an RGD sequence represented by the following formula (P1), and more preferably has a c-RGDfK sequence. In this case, the efficiency of molecular introduction into cells can be further increased. Furthermore, the ability of cells to adhere to the cell scaffold material can be improved, and the efficiency of cell proliferation can be increased. The c-RGDfK sequence is an amino acid sequence of Arg-Gly-Asp-Phe-Lys, in which Arg and Lys are bound together to form a cyclic peptide skeleton, and Phe is the D-isomer.

[0124] Arg-Gly-Asp-X...Formula (P1)

[0125] In the above formula (P1), X represents Gly, Ala, Val, Ser, Thr, Phe, Met, Pro, or Asn.

[0126] The second peptide portion described above preferably has an EILDV sequence (Glu-Ile-Leu-Asp-Val). In this case, the efficiency of molecular introduction into cells can be further increased. Furthermore, the ability of cells to adhere to the cell scaffold material can be improved, and the efficiency of cell proliferation can be increased.

[0127] In the peptide-containing resin (A) described above, the content P1 of the first peptide portion is preferably 0.01 mol% or more, more preferably 0.02 mol% or more, even more preferably 0.03 mol% or more, particularly preferably 0.04 mol% or more, preferably 2.0 mol% or less, more preferably 1.7 mol% or less, even more preferably 1.3 mol% or less, and particularly preferably 1.0 mol% or less. When the content P1 is above the lower limit and below the upper limit, the efficiency of molecular introduction into cells can be further increased. In addition, the ability of cells to adhere to the cell scaffold material can be improved, and the efficiency of cell proliferation can be increased.

[0128] In the peptide-containing resin (1) described above, the content P1 of the first peptide portion is preferably 0.01 mol% or more, more preferably 0.02 mol% or more, even more preferably 0.03 mol% or more, particularly preferably 0.04 mol% or more, preferably 2.0 mol% or less, more preferably 1.7 mol% or less, even more preferably 1.5 mol% or less, and particularly preferably 1.2 mol% or less. When the content P1 is above the lower limit and below the upper limit, the efficiency of molecular introduction into cells can be further increased. In addition, the ability of cells to adhere to the cell scaffold material can be improved, and the efficiency of cell proliferation can be increased.

[0129] In the peptide-containing resin (A) described above, the content P2 of the second peptide portion is preferably 0.01 mol% or more, more preferably 0.03 mol% or more, even more preferably 0.04 mol% or more, particularly preferably 0.06 mol% or more, preferably 2.0 mol% or less, more preferably 1.8 mol% or less, even more preferably 1.5 mol% or less, and particularly preferably 1.2 mol% or less. When the content P2 is above the lower limit and below the upper limit, the efficiency of molecular introduction into cells can be further increased. In addition, the ability of cells to adhere to the cell scaffold material can be improved, and the efficiency of cell proliferation can be increased.

[0130] In the peptide-containing resin (2) described above, the content P2 of the second peptide portion is preferably 0.01 mol% or more, more preferably 0.03 mol% or more, even more preferably 0.04 mol% or more, particularly preferably 0.06 mol% or more, preferably 2.0 mol% or less, more preferably 1.8 mol% or less, even more preferably 1.5 mol% or less, and particularly preferably 1.2 mol% or less. When the content P2 is above the lower limit and below the upper limit, the efficiency of molecular introduction into cells can be further increased. In addition, the ability of cells to adhere to the cell scaffold material can be improved, and the efficiency of cell proliferation can be increased.

[0131] In the peptide-containing resin (A) described above, the sum of the content P1 of the first peptide portion and the content P2 of the second peptide portion is preferably 0.02 mol% or more, more preferably 0.05 mol% or more, even more preferably 0.07 mol% or more, particularly preferably 0.1 mol% or more, preferably 4.0 mol% or less, more preferably 3.5 mol% or less, even more preferably 3.3 mol% or less, and particularly preferably 3.0 mol% or less. If the sum is above the lower limit, the ability of cells to adhere to the cell scaffold material can be improved, and the efficiency of cell proliferation can be increased. If the sum is below the upper limit, good solubility in solvents can be achieved.

[0132] The sum of the content P1 of the first peptide portion in the peptide-containing resin (1) and the content P2 of the second peptide portion in the peptide-containing resin (2) is preferably 0.02 mol% or more, more preferably 0.05 mol% or more, even more preferably 0.07 mol% or more, particularly preferably 0.1 mol% or more, preferably 4.0 mol% or less, more preferably 3.5 mol% or less, even more preferably 3.3 mol% or less, and particularly preferably 3.0 mol% or less. If the sum is above the lower limit, the ability of cells to adhere to the cell scaffold material can be improved, and the efficiency of cell proliferation can be increased. If the sum is below the upper limit, the solubility in the solvent can be improved.

[0133] In the peptide-containing resin (A) described above, the molar ratio (content P1 / content P2) of the content P1 of the first peptide portion to the content P2 of the second peptide portion is preferably 0.03 or more, more preferably 0.07 or more, even more preferably 0.14 or more, preferably 25 or less, even more preferably 15 or less, and even more preferably 7 or less. When the molar ratio (content P1 / content P2) is above the lower limit and below the upper limit, the efficiency of molecular introduction into cells can be further increased. In addition, the ability of cells to adhere to the cell scaffold material can be improved, and the efficiency of cell proliferation can be increased.

[0134] The molar ratio (content P1 / content P2) of the content P1 of the first peptide portion in the peptide-containing resin (1) to the content P2 of the second peptide portion in the peptide-containing resin (2) is preferably 0.03 or more, more preferably 0.07 or more, even more preferably 0.14 or more, preferably 25 or less, even more preferably 15 or less, and even more preferably 7 or less. When the molar ratio (content P1 / content P2) is above the lower limit and below the upper limit, the efficiency of molecular introduction into cells can be further increased. In addition, the ability of cells to adhere to the cell scaffold material can be improved, and the efficiency of cell proliferation can be increased.

[0135] In the peptide-containing resin (A) described above, the molar ratio ((content P1 + content P2) / content P) of the sum of the content P1 of the first peptide portion and the content P2 of the second peptide portion to the content P of the peptide portion is preferably 0.5 or more, more preferably 0.6 or more, even more preferably 0.7 or more, still more preferably 0.8 or more, particularly preferably 0.9 or more, and most preferably 1. When the molar ratio ((content P1 + content P2) / content P) is above the lower limit, the efficiency of molecular introduction into cells can be further increased. In addition, the ability of cells to adhere to the cell scaffold material can be improved, and the efficiency of cell proliferation can be increased. Note that in the peptide-containing resin (A) described above, the molar ratio ((content P1 + content P2) / content P) may be 1 or less, or less than 1.

[0136] The above content P is the content (mol%) of the peptide portion relative to the total content (100 mol%) of the structural units constituting the peptide-containing resin. The above content P1 is the content (mol%) of the first peptide portion relative to the total content (100 mol%) of the structural units constituting the peptide-containing resin. The above content P2 is the content (mol%) of the second peptide portion relative to the total content (100 mol%) of the structural units constituting the peptide-containing resin.

[0137] The content P of the peptide portion, the content P1 of the first peptide portion, and the content P2 of the second peptide portion can be measured, for example, by NMR (nuclear magnetic resonance).

[0138] In the case where the peptide-containing resin (A) described above is a peptide-containing resin (peptide-containing polyvinyl acetal resin) having a polyvinyl acetal resin portion and a linker portion, a preferred configuration of the peptide portion content will be further explained.

[0139] As described above, in the peptide-containing polyvinyl acetal resin, the degree of acetalization of the polyvinyl acetal resin portion is denoted as degree of acetalization A, the hydroxyl group content of the polyvinyl acetal resin portion is denoted as hydroxyl group content B, and the degree of acetylation of the polyvinyl acetal resin portion is denoted as degree of acetylation C. Also, as described above, in the peptide-containing polyvinyl acetal resin, the content of structural units in the linker portion (for example, the content of structural units derived from polymerizable monomers) is denoted as content L. Also, as described above, in the peptide-containing polyvinyl acetal resin, the content of the first peptide portion is denoted as content P1, and the content of the second peptide portion is denoted as content P2.

[0140] In the peptide-containing polyvinyl acetal resin (peptide-containing resin (A)) described above, of the total of acetalization degree A, hydroxyl group amount B, acetylation degree C, content L, content P1, and content P2 in 100 mol%, content P1 is preferably 0.01 mol% or more, more preferably 0.02 mol% or more, even more preferably 0.03 mol% or more, particularly preferably 0.04 mol% or more, preferably 2.0 mol% or less, more preferably 1.7 mol% or less, even more preferably 1.5 mol% or less, and particularly preferably 1.2 mol% or less. When the above content P1 is above the lower limit and below the upper limit, the efficiency of molecular introduction into cells can be further increased. In addition, the ability of cells to adhere to the cell scaffold material can be improved, and the efficiency of cell proliferation can be increased.

[0141] In the peptide-containing polyvinyl acetal resin (peptide-containing resin (A)) described above, of the total of acetalization degree A, hydroxyl group amount B, acetylation degree C, content L, content P1, and content P2 in 100 mol%, content P2 is preferably 0.01 mol% or more, more preferably 0.03 mol% or more, even more preferably 0.05 mol% or more, particularly preferably 0.07 mol% or more, preferably 2.0 mol% or less, more preferably 1.8 mol% or less, even more preferably 1.6 mol% or less, and particularly preferably 1.4 mol% or less. When the above content P2 is above the lower limit and below the upper limit, the efficiency of molecular introduction into cells can be further increased. In addition, the adhesion of cells to the cell scaffold material can be improved, and the efficiency of cell proliferation can be increased.

[0142] In the peptide-containing polyvinyl acetal resin (peptide-containing resin (A)) described above, out of a total of 100 mol% of the acetalization degree A, hydroxyl group amount B, acetylation degree C, content L, content P1, and content P2, the sum of content P1 and content P2 is preferably 0.02 mol% or more, more preferably 0.05 mol% or more, even more preferably 0.07 mol% or more, particularly preferably 0.10 mol% or more, preferably 4.0 mol% or less, more preferably 3.5 mol% or less, even more preferably 3.0 mol% or less, and particularly preferably 2.5 mol% or less. When the above sum is above the lower limit and below the upper limit, the efficiency of molecular introduction into cells can be further increased. In addition, the adhesion of cells to the cell scaffold material can be improved, and the efficiency of cell proliferation can be increased.

[0143] The preferred configuration of the linker portion in the peptide-containing resins (A), (1), and (2) described above will be explained below. Note that the linker portion in peptide-containing resin (A), the linker portion in peptide-containing resin (1), and the linker portion in peptide-containing resin (2) will sometimes be simply referred to as the "linker portion."

[0144] <Linker portion> The linker portion is a structural portion derived from the linker. The linker portion is formed by the linker. The peptide-containing resin (A) preferably has a linker portion. The peptide-containing resin (1) preferably has a linker portion. The peptide-containing resin (2) preferably has a linker portion. Only one type of linker may be used, or two or more types may be used in combination.

[0145] The linker is preferably a polymerizable monomer. Therefore, the linker portion in the peptide-containing resin (A) preferably has structural units derived from a polymerizable monomer. The linker portion in the peptide-containing resin (1) preferably has structural units derived from a polymerizable monomer. The linker portion in the peptide-containing resin (2) preferably has structural units derived from a polymerizable monomer. Only one polymerizable monomer may be used, or two or more may be used in combination.

[0146] Examples of polymerizable monomers include (meth)acrylate compounds. Examples of (meth)acrylate compounds include the (meth)acrylate compound (A) and the (meth)acrylate compound (B) described above.

[0147] From the viewpoint of good reaction with peptides, the polymerizable monomer is preferably a (meth)acrylate compound. The linker portion in the peptide-containing resin (A) preferably has structural units derived from a (meth)acrylate compound. The linker portion in the peptide-containing resin (1) preferably has structural units derived from a (meth)acrylate compound. The linker portion in the peptide-containing resin (2) preferably has structural units derived from a (meth)acrylate compound.

[0148] The polymerizable monomer described above may or may not have an aromatic skeleton.

[0149] The polymerizable monomer is preferably a compound having a functional group that can bind to the peptide, and more preferably a compound having a functional group that can condense with the carboxyl group or amino group of the peptide.

[0150] Functional groups that can condense with the carboxyl group or amino group of the above peptide include carboxyl groups, thiol groups, amino groups, hydroxyl groups, and cyano groups.

[0151] From the viewpoint of reacting well with peptides, the polymerizable monomer preferably contains a compound having a carboxyl group or an amino group, and more preferably contains a compound having a carboxyl group. The linker portion in the peptide-containing resin (A) preferably has structural units derived from a compound having a carboxyl group. The linker portion in the peptide-containing resin (1) preferably has structural units derived from a compound having a carboxyl group. The linker portion in the peptide-containing resin (2) preferably has structural units derived from a compound having a carboxyl group.

[0152] When obtaining a peptide-containing resin having a polyvinyl acetal resin portion, examples of the carboxyl group-containing compound (carboxylic acid monomer) include (meth)acrylic acid and carboxyl group-containing acrylamide. By using the above carboxyl group-containing compound (carboxylic acid monomer), the carboxylic acid monomer can be polymerized by graft polymerization when a linker is introduced, thereby increasing the number of carboxyl groups that can react with the peptide.

[0153] In the peptide-containing resin (A) described above, the linker portion preferably has structural units derived from a (meth)acrylate compound that does not have a carboxyl group and structural units derived from a (meth)acrylate compound that has a carboxyl group. In the peptide-containing resin (1) described above, the linker portion preferably has structural units derived from a (meth)acrylate compound that does not have a carboxyl group and structural units derived from a (meth)acrylate compound that has a carboxyl group. In the peptide-containing resin (2) described above, the linker portion preferably has structural units derived from a (meth)acrylate compound that does not have a carboxyl group and structural units derived from a (meth)acrylate compound that has a carboxyl group. In this case, the dispersion component γ of the surface free energy ｄ and dipole component γ ｐ This makes it easier to adjust the range to the preferred range mentioned above.

[0154] In the peptide-containing resin (A) described above, it is preferable that the structural units derived from the (meth)acrylate compound that does not have a carboxyl group include structural units derived from butyl (meth)acrylate. In the peptide-containing resin (1) described above, it is preferable that the structural units derived from the (meth)acrylate compound that does not have a carboxyl group include structural units derived from butyl (meth)acrylate. In the peptide-containing resin (2) described above, it is preferable that the structural units derived from the (meth)acrylate compound that does not have a carboxyl group include structural units derived from butyl (meth)acrylate. In this case, the dispersion component γ of the surface free energy ｄ and dipole component γ ｐ This makes it easier to adjust the range to the preferred range mentioned above.

[0155] In the peptide-containing resin (A) described above, the structural units derived from the (meth)acrylate compound having a carboxyl group are more preferably derived from (meth)acrylic acid, and even more preferably from acrylic acid. In the peptide-containing resin (1) described above, the structural units derived from the (meth)acrylate compound having a carboxyl group are more preferably derived from (meth)acrylic acid, and even more preferably from acrylic acid. In the peptide-containing resin (2) described above, the structural units derived from the (meth)acrylate compound having a carboxyl group are more preferably derived from (meth)acrylic acid, and even more preferably from acrylic acid. In this case, the synthetic resin portion and the peptide portion can be well bonded via the linker portion.

[0156] When obtaining a peptide-containing resin having a (meth)acrylic polymer portion, it is preferable that the polymerizable monomer has a functional group capable of binding to a (meth)acrylic acid ester. Examples of functional groups capable of binding to a (meth)acrylic acid ester include vinyl groups, (meth)acryloyl groups, and allyl groups. It is preferable that the polymerizable monomer has a (meth)acryloyl group, and more preferably has a carboxyl group or an amino group, and also has a (meth)acryloyl group.

[0157] Examples of polymerizable monomers used to obtain a peptide-containing resin having a (meth)acrylic polymer portion include (meth)acrylic acid, itaconic acid, and acrylamide.

[0158] From the viewpoint of effectively bonding the (meth)acrylic polymer and the peptide via the linker portion, the polymerizable monomer is preferably (meth)acrylic acid or itaconic acid, and more preferably (meth)acrylic acid.

[0159] <Further Details of Peptide-Containing Resins> The number average molecular weight of the peptide-containing resins (peptide-containing resins (A), (1), (2)) 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. If the number average molecular weight is above the lower limit, the cell scaffold material will be less likely to dissolve into the liquid culture medium during cell culture, and the cell scaffold material will be less likely to peel off the main body of the substrate during cell culture. If the number average molecular weight is below the upper limit, the solubility in alcohol solvents can be increased, making it easier to coat the main body of the substrate with a composition containing the cell scaffold material and alcohol solvent.

[0160] The number-average molecular weight of the peptide-containing resin can be measured, for example, by the following method. The peptide-containing resin is dissolved in tetrahydrofuran (THF) to prepare a 0.2% by weight solution of the peptide-containing resin. Next, it is evaluated using a gel permeation chromatography (GPC) analyzer (APC system, Waters Corporation) under the following measurement conditions.

[0161] Column: HSPgel HR MB-M 6.0 × 150 mm Flow rate: 0.5 mL / min Column temperature: 40°C Injection volume: 10 μL Detector: RI, PDA Standard sample: Polystyrene

[0162] Examples of methods for obtaining a peptide-containing resin (peptide-containing polyvinyl acetal resin) having the above-mentioned polyvinyl acetal resin portion include the following methods.

[0163] A polyvinyl acetal resin is reacted with a linker (polymerizable monomer) to obtain a reaction product in which the polyvinyl acetal resin and the linker are bonded. The obtained reaction product is then reacted with a peptide to obtain a peptide-containing polyvinyl acetal resin.

[0164] Methods for obtaining a peptide-containing resin (peptide-containing (meth)acrylic polymer) having the above-mentioned (meth)acrylic polymer portion include, for example, the following methods (1) and (2).

[0165] Method (1): A (meth)acrylic polymer is reacted with a linker (polymerizable monomer) to obtain a reaction product in which the (meth)acrylic polymer and the linker are bonded. The obtained reaction product is reacted with a peptide to obtain a peptide-containing (meth)acrylic polymer.

[0166] Method (2): A peptide-containing (meth)acrylic polymer is obtained by reacting a (meth)acrylic polymer with a peptide.

[0167] From the viewpoint of further improving the efficiency of molecular introduction into cells, it is preferable that the peptide-containing resin (A) has a main chain and a graft chain. In this case, it is preferable that the synthetic resin portion constitutes the main chain. It is also preferable that the graft chain has a structural unit derived from the polymerizable monomer and the peptide portion. It is preferable that the graft chain has a linker portion bound to the main chain and a peptide portion bound to the linker portion. It is preferable that the graft chain has a linker portion bound to the main chain, a first peptide portion bound to the linker portion and a second peptide portion bound to the linker portion.

[0168] From the viewpoint of further improving the efficiency of molecular introduction into cells, it is preferable that the peptide-containing resin (1) has a main chain and a graft chain. In this case, it is preferable that the synthetic resin portion constitutes the main chain. It is also preferable that the graft chain has a structural unit derived from the polymerizable monomer and the first peptide portion. It is preferable that the graft chain has a linker portion bound to the main chain and the first peptide portion bound to the linker portion.

[0169] From the viewpoint of further improving the efficiency of molecular introduction into cells, it is preferable that the peptide-containing resin (2) has a main chain and a graft chain. In this case, it is preferable that the synthetic resin portion constitutes the main chain. It is also preferable that the graft chain has a structural unit derived from the polymerizable monomer and the second peptide portion. It is preferable that the graft chain has a linker portion bound to the main chain and the second peptide portion bound to the linker portion.

[0170] Figure 1 is a schematic diagram showing an example of a peptide-containing resin (A) that can be used in the present invention.

[0171] The peptide-containing resin 10 shown in Figure 1 is the peptide-containing resin (A) described above. The peptide-containing resin 10 has a synthetic resin portion 11, a linker portion 12, and a peptide portion 15. The peptide portion 15 has a first peptide portion 13 and a second peptide portion 14. Therefore, the peptide-containing resin 10 has a synthetic resin portion 11, a linker portion 12, a first peptide portion 13, and a second peptide portion 14. In Figure 1, to facilitate understanding the similarities and differences between the linker portion 12, the first peptide portion 13, and the second peptide portion 14, the first peptide portion 13 is represented by a circle and the second peptide portion 14 is represented by a triangle.

[0172] The first peptide portion 13 has an RGD sequence. The second peptide portion 14 has an LDV sequence. The peptide-containing resin 10 has a main chain and a graft chain. The synthetic resin portion 11 constitutes the main chain of the peptide-containing resin 10. The graft chain has a linker portion 12, a first peptide portion 13, and a second peptide portion 14. The linker portion 12 is bound to the synthetic resin portion 11. The first peptide portion 13 is bound to the linker portion 12. The second peptide portion 14 is bound to the linker portion 12. The synthetic resin portion 11 and the first peptide portion 13 are bound via the linker portion 12, and the synthetic resin portion 11 and the second peptide portion 14 are bound via the linker portion 12. In the peptide-containing resin 10, the first peptide portion 13 and the second peptide portion 14 are bound to the same linker portion 12.

[0173] Figure 2(a) is a schematic diagram showing an example of a peptide-containing resin (1) that can be used in the present invention, and Figure 2(b) is a schematic diagram showing an example of a peptide-containing resin (2) that can be used in the present invention.

[0174] The peptide-containing resin 10a shown in Figure 2(a) is the peptide-containing resin (1) described above. The peptide-containing resin 10a has a synthetic resin portion 11a, a linker portion 12a, and a first peptide portion 13a. In Figure 2(a), the first peptide portion 13a is shown as a circle to facilitate understanding the similarities and differences between the linker portion 12a and the first peptide portion 13a.

[0175] The first peptide portion 13a has an RGD sequence. The peptide-containing resin 10a has a main chain and a graft chain. The synthetic resin portion 11a constitutes the main chain of the peptide-containing resin 10a. The graft chain has a linker portion 12a and the first peptide portion 13a. The linker portion 12a is bound to the synthetic resin portion 11a. The first peptide portion 13a is bound to the linker portion 12a. The synthetic resin portion 11a and the first peptide portion 13a are linked via the linker portion 12a.

[0176] The peptide-containing resin 10b shown in Figure 2(b) is the peptide-containing resin (2) described above. The peptide-containing resin 10b has a synthetic resin portion 11b, a linker portion 12b, and a second peptide portion 14b. In Figure 2(b), the second peptide portion 14b is represented by a triangle to facilitate understanding the similarities and differences between the linker portion 12b and the second peptide portion 14b.

[0177] The second peptide portion 14b has an LDV sequence. The peptide-containing resin 10b has a main chain and a graft chain. The synthetic resin portion 11b constitutes the main chain of the peptide-containing resin 10b. The graft chain has a linker portion 12b and the second peptide portion 14b. The linker portion 12b is bound to the synthetic resin portion 11b. The second peptide portion 14b is bound to the linker portion 12b. The synthetic resin portion 11b and the second peptide portion 14b are linked via the linker portion 12b.

[0178] (Further details of cell scaffold material) The cell scaffold material (A) above comprises the peptide-containing resin (A). The cell scaffold material (B) above comprises the peptide-containing resin (1) and the peptide-containing resin (2). The cell scaffold material may or may not contain other components other than the peptide-containing resin. The cell scaffold material may or may not contain, for example, polyvinyl acetal resin (polyvinyl acetal resin without peptides). The cell scaffold material may or may not contain, for example, (meth)acrylic polymer ((meth)acrylic polymer without peptides). Other components may include polyolefin resin, polyether resin, polyester, epoxy resin, polyamide resin, polyimide resin, polyurethane resin, polycarbonate resin, cellulose, and polypeptide. Only one of the other components may be used, or two or more may be used in combination.

[0179] From the viewpoint of improving the adhesion of cells to the cell scaffold material, cell scaffold material (A) containing peptide-containing resin (A) is more preferable to cell scaffold material (B) containing peptide-containing resins (1) and (2). This is because cell scaffold material (A) contains peptide-containing resin (A) which has a first and second peptide portion in one molecule, and therefore these first and second peptide portions are likely to be adjacent to each other.

[0180] In 100% by weight of the cell scaffold material (A), the content of the peptide-containing resin (A) is preferably 90% by weight or more, more preferably 95% by weight or more, even more 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 (A) is above the lower limit, the effects of the present invention can be exhibited even more effectively. In 100% by weight of the cell scaffold material (A), the content of the peptide-containing resin (A) may be 100% by weight or less, or less than 100% by weight.

[0181] In 100% by weight of the cell scaffold material (B), the total content of the peptide-containing resin (1) and the peptide-containing resin (2) is preferably 90% by weight or more, more preferably 95% by weight or more, even more 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 above the lower limit, the effects of the present invention can be exhibited even more effectively. In 100% by weight of the cell scaffold material (B), the total content of the peptide-containing resin (1) and the peptide-containing resin (2) may be 100% by weight or less, or less than 100% by weight.

[0182] The above-mentioned cell scaffold material is a cell scaffold material for introducing molecules into cells. The above-mentioned cell scaffold material is a cell scaffold material that serves as a scaffold for introducing molecules into cells. The above-mentioned cell scaffold material is used at least when introducing molecules into cells.

[0183] The above-mentioned cell scaffold material is preferably a cell scaffold material for gene transfer into cells.

[0184] Examples of the cells mentioned above include animal cells from humans, mice, rats, pigs, cattle, and monkeys. Other examples of the cells include somatic cells, such as stem cells, progenitor cells, and mature cells. These somatic cells may also be cancer cells. Only one type of cell may be used, or two or more types may be used in combination.

[0185] Examples of the above-mentioned stem cells include somatic stem cells and embryonic stem cells, such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells (MSCs), iPS cells, ES cells, Muse cells, embryonic cancer cells, embryonic germ stem cells, and mGS cells.

[0186] Examples of the mature cells mentioned above include nerve cells, cardiomyocytes, retinal cells, and hepatocytes.

[0187] The above cells are preferably CD3-positive cells including T cells, NK cells, monocytes, or macrophage cells.

[0188] Examples of molecules introduced into the above-mentioned cells include DNA (deoxyribonucleic acid), RNA (ribonucleic acid), and proteins.

[0189] The molecule introduced into the cells is preferably DNA, RNA, or protein, and more preferably DNA or RNA. In other words, the cell scaffold material is preferably a cell scaffold material for introducing DNA, RNA, or protein into cells, and more preferably a cell scaffold material for introducing DNA or RNA into cells. In this case, the efficiency of molecule introduction into cells can be further increased.

[0190] The length of the DNA is preferably 3000 bp or more, more preferably 4000 bp or more, more preferably 18000 bp or less, and more preferably 12000 bp or less. In this case, the efficiency of molecular introduction into cells can be further increased.

[0191] The RNA length is preferably 500 nt or more, more preferably 700 nt or more, more preferably 5000 nt or less, and more preferably 3000 nt or less. In this case, the efficiency of molecular introduction into cells can be further increased.

[0192] It is preferable to introduce molecules into cells using cellular endocytosis. In other words, the cell scaffold material is preferably a cell scaffold material for introducing molecules into cells using endocytosis. In this case, the efficiency of molecule introduction into cells can be further increased.

[0193] Methods for introducing molecules into cells using cellular endocytosis include non-viral introduction methods and viral introduction methods.

[0194] Examples of non-viral introduction methods include transfection and plasma methods.

[0195] Examples of the above-mentioned methods for introducing viruses include methods using lentiviruses, AAV viruses, retroviruses, and Sendai viruses.

[0196] The above-mentioned cell scaffold material is preferably a cell scaffold material for molecular introduction into cells using a non-viral introduction method, and is also preferably a cell scaffold material for molecular introduction into cells using a viral introduction method.

[0197] The above-mentioned cell scaffold material is more preferably a cell scaffold material for molecular introduction into cells using a non-viral introduction method, and even more preferably a cell scaffold material for molecular introduction into cells using a transfection method or a plasma method. In this case, safety can be enhanced while further increasing the efficiency of molecular introduction into cells.

[0198] [Cell Culture Substrate] The cell culture substrate according to the present invention comprises a substrate body and a cell scaffold material disposed on the first surface of the substrate body. In the cell culture substrate according to the present invention, the cell scaffold material is the cell scaffold material described above.

[0199] The material, size, and shape of the substrate body described above are not particularly limited. Conventional known substrate bodies can be used as the substrate body.

[0200] Examples of materials for the main body of the substrate include synthetic resins, metals, and glass. Examples of synthetic resins include polystyrene, polyethylene, polyethylene terephthalate, polypropylene, polyethersulfone, polycarbonate, polyester, polyisoprene, cycloolefin polymer, polyimide, polyamide, polyamideimide, (meth)acrylic resin, epoxy resin, and silicone.

[0201] The substrate body can take the form of fibrous material, particulate material, plate-like material, film-like material, porous membrane-like material, or bag-like material. The substrate body may also be a container.

[0202] The above-mentioned substrate body is preferably a container. The shape and size of the above-mentioned container (substrate body) are not particularly limited. Examples of the above-mentioned container (substrate body) include 2 to 384 well plates, single-walled flasks, multi-walled flasks, multifaceted flasks, dishes, roller bottles, bags, insert cups, and microfluidic tips.

[0203] Figure 3 is a schematic cross-sectional view showing a cell culture substrate according to one embodiment of the present invention.

[0204] The cell culture substrate 1 comprises a substrate body 2 and a cell scaffold material 3. The substrate body 2 is a container. The cell culture substrate 1 is a cell culture vessel. The cell scaffold material 3 is arranged on the first surface 2a of the substrate body 2.

[0205] [Method for introducing molecules into cells] The method for introducing molecules into cells according to the present invention comprises a culture step of culturing cells on the surface of the cell scaffold material described above, and a molecular introduction step of introducing molecules into cells adhered to the surface of the cell scaffold material.

[0206] The above-described method for introducing molecules into cells is a method using the cell culture substrate described above, and preferably comprises a culture step of culturing cells on the surface of the cell scaffold material in the cell culture substrate, and a molecule introduction step of introducing molecules into cells adhered to the surface of the cell scaffold material in the cell culture substrate.

[0207] <Culturing Process> The above culturing process is a process of culturing cells on the surface of the cell scaffold material. The above culturing process is a process of adhering and culturing cells. In the above culturing process, cells can be cultured under conventionally known conditions, except that the cell scaffold material according to the present invention is used as the cell scaffold material. The above culturing process may also be a process of pre-culturing cells on the surface of the cell scaffold material.

[0208] Examples of the above-mentioned cells include those described above. Preferably, the above-mentioned cells are CD3-positive cells including T cells, NK cells, monocytes, or macrophages.

[0209] <Molecular Introduction Process> The molecular introduction process described above is a process of introducing molecules into cells that are attached to the surface of the cell scaffold material described above. In the molecular introduction process described above, molecules can be introduced into cells under conventionally known conditions.

[0210] Examples of the above-mentioned molecules include those described above. The above-mentioned molecules are preferably DNA, RNA, or protein, and more preferably DNA or RNA. In this case, the efficiency of molecule introduction into cells can be further increased. The preferred configurations of DNA length and RNA length are as described above.

[0211] In the above molecular introduction step, it is preferable to introduce molecules into cells using endocytosis, more preferably using a non-viral or viral introduction method, even more preferably using a non-viral introduction method, and particularly preferable to introduce molecules into cells using a transfection method or plasma method. In this case, the efficiency of molecular introduction into cells can be further increased.

[0212] The present invention will be specifically described below with reference to examples and comparative examples. The present invention is not limited to the following examples. The content of structural units in the obtained peptide-containing resin, for example, the degree of acetalization A, the amount of hydroxyl groups B, the degree of acetylation C, the content of structural units derived from polymerizable monomers, the content of the first peptide portion P1, and the content of the second peptide portion P2 are determined by dissolving the peptide-containing resin in DMSO-d6 (dimethyl sulfoxide). １ The measurements were performed using 1H-NMR (nuclear magnetic resonance spectroscopy).

[0213] (Example 1) (Preparation of Polyvinyl Acetal Resin X) 2700 mL of deionized water and 300 g of polyvinyl alcohol with an average degree of polymerization of 250 and a degree of saponification of 99 mol% were added to a reactor equipped with a stirring device, and the mixture was heated and dissolved while stirring to obtain a solution. Next, 35 wt% hydrochloric acid was added to this solution as a catalyst to a hydrochloric acid concentration of 0.2 wt%, and the temperature was adjusted to 15°C. Then, 22 g of n-butyraldehyde (n-BA) was added while stirring. Subsequently, 148 g of n-butyraldehyde (n-BA) was added, and white particulate polyvinyl acetal resin precipitated. Fifteen minutes after precipitation, 35 wt% hydrochloric acid was added to a hydrochloric acid concentration of 1.8 wt%, the mixture was heated to 50°C, and aged at 50°C for 2 hours. Next, the solution was cooled and neutralized, then the polyvinyl acetal resin was washed with water and dried to obtain polyvinyl acetal resin (polyvinyl butyral resin). A mixed solution was obtained by dissolving 6 parts by weight of acrylic acid, 21 parts by weight of butyl acrylate, and 70 parts by weight of the obtained polyvinyl acetal resin in 255 parts by weight of tetrahydrofuran. 0.015 parts by weight of perbutyl O (manufactured by NOF Corporation) was dissolved in the obtained mixed solution and reacted at 90°C for 6 hours. Next, the reaction solution was mixed with 30,000 parts by weight of water. The obtained precipitate was vacuum dried at 80°C for 3 hours to produce polyvinyl acetal resin X (polyvinyl acetal resin with a linker portion) having structural units derived from acrylic acid and structural units derived from butyl acrylate.

[0214] (Preparation Steps) DMF was prepared as the first solvent. DMF was prepared as the second solvent. A cyclic peptide having the amino acid sequence Arg-Gly-Asp-Phe-Lys (5 amino acid residues, Arg and Lys combine to form a cyclic skeleton, Phe is the D-isomer, c-RGDfK) was prepared as the first peptide. A peptide having the amino acid sequence Glu-Ile-Leu-Asp-Val (5 amino acid residues, EILDV) was prepared as the second peptide. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was prepared as the condensing agent. The first solution was prepared by mixing 50 parts by weight of polyvinyl acetal resin X, 3 parts by weight of the first peptide, and 3 parts by weight of the second peptide with 1000 parts by weight of the first solvent. The second solution was prepared by mixing 10 parts by weight of the condensing agent with 1000 parts by weight of the second solvent. The first solution and the second solution were mixed to prepare a solution containing polyvinyl acetal resin X, the first peptide, the second peptide, and a condensing agent.

[0215] (Reaction step) The obtained solution was reacted at 40°C for 2 hours to dehydrate and condense the carboxyl groups in the structural units derived from acrylic acid of polyvinyl acetal resin X with the amino groups of Lys of the first peptide or Glu of the second peptide, thereby obtaining a solution containing peptide-containing polyvinyl acetal resin (A).

[0216] (Purification Process) The solution containing the obtained peptide-containing polyvinyl acetal resin (A) was diluted 100-fold with DMF and washed by dropping it onto a column packed with ion exchange resin (manufactured by Organo) at a rate of 0.3 mL / min. The washed solution was vacuum-dried at 60°C for 3 hours to obtain a dry product. The obtained dry product was dissolved in butanol (alcohol solvent), and 5 parts by weight of acetic acid (pH adjuster) was added to 100 parts by weight of butanol to obtain a coating solution containing peptide-containing polyvinyl acetal resin (A) and butanol. The content of peptide-containing polyvinyl acetal resin (A) in the coating solution was 0.1% by weight.

[0217] (Preparation of cell culture substrate) 15 μL of the obtained coating solution was coated onto each well of a 48-well plate by cast coating, and then vacuum-dried at 60°C for 3 hours to remove the alcohol solvent. In this way, a cell culture substrate (cell culture container) was obtained in which a cell scaffold material (resin film), which is a dried layer of the coating solution, was placed on the bottom surface of each well of the 48-well plate.

[0218] (Example 2) Except that the amount of the first peptide used was changed to 6 parts by weight and the amount of the second peptide used was changed to 6 parts by weight in the preparation step, a cell scaffold material and a cell culture substrate were obtained in the same manner as in Example 1.

[0219] (Example 3) Except that the amount of the first peptide used in the preparation step was changed to 11.3 parts by weight and the amount of the second peptide used to 0.8 parts by weight, a cell scaffold material and a cell culture substrate were obtained in the same manner as in Example 1.

[0220] (Example 4) Except that the amount of the first peptide used was changed to 10.5 parts by weight and the amount of the second peptide used to 1.5 parts by weight in the preparation step, a cell scaffold material and a cell culture substrate were obtained in the same manner as in Example 1.

[0221] (Example 5) Except that the amount of the first peptide used was changed to 7.5 parts by weight and the amount of the second peptide used to 4.5 parts by weight in the preparation step, a cell scaffold material and a cell culture substrate were obtained in the same manner as in Example 1.

[0222] (Example 6) Except that the amount of the first peptide used was changed to 4.5 parts by weight and the amount of the second peptide used to 7.5 parts by weight in the preparation step, a cell scaffold material and a cell culture substrate were obtained in the same manner as in Example 1.

[0223] (Example 7) Except that the amount of the first peptide used was changed to 1.5 parts by weight and the amount of the second peptide used to 10.5 parts by weight in the preparation step, a cell scaffold material and a cell culture substrate were obtained in the same manner as in Example 1.

[0224] (Example 8) Except that the amount of the first peptide used was changed to 0.8 parts by weight and the amount of the second peptide used to 11.3 parts by weight in the preparation step, a cell scaffold material and a cell culture substrate were obtained in the same manner as in Example 1.

[0225] (Example 9) A cell scaffold material and a cell culture substrate were obtained in the same manner as in Example 1, except that a peptide having the amino acid sequence Arg-Gly-Asp-Ser (4 amino acid residues, RGDS) was used as the first peptide.

[0226] (Example 10) A cell scaffold material and a cell culture substrate were obtained in the same manner as in Example 1, except that a peptide having the amino acid sequence Gly-Arg-Gly-Asp-Ser (5 amino acid residues, GRGDS) was used as the first peptide.

[0227] (Example 11) A cell scaffold material and a cell culture substrate were obtained in the same manner as in Example 1, except that a peptide having the amino acid sequence Leu-Asp-Val (3 amino acid residues, LDV) was used as the second peptide.

[0228] (Example 12) A cell scaffold material and a cell culture substrate were obtained in the same manner as in Example 1, except that the amount of the first peptide used was changed to 15.0 parts by weight and the amount of the second peptide used was changed to 15.0 parts by weight in the preparation step.

[0229] (Example 13) A cell scaffold material and a cell culture substrate were obtained in the same manner as in Example 1, except that the amount of the first peptide used was changed to 22.5 parts by weight and the amount of the second peptide used was changed to 22.5 parts by weight in the preparation step.

[0230] (Example 14) Except that the amount of the first peptide used was changed to 27 parts by weight and the amount of the second peptide used was changed to 27 parts by weight in the preparation step, a cell scaffold material and a cell culture substrate were obtained in the same manner as in Example 1.

[0231] (Example 15) Except that the amount of the first peptide used was changed to 0.3 parts by weight and the amount of the second peptide used was changed to 0.3 parts by weight in the preparation step, a cell scaffold material and a cell culture substrate were obtained in the same manner as in Example 1.

[0232] (Example 16) Cell scaffold material and cell culture substrate were obtained in the same manner as in Example 1, except that the amount of n-butyraldehyde (n-BA) added in the second step of preparing polyvinyl acetal resin X was changed from 148 g to 89 g.

[0233] (Example 17) 75 parts by weight of N-isopropylacrylamide and 25 parts by weight of butyl methacrylate were dissolved in 300 parts by weight of tetrahydrofuran to obtain an acrylic monomer solution. 2 parts by weight of Irgacure 184 (manufactured by BASF) were dissolved in the obtained acrylic monomer solution and coated onto a PET film. The coated object was exposed to light at a wavelength of 365 nm with an integrated light intensity of 2000 mJ / cm using an I-Graphics UV conveyor system "ECS301G1" at 25°C. ２ An acrylic resin solution was obtained by irradiation. The obtained acrylic resin solution was vacuum-dried at 80°C for 3 hours to obtain acrylic resin. Cell scaffold material and cell culture substrate were obtained in the same manner as in Example 1, except that the obtained acrylic resin was used instead of polyvinyl acetal resin.

[0234] (Example 18) An acrylic resin was obtained in the same manner as in Example 17, except that 90 parts by weight of methoxyethyl acrylate and 10 parts by weight of butyl methacrylate were used instead of 75 parts by weight of N-isopropylacrylamide and 25 parts by weight of butyl methacrylate. Furthermore, a cell scaffold material and a cell culture substrate were obtained in the same manner as in Example 17, except that this acrylic resin was used.

[0235] (Example 19) An acrylic resin was obtained in the same manner as in Example 17, except that 75 parts by weight of methoxyethyl acrylate and 25 parts by weight of butyl methacrylate were used instead of 75 parts by weight of N-isopropylacrylamide and 25 parts by weight of butyl methacrylate. Furthermore, a cell scaffold material and a cell culture substrate were obtained in the same manner as in Example 17, except that this acrylic resin was used.

[0236] (Example 20) An acrylic resin was obtained in the same manner as in Example 17, except that 2 parts by weight of butyl methacrylate and 98 parts by weight of ethyl acrylate were used instead of 75 parts by weight of N-isopropylacrylamide and 25 parts by weight of butyl methacrylate. Furthermore, a cell scaffold material and a cell culture substrate were obtained in the same manner as in Example 17, except that this acrylic resin was used.

[0237] (Example 21) 100 parts by weight of ethyl acrylate, 75 parts by weight of ethyl acetate, and 0.5 parts by weight of azobisisobutyronitrile were mixed and polymerized at 65°C for 8 hours under a nitrogen atmosphere to obtain a polyethyl acrylate resin. A cell scaffold material and a cell culture substrate were obtained in the same manner as in Example 1, except that the obtained polyethyl acrylate resin was used instead of polyvinyl acetal resin.

[0238] (Example 22) A cell scaffold material and a cell culture substrate were obtained in the same manner as in Example 21, except that 100 parts by weight of butyl methacrylate were used instead of 100 parts by weight of ethyl acrylate.

[0239] (Example 23) (Preparation of coating solution X) A polyvinyl acetal resin X (polyvinyl acetal resin bonded with acrylic acid and butyl acrylate) prepared in Example 1 was prepared. DMF was prepared as the first solvent. A cyclic peptide having the amino acid sequence Arg-Gly-Asp-Phe-Lys (5 amino acid residues, Arg and Lys bond to form a cyclic skeleton, Phe is the D-isomer, c-RGDfK) was prepared as the first peptide. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was prepared as the condensing agent. 50 parts by weight of polyvinyl acetal resin X and 3 parts by weight of the first peptide were mixed with 1000 parts by weight of the first solvent to prepare the first solution. A second solution was prepared by mixing 10 parts by weight of the condensing agent with 10 parts by weight of the first solvent. The first solution and the second solution were mixed to prepare a solution containing polyvinyl acetal resin X, the first peptide, and a condensing agent.

[0240] The obtained solution was reacted at 40°C for 2 hours to dehydrate and condense the carboxyl groups in the structural units derived from acrylic acid of polyvinyl acetal resin X with the amino groups of Lys in the first peptide, thereby obtaining a solution containing peptide-containing polyvinyl acetal resin (1).

[0241] The solution containing the obtained peptide-containing polyvinyl acetal resin (1) was diluted 100-fold with DMF and washed by dropping it dropwise into a column packed with ion exchange resin (manufactured by Organo) at a rate of 0.3 mL / min. The washed solution was vacuum-dried at 60°C for 3 hours to obtain a dry product. The obtained dry product was dissolved in butanol (alcohol solvent), and 5 parts by weight of acetic acid (pH adjuster) was added to 100 parts by weight of butanol to obtain a coating solution X containing peptide-containing polyvinyl acetal resin (1) and butanol. The content of peptide-containing polyvinyl acetal resin (1) in the coating solution X was 0.1% by weight.

[0242] (Preparation of coating solution Y) A polyvinyl acetal resin X (polyvinyl acetal resin bonded with acrylic acid and butyl acrylate) prepared in Example 1 was prepared. DMF was prepared as the second solvent. A peptide having the amino acid sequence Glu-Ile-Leu-Asp-Val (5 amino acid residues, EILDV) was prepared as the second peptide. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was prepared as the condensing agent. 50 parts by weight of polyvinyl acetal resin X and 3 parts by weight of the second peptide were mixed with 1000 parts by weight of the second solvent to prepare the first solution. Also, 10 parts by weight of the condensing agent were mixed with 1000 parts by weight of the second solvent to prepare the second solution. The first solution and the second solution were mixed to prepare a solution containing polyvinyl acetal resin X, the second peptide and the condensing agent.

[0243] The obtained solution was reacted at 40°C for 2 hours to dehydrate and condense the carboxyl groups in the structural units derived from acrylic acid of polyvinyl acetal resin X with the amino groups of Glu in the second peptide, thereby obtaining a solution containing peptide-containing polyvinyl acetal resin (2).

[0244] The solution containing the obtained peptide-containing polyvinyl acetal resin (2) was diluted 100-fold with DMF and washed by dropping it dropwise into a column packed with ion exchange resin (manufactured by Organo) at a rate of 0.3 mL / min. The washed solution was vacuum-dried at 60°C for 3 hours to obtain a dry product. The obtained dry product was dissolved in butanol (alcohol solvent), and 5 parts by weight of acetic acid (pH adjuster) was added to 100 parts by weight of butanol to obtain a coating solution Y containing peptide-containing polyvinyl acetal resin (2) and butanol. The content of peptide-containing polyvinyl acetal resin (2) in the coating solution Y was 0.1% by weight.

[0245] (Preparation of cell scaffold material and cell culture substrate) Coating solution X was mixed with coating solution Y, 50 parts by weight to obtain coating solution Z. Except for using coating solution Z, a cell scaffold material and a cell culture substrate were obtained in the same manner as in Example 1.

[0246] (Example 24) A cell scaffold material and a cell culture substrate were obtained in the same manner as in Example 23, except that the amount of the first peptide used in the preparation step of coating solution X was changed to 11.3 parts by weight, and the amount of the second peptide used in the preparation step of coating solution Y was changed to 0.8 parts by weight.

[0247] (Example 25) A cell scaffold material and a cell culture substrate were obtained in the same manner as in Example 23, except that the amount of the first peptide used in the preparation step of coating solution X was changed to 0.8 parts by weight, and the amount of the second peptide used in the preparation step of coating solution Y was changed to 11.3 parts by weight.

[0248] (Comparative Example 1) A cell scaffold material and a cell culture substrate were obtained in the same manner as in Example 1, except that the first peptide was not used.

[0249] (Comparative Example 2) A cell scaffold material and a cell culture substrate were obtained in the same manner as in Example 1, except that the second peptide was not used.

[0250] (Evaluation) (1) (Surface Free Energy) The surface free energy of the obtained cell scaffold material (resin film) was measured using a contact angle meter (Kyowa Interface Chemical Co., Ltd., DMo-701). The contact angle of pure water was obtained by dropping 1 μL of pure water onto the resin film and taking a picture of the droplet after 10 seconds. The contact angle of diiodomethane was obtained by dropping 1 μL of diiodomethane onto the resin film and taking a picture of the droplet after 10 seconds. From the obtained contact angles, the dispersion component γ of the surface free energy was calculated using the Kaelble-Uy theoretical formula. ｄ and dipole component γ ｐ The result was calculated.

[0251] (2) The following were prepared for the efficiency of molecular introduction into cells.

[0252] Human PBMC (“CTL-UP1” manufactured by Cellular Technology Ltd) ALyS705 IL-2(-) (“120P10” manufactured by Cell Science Institute (CSTI)) IL-7 (“130-095-367” manufactured by Miltenyi Biotec) Human IL-15 (“130-095-764” manufactured by Miltenyi Biotec) Artificial serum (Animal-free) (“A2G20P2CC” manufactured by Cell Science Institute (CSTI)) TransAct (“130-111-160” manufactured by Miltenyi Biotec)

[0253] A culture medium was prepared by adding 2.5 mL of artificial serum, IL-7 at 10 ng / ml, and IL-15 at 5 ng / ml to 47.5 mL of AlyS 705 medium.

[0254] (2-1) DNA introduction test into cells using plasma method (non-viral introduction method) Approximately 1 mL of thawed human PBMC cells (CTL-UP1, manufactured by CTL Corporation) was added to 9 mL of culture medium and suspended. Next, the cells were centrifuged at room temperature at 200 × g for 10 minutes, and the supernatant was aspirated. The cell pellet was suspended in 2 mL of culture medium, and the number of cells was counted using a cell counter (BioRAD Corporation). The cell suspension solution was 12.5 × 10 ５ The cell solution was adjusted to a cell / mL ratio, and 1 / 100th the volume of TransAct was added relative to the volume of the suspension. 5 × 10⁻⁶ of this cell solution was prepared. ５Cells were seeded into the prepared cell culture substrate (a cell culture vessel equipped with a cell scaffold material containing peptide-containing resin, a 48-well plate) to a cell / 400 μL / well ratio, and then cultured using AlyS705 medium (manufactured by Cell Science Institute Co., Ltd.) with CO2. ２ Incubator at 37°C and 5% CO2 ２ The eggs were incubated for two days under these conditions.

[0255] Next, the culture medium and supernatant were removed from the wells, and DNA (GFP gene) was added to the wells. Plasma irradiation was performed under the following conditions, and 0.4 mL of AlyS705 medium (manufactured by Cell Science Institute Co., Ltd.) was added as a recovery medium. Then, CO ２ Incubator at 37°C and 5% CO2 ２ The cells were cultured under the following conditions. Cells (3 × 10) one day after plasma irradiation. ４ The number of cells was measured using a flow cytometer. Gates (R1) presumed to be "live cells" and "GFP-positive cells" were measured. The number of live cells was determined from the gates presumed to be live cells using the flow cytometer, and the number of cells showing positive fluorescence for GFP, the "GFP-positive cell count," was determined. The transmission efficiency (%) was calculated by dividing the "GFP-positive cell count" by the "live cell count" and multiplying it by 100.

[0256] <Plasma Irradiation Conditions> Fabrication of the first electrode: One end of a 3 mm diameter stainless steel cylinder was covered with a 0.5 mm thick alumina cap. A 0.1 m diameter through-hole was formed on the surface of the alumina cap facing the second electrode by laser processing to fabricate the first electrode. Second electrode: 3 mm thick copper plate Power supply frequency: 50 kHz Discharge voltage: 16 kV ｐ－ｐ Discharge time: 80 ms; Number of discharge points per well: 1; Distance between electrode end and top of container: 1 mm; Amount of DNA (GFP): 2 μg

[0257] [Criteria for determining implementation efficiency] ○○○: Implementation efficiency exceeds 9% ○○: Implementation efficiency is 8% or more and 9% or less ○: Implementation efficiency is 7% or more and less than 8% ×: Implementation efficiency is less than 7%

[0258] (2-2) RNA Introduction Test into Cells Using the Plasma Method (Non-viral Introduction Method) The test was conducted in the same manner as the above-mentioned “(2-1) DNA Introduction Test into Cells Using the Plasma Method (Non-viral Introduction Method)”, except that RNA (GFP gene) was used instead of DNA (GFP gene). Using a flow cytometer, the “number of viable cells” was determined from the gate estimated to be viable cells, and the number of cells showing positive fluorescence for GFP, the “number of GFP-positive cells”, was determined. The introduction efficiency (%) was calculated as (“number of GFP-positive cells” ÷ “number of viable cells”) × 100.

[0259] [Criteria for Judging Introduction Efficiency] ○○○: Introduction efficiency exceeds 9% ○○: Introduction efficiency is 8% or more and 9% or less ○: Introduction efficiency is 7% or more and less than 8% ×: Introduction efficiency is less than 7%

[0260] (2-3) DNA Introduction Test into Cells Using the Transfection Method (Non-viral Introduction Method) Approximately 1 mL of thawed human PBMC cells (“CTL-UP1” manufactured by CTL) was added to 9 mL of culture medium and suspended. Next, centrifugation was performed at 200×g for 10 minutes at room temperature, and the supernatant was aspirated. The cell pellet was suspended in 2 mL of culture medium, and the cell count was performed using a cell counter (BioRAD). The solution in which the cells were suspended was adjusted to 12.5×10 ５ cells / mL, and 1 / 100 volume of TransAct was added to the volume of the suspension solution. This cell solution was seeded into the prepared cell culture substrate (a cell culture container equipped with a cell scaffold material containing a peptide-containing resin, a 48-well plate) so as to be 5×10 ５ cells / 400 μL / well, and using AlyS705 medium (manufactured by Cell Science Institute), it was incubated in a CO ２ incubator at 37°C and 5% by volume of CO ２ for 2 days.

[0261] Solution 1 was prepared by adding 125 μL of Opti-MEM (registered trademark, Thermo Fisher Scientific) and 3.75 μL of Lipofectamine (registered trademark) 2000 Reagent (Thermo Fisher Scientific) to a 1.5 mL tube and mixing them. Solution 2 was prepared by adding 1 μL of DNA (GFP gene) adjusted to 1 μg / μL to 125 μL of Opti-MEM (registered trademark) Medium (Thermo Fisher Scientific). Solution 1 and Solution 2 were mixed in equal volumes and allowed to stand at room temperature for 10 to 15 minutes. The solutions were then dispensed into 48-well plates to a concentration of 250 μL / well. The well plates were gently shaken up and down and side to side to mix, and the plates were incubated at 37°C for 24 to 72 hours.

[0262] Using a flow cytometer, the "number of viable cells" was determined from gates presumed to contain viable cells, and the "number of GFP-positive cells" (cells showing positive fluorescence for GFP) was determined. The transmission efficiency (%) was calculated by dividing the "number of GFP-positive cells" by the "number of viable cells" and multiplying by 100.

[0263] [Criteria for determining implementation efficiency] ○○○: Implementation efficiency exceeds 6.5% ○○: Implementation efficiency is 5.5% or more and 6.5% or less ○: Implementation efficiency is 4.5% or more and less than 5.5% ×: Implementation efficiency is less than 4.5%

[0264] (2-4) RNA introduction test into cells using transfection method (non-viral introduction method) The test was conducted in the same manner as in "(2-3) DNA introduction test into cells using transfection method (non-viral introduction method)" above, except that RNA (GFP gene) was used instead of DNA (GFP gene). The "number of viable cells" was determined from gates presumed to be viable cells using a flow cytometer, and the "number of GFP-positive cells" was determined, which is the number of cells that showed positive fluorescence for GFP. The introduction efficiency (%) was calculated by "number of GFP-positive cells" ÷ "number of viable cells" × 100.

[0265] [Criteria for determining implementation efficiency] ○○○: Implementation efficiency exceeds 6.5% ○○: Implementation efficiency is 5.5% or more and 6.5% or less ○: Implementation efficiency is 4.5% or more and less than 5.5% ×: Implementation efficiency is less than 4.5%

[0266] (2-5) DNA introduction test into cells using the virus introduction method Approximately 1 mL of thawed human PBMC cells (CTL Corporation's "CTL-UP1") was added to 9 mL of culture medium and suspended. This cell solution was divided into 2 × 10⁻⁶ units. ５ Cells were seeded into the prepared cell culture substrate (a cell culture vessel equipped with a cell scaffold material containing peptide-containing resin, a 48-well plate) at a cell / 400 μL / well ratio, and then cultured using AlyS705 medium with CO2. ２ Incubator at 37°C and 5% CO2 ２ The eggs were incubated for two days under these conditions.

[0267] A retroviral vector encoding the GFP gene (VectorBuilder, pMMLV[Exp]-EGFP / Puro (Vector ID:VB010000-9307ddn)) was prepared. This retroviral vector was diluted fourfold in AlyS705 medium to obtain a virus-containing solution. After removing the medium (supernatant) from the wells so that approximately 100 μL of the medium remained in each well, 50 μL of the virus-containing solution was added to the wells to achieve an MOI of 5. The cell culture vessels were then left to stand for 2 hours in an incubator set to 37°C. Next, 100 μL of the medium was added to the wells, and CO2 was added. ２ Incubator at 37°C and 5% CO2 ２ The eggs were incubated for two days under these conditions.

[0268] After two days of incubation (two days after infection), CD3 staining was performed using CD3-APC (BD Corporation). Subsequently, the number of CD3-positive cells ("CD3-positive cell count") and the number of CD3-positive and GFP-positive cells ("CD3-positive and GFP-positive cell count") were determined using a flow cytometer. The transmission efficiency (%) was calculated as "CD3-positive and GFP-positive cell count" ÷ "CD3-positive cell count" × 100, and this was defined as the transmission efficiency (A).

[0269] Furthermore, a DNA introduction test using a retroviral vector was performed in the same manner as described above, except that a cell culture substrate without cell scaffold material (a 48-well plate before coating with the coating solution) was used instead of the prepared cell culture substrate. The introduction efficiency was calculated using the formula described above and was defined as introduction efficiency (B).

[0270] [Criteria for determining implementation efficiency] ○○○: Implementation efficiency (A) is more than 1.8 times that of implementation efficiency (B) ○○: Implementation efficiency (A) is 1.1 times or more but 1.8 times or less that of implementation efficiency (B) ○: Implementation efficiency (A) is 1.0 times or more but less than 1.1 times that of implementation efficiency (B) ×: Implementation efficiency (A) is less than 1.0 times that of implementation efficiency (B)

[0271] (2-6) RNA introduction test into cells using the viral introduction method The test was conducted in the same manner as in "(2-5) DNA introduction test into cells using the viral introduction method" above, except that a Sendai virus vector encoding the GFP gene (manufactured by Tokiwa Bio Co., Ltd.) was used instead of a retroviral vector. The number of cells showing CD3 positivity, "CD3-positive cell count," was determined using a flow cytometer and defined as CD3-positive cell count (A).

[0272] Furthermore, an RNA introduction test using a Sendai virus vector was performed in the same manner as described above, except that a cell culture substrate without cell scaffold material (a 48-well plate before coating with the coating solution) was used instead of the prepared cell culture substrate. In addition, the number of cells showing CD3 positivity, "CD3-positive cell count," was determined using a flow cytometer and was defined as CD3-positive cell count (B).

[0273] [Criteria for determining introduction efficiency] ○○○: Number of GFP-positive cells (A) is more than 2.5 times the number of CD3-positive cells (B) ○○: Number of GFP-positive cells (A) is 1.5 times or more but less than 2.5 times the number of CD3-positive cells (B) ○: Number of GFP-positive cells (A) is 1.0 times or more but less than 1.5 times the number of CD3-positive cells (B) ×: Number of GFP-positive cells (A) is less than 1.0 times the number of CD3-positive cells (B)

[0274] (3) The solubility of peptide-containing resin in the coating solution was quantified using a dynamic light scattering (DLS) measuring device ("NANOTRAC WAVE I" manufactured by Microtrac-Bell). First, SetZ (SZ) was performed with a solution of ethanol (95 wt%) / acetic acid (5 wt%). The coating solution was filled into the measuring device and processed in the order of SL (Sample Load) and Run. The average value of the results of a total of three measurements was taken as the Loading Index. A smaller Loading Index indicates higher transparency of the coating solution and better solubility of the peptide-containing resin in the coating solution.

[0275] [Criteria for determining solubility] A: Loading Index is 0.1 or less B: Loading Index is greater than 0.1 and 0.5 or less C: Loading Index is greater than 0.5

[0276] The configuration and results are shown in Tables 1 to 17 below.

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294] 1...Cell culture substrate 2...Substrate body 2a...First surface 3...Cell scaffold material 10...Peptide-containing resin (A) 10a...Peptide-containing resin (1) 10b...Peptide-containing resin (2) 11, 11a, 11b...Synthetic resin part 12, 12a, 12b...Linker part 13, 13a...First peptide part 14, 14b...Second peptide part 15...Peptide part

Claims

1. A cell scaffold material for introducing molecules into cells, comprising a peptide-containing resin (A) having a synthetic resin portion and a peptide portion, wherein the peptide portion has a first peptide portion and a second peptide portion, the first peptide portion and the second peptide portion have different amino acid sequences, the first peptide portion is capable of binding to a first integrin, and the second peptide portion is capable of binding to a second integrin.

2. The cell scaffold material according to claim 1, wherein the first integrin is VLA-5 and the second integrin is VLA-4.

3. The cell scaffold material according to claim 1 or 2, wherein the first peptide portion has an RGD sequence and the second peptide portion has an LDV sequence.

4. The cell scaffold material according to any one of claims 1 to 3, wherein the first peptide portion has a c-RGDfK sequence.

5. The cell scaffold material according to any one of claims 1 to 4, wherein the second peptide portion has an EILDV sequence.

6. The cell scaffold material according to any one of claims 1 to 5, wherein in the peptide-containing resin (A), the sum of the content P1 of the first peptide portion and the content P2 of the second peptide portion is 0.05 mol% or more and 3.5 mol% or less.

7. The cell scaffold material according to any one of claims 1 to 6, wherein the peptide-containing resin (A) has a content P1 of the first peptide portion of 0.02 mol% or more and a content P2 of the second peptide portion of 0.03 mol% or more and a content P2 of 1.8 mol% or less.

8. The cell scaffold material according to any one of claims 1 to 7, wherein the synthetic resin portion is a polyvinyl acetal resin portion or a (meth)acrylic polymer portion.

9. The cell scaffold material according to any one of claims 1 to 8, wherein the synthetic resin portion is a polyvinyl acetal resin portion.

10. The cell scaffold material according to any one of claims 1 to 9, wherein the peptide-containing resin (A) has a linker portion, and the synthetic resin portion and the peptide portion are bonded via the linker portion.

11. The cell scaffold material according to claim 10, wherein the linker portion has structural units derived from polymerizable monomers.

12. The cell scaffold material according to claim 11, wherein the polymerizable monomer is a (meth)acrylate compound.

13. The cell scaffold material according to claim 11 or 12, wherein the polymerizable monomer comprises a compound having a carboxyl group.

14. A cell scaffold material for introducing molecules into cells, comprising a peptide-containing resin (1) having a synthetic resin portion and a first peptide portion, and a peptide-containing resin (2) having a synthetic resin portion and a second peptide portion, wherein the first peptide portion and the second peptide portion have different amino acid sequences, the first peptide portion is capable of binding to a first integrin, and the second peptide portion is capable of binding to a second integrin.

15. The cell scaffold material according to claim 14, wherein the first integrin is VLA-5 and the second integrin is VLA-4.

16. The cell scaffold material according to claim 14 or 15, wherein the first peptide portion has an RGD sequence and the second peptide portion has an LDV sequence.

17. The cell scaffold material according to any one of claims 14 to 16, wherein the first peptide portion has a c-RGDfK sequence.

18. The cell scaffold material according to any one of claims 14 to 17, wherein the second peptide portion has an EILDV sequence.

19. The cell scaffold material according to any one of claims 14 to 18, wherein the sum of the content P1 of the first peptide portion in the peptide-containing resin (1) and the content P2 of the second peptide portion in the peptide-containing resin (2) is 0.05 mol% or more and 3.5 mol% or less.

20. The cell scaffold material according to any one of claims 14 to 19, wherein in the peptide-containing resin (1), the content P1 of the first peptide portion is 0.02 mol% or more and 1.7 mol% or less, and in the peptide-containing resin (2), the content P2 of the second peptide portion is 0.03 mol% or more and 1.8 mol% or less.

21. The cell scaffold material according to any one of claims 14 to 20, wherein the synthetic resin portion in the peptide-containing resin (1) is a polyvinyl acetal resin portion or a (meth)acrylic polymer portion, and the synthetic resin portion in the peptide-containing resin (2) is a polyvinyl acetal resin portion or a (meth)acrylic polymer portion.

22. The cell scaffold material according to any one of claims 14 to 21, wherein the synthetic resin portion in the peptide-containing resin (1) is a polyvinyl acetal resin portion, and the synthetic resin portion in the peptide-containing resin (2) is a polyvinyl acetal resin portion.

23. The cell scaffold material according to any one of claims 14 to 22, wherein the peptide-containing resin (1) has a linker portion, and in the peptide-containing resin (1), the synthetic resin portion and the first peptide portion are linked via the linker portion, and the peptide-containing resin (2) has a linker portion, and in the peptide-containing resin (2), the synthetic resin portion and the second peptide portion are linked via the linker portion.

24. The cell scaffold material according to claim 23, wherein the linker portion in the peptide-containing resin (1) has structural units derived from polymerizable monomers, and the linker portion in the peptide-containing resin (2) has structural units derived from polymerizable monomers.

25. The cell scaffold material according to claim 23 or 24, wherein the linker portion in the peptide-containing resin (1) has structural units derived from a (meth)acrylate compound, and the linker portion in the peptide-containing resin (2) has structural units derived from a (meth)acrylate compound.

26. The cell scaffold material according to any one of claims 23 to 25, wherein the linker portion in the peptide-containing resin (1) has structural units derived from a compound having a carboxyl group, and the linker portion in the peptide-containing resin (2) has structural units derived from a compound having a carboxyl group.

27. Dispersion component γ of the surface free energy of the cell scaffold material ｄ However, 24.5 mJ / m ２ 45.0mJ / m or more ２ The dipole component γ of the surface free energy of the cell scaffold material is as follows: ｐ However, 1.0 mJ / m ２ 20.0mJ / m or more ２ The cell scaffold material according to any one of claims 1 to 26, which is as follows:

28. A cell scaffold material for introducing molecules into cells using endocytosis, as described in any one of claims 1 to 27.

29. A cell scaffold material for introducing molecules into cells using a nonviral introduction method, as described in any one of claims 1 to 28.

30. A cell scaffold material for introducing molecules into cells using a virus introduction method, as described in any one of claims 1 to 28.

31. A cell scaffold material according to any one of claims 1 to 30, which is a cell scaffold material for introducing DNA or RNA into cells.

32. A cell culture substrate comprising a substrate body and a cell scaffold material disposed on a first surface of the substrate body, wherein the cell scaffold material is the cell scaffold material according to any one of claims 1 to 31.

33. The cell culture substrate according to claim 32, wherein the substrate body is a container.

34. A method for introducing molecules into cells, comprising: a culture step of culturing cells on the surface of a cell scaffold material according to any one of claims 1 to 31; and a molecule introduction step of introducing molecules into cells adhered to the surface of the cell scaffold material.

35. The method for introducing molecules into cells according to claim 34, wherein in the molecular introduction step, molecules are introduced into cells using endocytosis.

36. The method for introducing a molecule into a cell according to claim 34 or 35, wherein the molecule is DNA or RNA.

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