Holder for cells, method for holding floating cells, and method for recovering floating cells

Abstract

A cell holder has a substrate and a plurality of spots arranged at intervals on the surface of the substrate. Each of the plurality of spots has a cell holding base film containing a polymer and is used to hold floating cells in the spot.

Description

Cell retainer, method for retaining suspended cells, and method for recovering suspended cells

[0001] The present invention relates to a cell retainer, a method for retaining suspended cells, and a method for recovering suspended cells.

[0002] For suspension cells, such as lymphocytes and other hematopoietic cells, which are often cultured in a suspension state, closed-system cell culture devices have been proposed, particularly for regenerative medicine applications, to reduce contamination risk and ensure stable production. In these closed-system cell culture devices, the state of the cells in the culture vessel is observed for process control and other purposes. For example, a microscopic observation system for closed-system cell culture devices has been proposed (Patent Document 1).

[0003] While such closed-system cell culture devices have the advantage of reducing the risk of contamination, they tend to make it difficult to change the culture medium when it becomes necessary due to deterioration of the medium. From this perspective, a cell culture device that facilitates culture medium change in a closed-system cell culture device has been proposed (Patent Document 2).

[0004] Furthermore, genetically modified cell therapies for the treatment of cancers such as leukemia are being manufactured at an industrial level. Generally, closed-system culture equipment is used as the manufacturing equipment, and multiple processes can be automated (Non-Patent Documents 1 and 2). However, the equipment described in Non-Patent Documents 1 and 2 does not have an observation unit, making it difficult to monitor and control quality in real time during culture.

[0005] Furthermore, when using damaged cells with impaired function, there is a method of culturing them on a plate for several days (hereinafter referred to as "recovery culture") to restore their original function, and then transferring them to a flask for large-scale culture. Examples of damaged cells with impaired function are not limited to the following, but include, for example, cells that have just been collected from a patient, cells that have just been thawed after cryopreservation (e.g.,

[0057] in Patent Document 3), and cells that have just had nucleic acids introduced by transfection.

[0006] International Publication No. 2021 / 186648 Pamphlet, Japanese Patent Publication No. 2020-78356, Japanese Patent Publication No. 2013-215141

[0007] Cytotherapy, December 2023, Vol. 25, Issue 12, p.1349-1360Heinz et al, “Automated production of specific T cells for treatment of refractory viral infections after allogeneic stem cell transplantation”, Haematologica, August 2023, Vol. 108, No. 8

[0008] However, the above proposal requires a culture apparatus equipped with special means such as a transport mechanism for transporting a microscope device for observing cells, and a partition wall to divide the internal space of the culture medium tank into an upper and lower space. Furthermore, if the cells to be recovered are suspension cells, when the culture medium needs to be changed due to deterioration of the medium, it becomes necessary to separate the medium and cells by centrifugation or by repeating pipetting operations, which not only makes the work complicated but also has the potential to cause mechanical damage to the cells.

[0009] The present invention aims to provide a cell holder that enables observation of suspended cells and is applicable to closed-system cell culture devices. Furthermore, the present invention provides a cell holder that facilitates the exchange of culture medium for suspended cells and also allows for their retrieval.

[0010] The inventors of this invention have found that the aforementioned problems can be solved and have completed the present invention.

[0011] In other words, the present invention encompasses the following embodiments: [1] A cell holder comprising a substrate and a cell-retaining underlayer disposed on the surface of the substrate, wherein the cell-retaining underlayer contains a polymer, and is used to retain suspended cells on the cell-retaining underlayer. [2] The cell holder according to [1], wherein the surface of the cell-retaining underlayer has a charge opposite to that of the suspended cells. [3] The cell holder according to [1] or [2], wherein the zeta potential of the surface of the cell-retaining underlayer is +25 mV to +80 mV. [4] The cell holder according to [2], wherein the suspended cells can be retained on the cell-retaining underlayer by electrical attraction, and the suspended cells can be detached from the cell-retaining underlayer when a force greater than the attractive force is applied to the suspended cells. [5] The cell holder according to any one of [1] to [4], further comprising suspended cells held on a cell-retaining base membrane, wherein the suspended cells are held without detaching from the cell-retaining base membrane when the shear stress on the suspended cells is 0.8 Pa or less. [6] The cell holder according to [4], further comprising suspended cells held on a cell-retaining base membrane, wherein the suspended cells are detached from the cell-retaining base membrane when the shear stress on the suspended cells is 15 Pa or less. [7] The cell holder according to [6], wherein the suspended cells are held without detaching from the cell-retaining base membrane when the shear stress on the suspended cells is 0.8 Pa or less. [8] The cell holder according to any one of [1] to [7], wherein the polymer comprises at least one of a polymer (A) containing repeating units represented by the following formula (a-1), a polymer (B) containing repeating units represented by the following formula (b-1), and a polymer (C) containing repeating units represented by the following formula (c-1). (In formula (a-1), R ａ１ Z represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms. ａ This represents the following equation (Z1), equation (Z2), or equation (Z3). In equation (b-1), X ｂ This represents a linear alkylene group with 1 to 3 carbon atoms, Y ｂ (This represents a guanidino group or an imidazoyl group.) (In formula (Z1), Zｂ represents the following formula (Ia) or the following formula (Ia-2). In formula (Z2), R ａ２１ represents a linear or branched alkyl group having 1 to 5 carbon atoms. In formula (Z3), R ａ３１ and R ａ３２ each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms. In formulas (Z1) to (Z3), * represents a bond.) (In formula (Ia) and formula (Ia-2), R ａ２ represents a linear or branched alkylene group having 1 to 5 carbon atoms, U ａ１ and U ａ２ each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, U ａ３ represents a hydrogen atom, a linear or branched alkyl group having 1 to 5 carbon atoms, or an aralkyl group, X － represents a salt-forming anion, and * represents a bond.) [9] The cell retainer according to [8], wherein the polymer (A) contains a crosslinked structure.

[10] The cell retainer according to [8] or [9], wherein the polymer (A) contains at least any one of the structure represented by the following formula (IIIa), the structure represented by the following formula (IVa), and the structure represented by the following formula (Va). (In the formula, R ｃ and R ｄ each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, R ｅ(wherein represents a linear or branched alkylene group having 1 to 5 carbon atoms, and n represents a number from 1 to 50.)

[11] A cell holder according to any one of [1] to

[10] , wherein the surface of the substrate has a coating film having the ability to suppress cell adhesion, and the cell-holding base film is disposed on the coating film.

[12] A cell holder according to any one of [1] to

[11] , having a cell-adhering substance disposed on the cell-holding base film.

[13] A method for holding suspended cells, wherein the cell-holding base film of the cell holder according to any one of [1] to

[12] holds suspended cells.

[14] A method for holding suspended cells according to

[13] , wherein the cell holder holding the suspended cells is stirred with a shear stress of 0.8 Pa to 15 Pa to detach the suspended cells from the cell holder.

[15] A method for recovering suspended cells according to

[13] or

[14] from the cell-holding base film.

[0012] According to the present invention, a cell holder that enables observation of suspended cells and is applicable to closed-system cell culture devices can be provided. Furthermore, according to the present invention, a cell holder that facilitates the exchange of culture medium for suspended cells can be provided.

[0013] Figure 1 is a top view of an example of a cell holder before it holds cells. Figure 2 is a top view of another example of a cell holder before it holds cells. Figure 3 is a top view of another example of a cell holder before it holds cells. Figure 4 shows the results of a cell detachment force test on cell temporary adsorption substrates 4 and 5 and an untreated polystyrene substrate. Figure 5A shows the observation results 3 hours after cell seeding in Example 2. Figure 5B shows the observation results after medium exchange in Example 2. Figure 5C shows the observation results after pipetting and retrieval in Example 2. Figure 6A shows the observation results of the undercoat forming agent 1 in Test Example 9 of Example 3. Figure 6B shows the observation results of the undercoat forming agent 2 in Test Example 9 of Example 3. Figure 7A shows the observation results of the undercoat forming agent 1 before pipetting in Test Example 10 of Example 3. Figure 7B shows the observation results of the undercoat forming agent 1 after pipetting in Test Example 10 of Example 3. Figure 8A shows the observation results of the undercoat-forming agent 1 in Test Example 11 of Example 3 before cooling and shaking. Figure 8B shows the observation results of the undercoat-forming agent 1 in Test Example 11 of Example 3 before cooling and shaking. Figure 9A shows the observation results of Example 4 before changing the culture medium. Figure 9B shows the observation results of Example 4 after changing the culture medium. Figure 10A shows the observation results of Example 5 before changing the culture medium. Figure 10B shows the observation results of Example 5 after changing the culture medium. Figure 11 shows the observation results of Example 6 after changing the culture medium. Figure 12A shows the observation results of Production Example 6 in Test Example 16 of Example 8 21 hours after cell seeding. Figure 12B shows the observation results of Production Example 15 in Test Example 16 of Example 8 21 hours after cell seeding. Figure 12C shows the observation results of Production Example 6 in Test Example 16 of Example 8 after changing the culture medium. Figure 12D shows the observation results of Production Example 15 in Test Example 16 of Example 8 after changing the culture medium. Figure 12E shows the observation results after pipetting and collection of Preparation Example 6 in Test Example 17 of Example 8. Figure 12F shows the observation results after pipetting and collection of Preparation Example 15 in Test Example 17 of Example 8. Figure 13 shows the calculation results of the cell proliferation rate in the expanded culture (Day 3 to Day 10) of Test Example 21 of Example 11. Figure 14 shows the detection results of CD4 and CD8 positive cells by flow cytometry in Test Example 22 of Example 11.

[0014] (Cell Retainer) The cell retainer of the present invention comprises a substrate and a cell retaining underlayment disposed on the surface of the substrate. The cell retaining underlayment contains a polymer, and the cell retainer is used to retain suspended cells on the cell retaining underlayment. Here, "surface of the substrate" refers to the surface that comes into contact with a cell culture medium containing suspended cells.

[0015] Furthermore, in one embodiment, the cell retainer has a plurality of spots, which are spaced apart on the surface of the substrate. In one embodiment, the spots have a cell retaining underlayer. In another embodiment, the spots have a cell retaining underlayer and a cell adhesion substance placed on the cell retaining underlayer.

[0016] Furthermore, in one embodiment, the cell retainer has floating cells held on a cell retaining substrate. Furthermore, in another embodiment, the cell retainer has floating cells held on a cell adhesion substance arranged on a cell retaining substrate.

[0017] For example, as shown in Figure 1, the cell holder 100 has a cell-holding underlayer 3. In the cell holder 100, the shape of the cell-holding underlayer 3 disposed on the surface of the substrate 1 is not particularly limited. For example, the cell-holding underlayer 3 may cover the entire surface of the substrate 1, or it may cover only a part of the surface of the substrate 1. Also, as shown in Figure 2, the cell holder 100 may have a cell-adhesion substance 2 on top of the cell-holding underlayer 3.

[0018] The inventors of the present invention have found that the cell retainer of the present invention makes it possible to retain suspended cells. Thereupon, the inventors conducted further investigations and found that the suspended cells can be recovered by stirring the cell retainer containing the suspended cells with a specific strength of shear stress, or by cooling and shaking it.

[0019] <Suspended Cells> A cell is the most basic unit that makes up an animal or plant, and its elements include cytoplasm and various organelles inside a cell membrane. In this case, the nucleus, which contains DNA, may or may not be included inside the cell. For example, the animal-derived cells in this invention include germ cells such as sperm and eggs, somatic cells that make up living organisms, stem cells (pluripotent stem cells, etc.), progenitor cells, cancer cells isolated from living organisms, cells isolated from living organisms that acquire immortalization ability and are stably maintained outside the body (cell lines), cells isolated from living organisms that have been genetically modified, and cells isolated from living organisms that have had their nuclei replaced artificially. Examples of somatic cells that make up living organisms include, but are not limited to, fibroblasts, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, erythrocytes, platelets, macrophages, monocytes, osteocytes, pericytes, dendritic cells, keratinocytes, adipocytes, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, hepatocytes, chondrocytes, cumulus cells, nervous system cells, glial cells, neurons, oligodendrocytes, microglia, astrocytes, cardiac cells, esophageal cells, muscle cells (e.g., smooth muscle cells or skeletal muscle cells), pancreatic beta cells, melanocytes, hematopoietic progenitor cells (e.g., CD34-positive cells derived from umbilical cord blood), and mononuclear cells. These somatic cells include cells taken from any tissue, such as skin, kidneys, spleen, adrenal glands, liver, lungs, ovaries, pancreas, uterus, stomach, colon, small intestine, large intestine, bladder, prostate, testes, thymus, muscle, connective tissue, bone, cartilage, vascular tissue, blood (including umbilical cord blood), bone marrow, heart, myocardium, eyes, brain, nerve tissue, and hair. Furthermore, these somatic cells include cells differentiated from stem cells or progenitor cells.

[0020] Stem cells are cells that possess both the ability to replicate themselves and the ability to differentiate into multiple other cell lineages. Examples include, but are not limited to, embryonic stem cells (ES cells), embryonic tumor cells, embryonic germ cells, induced pluripotent stem cells (iPS cells), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, liver stem cells, pancreatic stem cells, muscle stem cells, germ stem cells, intestinal stem cells, cancer stem cells, and hair follicle stem cells. Pluripotent stem cells include ES cells, embryonic germ cells, and iPS cells among the aforementioned stem cells. Progenitor cells are cells that are in the intermediate stage of differentiating from the aforementioned stem cells into specific somatic cells or germ cells. Cancer cells are cells that have derived from somatic cells and acquired unlimited proliferative capacity. Cell lines are cells that have acquired unlimited proliferative capacity through artificial manipulation outside the body.

[0021] Among these, suspension cells refer to cultured cells that proliferate while suspended in the culture medium. Suspension cells are non-adhesion-dependent cells and do not require tissue culture treatment of the culture vessel during cultivation. Examples of suspension cells, though not limited to those listed below, include hematological cells such as red blood cells, platelets, and white blood cells. Furthermore, examples of white blood cells, though not limited to those listed below, include B lymphocytes, T lymphocytes, natural killer cells, neutrophils, eosinophils, basophils, and monocytes.

[0022] In the case of suspended cells in a cell holder, multiple cells may be aggregated, but in that case, the aggregate of multiple cells may be in a two-dimensional structural state (sheet-like) in which the cells are adhered to each other.

[0023] <Spot> A spot has a cell-retaining underlayer. A spot is the location in a cell retainer where suspended cells are held.

[0024] There are no particular limitations on the number of spots that are spaced apart on the surface of the substrate. For example, the number of spots could be 1 cm. ２The number of spots per area is preferably 50 to 5,000, and more preferably 100 to 2,000. The distance between adjacent spots is not particularly limited, but is preferably 100 μm to 6,000 μm, more preferably 150 μm to 4,000 μm, and particularly preferably 150 μm to 3,000 μm. Here, distance refers to the length between the centers of two spots. The size of the spot surface is not particularly limited, for example, the equivalent circle diameter is preferably 25 μm to 5,000 μm, and more preferably 50 μm to 3,000 μm. The equivalent circle diameter is the diameter of a perfect circle that corresponds to the area of ​​the surface in question. If the spot surface is a perfect circle, the equivalent circle diameter refers to the diameter of that perfect circle; if the spot surface is not a perfect circle, the equivalent circle diameter refers to the diameter of a perfect circle that has the same area as the surface.

[0025] The area ratio occupied by the spots on the substrate is not particularly limited, but it is preferably 30% or more, 40% or more, 50% or more, and preferably 99% or less.

[0026] The spots preferably have a cell adhesion substance. In this case, the cell adhesion substance is placed on the cell-retaining base film. In this case, the area occupied by the cell-retaining base film is the spot. Therefore, the surface of the cell-retaining base film corresponds to the spot. The size of the surface of the cell adhesion substance placed on the cell-retaining base film is not particularly limited as long as it is less than the size of the surface of the cell-retaining base film, but it is preferably 1 / 80 to 1 / 3 of the area of ​​the cell-retaining base film, and more preferably 1 / 50 to 1 / 4.

[0027] <<Cell Adhesion Substances>> Cell adhesion substances exist in one location per spot, for example.

[0028] Cell adhesion substances promote cell adhesion, extension, proliferation, and differentiation. Known substances such as extracellular matrix (ECM) proteins, glycoproteins, peptides, and synthetic compounds (low and high molecular weight) can be used as cell adhesion substances; however, compounds that are not of biological origin, such as synthetic compounds (low and high molecular weight), are preferred. Low molecular weight refers to compounds with a weight-average molecular weight of 2,000 or less, while high molecular weight refers to compounds with a weight-average molecular weight of 2,000 or more, with an upper limit of, for example, 1,000,000.

[0029] Examples of extracellular matrix (ECM) proteins include collagen (e.g., Merck's type I collagen (catalog numbers C9791, C7661, C1809, C2249, C2124), type II collagen (catalog number C9301), type IV collagen (catalog numbers C0543, C5533), elastin (e.g., Merck catalog numbers: E1625, E6527), fibronectin (e.g., Merck catalog numbers F1141, F0635, F2518, F0895, F4759, F2006), laminin (e.g., Merck catalog numbers: L6724, L2020, L4544), laminin fragments (e.g., Matrixome: 892011), and vitronectin (e.g., VTN-N (Gibco), Vitronectin, Human, Recombinant, Examples include Animal Free (PeproTech), Merck part numbers: V0132, V9881, V8379, 08-126, SRP3186).

[0030] The cell adhesion substance is preferably a glycoprotein. Specifically, it is preferably selected from vitronectin, integrin, cadherin, fibronectin, laminin, tenascin, osteopontin, and bone sialoprotein. Furthermore, it is preferably a protein having an RGD sequence as its amino acid sequence.

[0031] Examples of peptides include ECM peptide (MAPTrix®, a registered trademark of Kollodis Bio Sciences) and RGD peptide (manufactured by Fujifilm Wako Pure Chemical Industries: 180-01531).

[0032] Examples of synthetic compounds (high molecular weight) include polylysine (e.g., Merck products: P4707, P4832, P7280, P9155, P6407, P6282, P7405, P5899) and polyornithine (e.g., Merck product number P4975). Examples of synthetic compounds (low molecular weight) include adhesamine (e.g., manufactured by Nagase & Co., Ltd.: AD-00000-0201) and synthetic cyclic RGD peptide (e.g., manufactured by IRIS BIOTECH: LS-3920.0010).

[0033] <<Cell-retaining underlayer film>> The cell-retaining underlayer film contains a polymer. The polymer is a substance different from the cell adhesion substance and is typically a synthetic polymer. The cell-retaining underlayer film may further contain a cell adhesion substance.

[0034] The thickness of the cell-retaining underlayer is not particularly limited, but is, for example, in the range of 1 to 1000 nm, and preferably in the range of 5 to 500 nm.

[0035] It is preferable that the surface of the cell-retaining substrate has an opposite charge to that of the floating cells. By having an opposite charge to the floating cells, the floating cells can be held to the substrate by electrical attraction. Furthermore, applying a force greater than the attractive force to the floating cells can cause them to detach from the substrate. Here, the charge can be measured by methods such as the zeta potential measurement method described later. Having an opposite charge means, for example, that if the floating cells have a negative charge, the surface of the cell-retaining substrate has a positive charge.

[0036] The zeta potential of the surface of the cell-retaining substrate is preferably between +25 mV and +80 mV. When the zeta potential of the surface of the cell-retaining substrate is within this range, the adsorption rate of suspended cells tends to be high. From the viewpoint of making it easier to adsorb suspended cells, the zeta potential of the surface of the cell-retaining substrate is even more preferably +30 mV or higher, and particularly preferably +35 mV or higher. Furthermore, from the viewpoint of detaching adsorbed cells and improving recovery, the zeta potential of the surface of the cell-retaining substrate is even more preferably +70 mV or lower.

[0037] The zeta potential of the cell-retaining substrate surface can be measured as the average zeta potential at room temperature using the flow potential method. The zeta potential can also be measured using a general zeta potential measuring device capable of flow potential measurement. In the above zeta potential measurement, if the cell-retaining substrate exhibits cationic properties, the measurement was performed under conditions of immersion in cationic standard monitor particles. If the cell-retaining substrate exhibits neutral or anionic properties, the measurement was performed under conditions of immersion in anionic standard monitor particles. Whether the cell-retaining substrate exhibits cationic or anionic properties was determined by literature values ​​or simulations of the acid dissociation constant pKa under pH 7 conditions.

[0038] Furthermore, the cell holder further comprises suspended cells held on a cell-holding base membrane, and it is preferable that the suspended cells detach from the cell-holding base membrane when the shear stress on the suspended cells is 15 Pa or less. It is also preferable that the suspended cells remain held without detaching from the cell-holding base membrane when the shear stress on the suspended cells is 0.8 Pa or less. Suspended cells held in the cell holder of the present invention are less likely to detach with weak forces of 0.8 Pa or less shear stress, and are more likely to detach from the cell holder when a shear stress above a certain level (greater than 0.8 Pa) is applied. Here, the shear stress for detaching the suspended cells is preferably 15 Pa or less, more preferably 13 Pa or less, and even more preferably 10 Pa or less, from the viewpoint of suppressing damage to cells and foaming of the culture medium, and stable operation of the device, etc. (See Dynamics and Design Conference 2018 Proceedings 532). Shear stress is the maximum stress applied to suspended cells when they are detached. It can be calculated using spreadsheet software such as Excel or fluid analysis software by inputting appropriate values ​​for variables such as culture medium density, culture medium viscosity, culture medium kinematic viscosity coefficient, culture vessel shape (aspect ratio), and fluid flow velocity caused by shaking of the vessel or liquid flow.

[0039] The polymer is not particularly limited as long as it is a polymer that can hold floating cells, but from the viewpoint of suitably obtaining the effects of the present invention, a polymer containing at least one of the following is preferred: polymer (A) containing a repeating unit represented by the following formula (a-1), polymer (B) containing a repeating unit represented by the following formula (b-1), and polymer (C) containing a repeating unit represented by the following formula (c-1). Note that the following are not limited to representative examples of polymers. (In formula (a-1), R ａ１ Z represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms. ａ This represents the following equation (Z1), equation (Z2), or equation (Z3). In equation (b-1), X ｂ This represents a linear alkylene group with 1 to 3 carbon atoms, Y ｂ (This represents a guanidino group or an imidazoyl group.)

[0040] <<<Polymer (A)>>> Polymer (A) contains repeating units represented by the following formula (a-1). (In formula (a-1), R ａ１ Z represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms. ａ This represents the following formula (Z1), formula (Z2), or formula (Z3). (In formula (Z1), Z ｂ This represents the following equation (Ia) or equation (Ia-2). In equation (Z2), R ａ２１ R represents a linear or branched alkyl group having 1 to 5 carbon atoms. In formula (Z3), R ａ３１ and R ａ３２ Each of these independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms. In formulas (Z1) to (Z3), * represents a bond. (In equations (Ia) and (Ia-2), R ａ２ This represents a linear or branched alkylene group with 1 to 5 carbon atoms, U ａ１ and U ａ２ Each of these independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, U ａ３X represents a hydrogen atom, a linear or branched alkyl group having 1 to 5 carbon atoms, or an aralkyl group. － (where * represents a salt-forming anion, and * represents a bonding bond.)

[0041] The polymer (A) preferably further contains repeating units represented by the following formula (IIa). (In formula (IIb), R ｂ (This represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms.)

[0042] R ａ１ and R ｂ Preferably, each of these is independently selected from a hydrogen atom and a methyl group.

[0043] In this specification, unless otherwise defined, "linear or branched alkyl groups having 1 to 5 carbon atoms" include, for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, s-butyl group, t-butyl group, n-pentyl group, 1-methylbutyl group, 2-methylbutyl group, 3-methylbutyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, or 1-ethylpropyl group. Examples of aralkyl groups include benzyl group and phenethyl group.

[0044] R ａ２１ The group is preferably selected from isopropyl, isobutyl, and t-butyl groups, but isopropyl is the most preferred. ａ３１ and R ａ３２ Each of these is preferably independently selected from a hydrogen atom, a methyl group, and an ethyl group, but a hydrogen atom is the most preferred.

[0045] R ａ２ The group is preferably selected from a methyl group, an ethyl group, and an n-propyl group, but the ethyl group is the most preferred.

[0046] U ａ１ and U ａ２Each of these is preferably independently selected from a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, and an n-butyl group, but is preferably a methyl group or an ethyl group, with the methyl group being the most preferred. ａ３ The group is preferably selected from a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a benzyl group, and a phenethyl group, but is preferably a benzyl group or a phenethyl group, with the benzyl group being the most preferred.

[0047] In this specification, unless otherwise defined, "linear or branched alkylene groups having 1 to 5 carbon atoms" include, for example, methylene group, ethylene group, propylene group, trimethylene group, tetramethylene group, 1-methylpropylene group, 2-methylpropylene group, dimethylethylene group, ethylethylene group, pentamethylene group, 1-methyltetramethylene group, 2-methyltetramethylene group, 1,1-dimethyl-trimethylene group, 1,2-dimethyl-trimethylene group, 2,2-dimethyl-trimethylene group, 1-ethyl-trimethylene group, etc. Among these, R ａ２ Preferably, the group is selected from ethylene groups and propylene groups.

[0048] X － Examples include halogen ions such as chloride ions, bromide ions, and iodide ions, and 1 / 2 SO ４ ２－ NO ３ － ,CH ３ COO － ,CH ３ SO ４ － , C ２ H ５ SO ４ － These are some examples.

[0049] Examples of monomers that give the repeating unit of formula (a-1) above include 2-N,N-dimethylaminoethyl methacrylate, N-isopropylacrylamide (NIPAAm), methacryloyloxyethyltrimethylammonium chloride, methacryloyloxyethyldimethylbenzylammonium chloride, and 4-aminostyrene.

[0050] Examples of monomers (cationic monomers) that impart repeating units represented by formula (Ia) to polymer (A) include 2-N,N-dimethylaminoethyl methacrylate and N,N-dimethylaminomethyl methacrylate, with 2-N,N-dimethylaminoethyl methacrylate (2-(dimethylamino)ethyl methacrylate) being preferred. Examples of monomers (cationic monomers) that impart repeating units represented by formula (Ia-2) to polymer (A) include methacryloyloxyethyltrimethylammonium chloride and methacryloyloxyethyldimethylbenzylammonium chloride.

[0051] Examples of monomers (anionic monomers) that provide the polymer (A) with repeating units represented by formula (IIa) include acrylic acid and methacrylic acid, with methacrylic acid being preferred.

[0052] The molar ratio [(Ia) / (IIa)] of repeating units (Ia) represented by formula (Ia) to repeating units (IIa) represented by formula (IIa) in polymer (A) is, for example, 100 / 0 to 50 / 50. Preferably it is 98 / 2 to 50 / 50, more preferably 98 / 2 to 60 / 40, and particularly preferably 98 / 2 to 70 / 30. When the molar ratio [(Ia) / (IIa)] is 1 or more, the decrease in cell adhesion due to the anionic properties of the polymer can be suppressed. The preferred range for the molar ratio of repeating units (Ia-2) represented by formula (Ia-2) to repeating units (IIa) represented by formula (IIa) is the same as above.

[0053] Polymer (A) may have other repeating units. In polymer (A), the total of the repeating units represented by formula (Ia) and the repeating units represented by formula (IIa) relative to the total repeating units is, for example, 50 mol% or more, preferably 75 mol% or more, more preferably 80 mol% or more, and even more preferably 90 mol% or more. In polymer (A), the preferred range of mol% for the total of the repeating units represented by formula (Ia-2) and the repeating units represented by formula (IIa) relative to the total repeating units is the same as above.

[0054] From the viewpoint of suitably obtaining the effects of the present invention, polymer (A) preferably further includes a crosslinked structure. The crosslinked structure is obtained by using a monomer having two or more carbon-carbon unsaturated bonds when synthesizing polymer (A). Specifically, a monomer having two or more carbon-carbon unsaturated bonds is a monomer having two or more carbon-carbon double bonds, and examples include polyfunctional acrylate compounds, polyfunctional acrylamide compounds, polyfunctional polyesters, or isoprene compounds.

[0055] Polymer (A) includes a structure derived from, for example, at least one of a polyfunctional acrylate compound, a polyfunctional acrylamide compound, a polyfunctional polyester, and an isoprene compound. Polymer (A) preferably includes at least one of the following structures: the structure represented by formula (IIIa), the structure represented by formula (IVa), and the structure represented by formula (Va). Among these, the structure represented by formula (IIIa) is preferred. (In the formula, R ｃ and R ｄ Each of these independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, and R ｅ (where n represents a linear or branched alkylene group with 1 to 5 carbon atoms, and n represents a number from 1 to 50.)

[0056] R ｃ and R ｄ It is preferable that each of these be independently selected from a hydrogen atom and a methyl group. ｅThe group is preferably selected from a methylene group, an ethylene group, and a propylene group, with an ethylene group being more preferred. n is a number from 1 to 50, but is preferably a number from 1 to 30, and more preferably a number from 1 to 10.

[0057] The molar ratio of the structures represented by formulas (IIIa) to (Va) to the total polymer (A) is preferably 0 mol% to 50 mol%, and more preferably 2 mol% to 25 mol%. When the molar ratio of the structures represented by formulas (IIIa) to (Va) is 50% or less of the total polymer (A), gelation of the solid content during manufacturing due to excessive crosslinking and resulting high molecular weight can be suppressed, and manufacturing can be facilitated.

[0058] The method for synthesizing polymer (A) is not particularly limited and includes known radical polymerization methods. Alternatively, the method described in International Publication No. 2020 / 040247 may be used.

[0059] <<<Polymer (B)>>> Polymer (B) contains repeating units represented by the following formula (b-1). (In formula (b-1), X ｂ This represents a linear alkylene group with 1 to 3 carbon atoms, Y ｂ (This represents a guanidino group or an imidazoyl group.)

[0060] Examples of "linear alkylene groups with 1 to 3 carbon atoms" include the methyl group, ethyl group, and n-propyl group. The guanidino group is H ２ It is a monovalent group represented as N-(C=NH)-NH-. The imidazoline group is a heterocyclic amine containing nitrogen atoms at the 1st and 3rd positions of a five-membered ring.

[0061] Examples of repeating units in formula (b-1) include basic amino acids such as arginine and histidine. Examples of polymers (B) containing repeating units of formula (b-1) include polyamino acids containing basic amino acids.

[0062] <<<Polymer (C)>>> Polymer (C) contains repeating units represented by the following formula (c-1).

[0063] Polyethyleneimine is an example of a polymer containing the repeating unit of formula (c-1). Polymer C only needs to contain the repeating unit represented by formula (c-1) above, and may also contain other repeating unit structures. Polymer (C) may contain, for example, a three-dimensional structure represented by the following formula (C-2).

[0064] Polymers (A), (B), and (C) may be copolymerized with or have added components other than the repeating units described above. Other components include, for example, various components for adding functions such as solubility in solvents, coatability, adhesion to substrates, sterilization resistance, and cell affinity.

[0065] The number-average molecular weight (Mn) of the polymer is, for example, 20,000 to 1,000,000, preferably 50,000 to 800,000. The number-average molecular weight (Mw) of the polymer is, for example, 50,000 to 2,000,000, preferably 10,000 to 1,200,000. The ratio of the weight-average molecular weight (Mw) to the number-average molecular weight (Mn) of the polymer (Mw / Mn) is, for example, 1.01 to 10.00, preferably 1.2 to 8.0, more preferably 1.4 to 6.0, even more preferably 1.5 to 5.0, and particularly preferably 1.6 to 4.5. The number-average molecular weight (Mn) and weight-average molecular weight (Mw) can be determined, for example, by gel filtration chromatography.

[0066] <Substrate> There are no particular restrictions on the material, shape, size, or structure of the substrate. The surface shape of the substrate may be flat or uneven, but a flat shape is preferred.

[0067] Examples of substrate materials include glass, metal, metal-containing compounds or metalloid-containing compounds, activated carbon, or resin. Examples of metals include typical metals (aluminum group elements: Al, Ga, In; iron group elements: Fe, Co, Ni; chromium group elements: Cr, Mo, W, U; manganese group elements: Mn, Re; noble metals: Cu, Ag, Au, etc.). Examples of metal-containing compounds or metalloid-containing compounds include ceramics, which are sintered bodies whose basic component is a metal oxide and are hardened by heat treatment at high temperatures; semiconductors such as silicon; inorganic solid materials such as molded bodies of inorganic compounds such as metal oxides or metalloid oxides (silicon oxide, alumina, etc.), metal carbides or metalloid carbides, metal nitrides or metalloid nitrides (silicon nitride, etc.), metal borides or metalloid borides, etc.; aluminum, nickel titanium, stainless steel (SUS304, SUS316, SUS316L, etc.).

[0068] The resin may be a natural resin or its derivative, or a synthetic resin. Examples of natural resins or their derivatives include cellulose, cellulose triacetate (CTA), nitrocellulose (NC), and cellulose immobilized with dextran sulfate. Examples of synthetic resins include polyacrylonitrile (PAN), polyimide (PI), polyester polymer alloy (PEPA), polystyrene (PS), polysulfone (PSF), polyethylene terephthalate (PET), polymethyl methacrylate (PMMA), polyvinyl alcohol (PVA), and vinyl acetate-modified polyethylene. (EVA), polyurethane (PU), ethylene vinyl alcohol copolymer (EVOH), polyethylene (PE), polyester, polypropylene (PP), polyvinylidene fluoride (PVDF), polyethersulfone (PES), polycarbonate (PC), cycloolefin polymer (COP), polyvinyl chloride (PVC), polytetrafluoroethylene (PTFE), ultra-high molecular weight polyethylene (UHPE), polydimethylsiloxane (PDMS), acrylonitrile-butadiene-styrene resin (ABS), or Teflon (registered trademark) are preferably used.

[0069] The substrate may be in the form of a plate or a film. There are no particular restrictions on the thickness of the substrate. For example, the substrate may be a film with a thickness of 1 mm or less.

[0070] Furthermore, the substrate may be a substrate used in so-called cell culture devices. Examples include petri dishes or dishes such as petri dishes, tissue culture dishes, and multi-dishes commonly used for cell culture, flasks such as cell culture flasks, spinner flasks, and multi-stage flasks, bags such as plastic bags, Teflon® bags, and culture bags, plates such as microplates, microwell plates, multi-plates, and multi-well plates, chamber slides, tubes, trays, and bottles such as roller bottles.

[0071] <<Coating Film>> The surface of the substrate may have a coating film that inhibits cell adhesion. In this case, the spots are arranged on the coating film. The coating film can be obtained, for example, by applying a coating agent containing a polymer that inhibits cell adhesion to the substrate. The shape of the coating film on the surface of the substrate is not particularly limited as long as a cell-retaining underlayer film is placed on it, and one film may uniformly cover the entire surface of the substrate, one film may be formed within the plane of the surface of the substrate, or multiple films may be scattered on the surface of the substrate. For example, the cell retainer 100 may have a coating film 1A on the surface of the substrate 1, as shown in Figure 3.

[0072] Here, having cell adhesion inhibitory ability means that the relative absorbance (WST O.D. 450 nm) (%) ((absorbance of the example (WST O.D. 450 nm)) / (absorbance of the comparative example (WST O.D. 450 nm))) when measured with a fluorescence microscope using the method described in the examples of International Publication No. 2016 / 093293, for example, is 50% or less, preferably 30% or less, and more preferably 20% or less.

[0073] The coating film having the ability to inhibit cell adhesion is not particularly limited, but examples include the following coating films.

[0074] <<<Coating film of the first embodiment>>> The coating film of the first embodiment comprises a copolymer. The copolymer of the first embodiment comprises repeating units comprising an organic group represented by the following formula (a) and repeating units comprising an organic group represented by the following formula (b). The copolymer is, for example, the copolymer described in brochure WO2014 / 196652. The contents of brochure WO2014 / 196652 are incorporated herein to the same extent as if they were fully expressed.

[0075] (In the formula, U ａ１ , U ａ２ , U ｂ１ , U ｂ２ and U ｂ３ Each of these independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, and An － (This represents an anion selected from the group consisting of halide ions, inorganic acid ions, hydroxide ions, and isothiocyanate ions.)

[0076] The copolymer is not particularly limited as long as it is a copolymer containing repeating units containing an organic group represented by formula (a) and repeating units containing an organic group represented by formula (b). Preferably, the copolymer is obtained by radical polymerization of a monomer containing an organic group represented by formula (a) and a monomer containing an organic group represented by formula (b), but copolymers obtained by polycondensation or polyaddition reactions can also be used. Examples of copolymers include vinyl polymers obtained by the reaction of olefins, polyamides, polyesters, polycarbonates, polyurethanes, etc., but among these, vinyl polymers obtained by the reaction of olefins or (meth)acrylic polymers obtained by polymerizing (meth)acrylate compounds are particularly preferred. In this invention, (meth)acrylate compounds mean both acrylate compounds and methacrylate compounds. For example, (meth)acrylic acid means acrylic acid and methacrylic acid.

[0077] Preferably, the monomers containing the organic groups represented by the above formulas (a) and (b) are monomers represented by the following formulas (A) and (B), respectively. (In the formula, T ａ , T ｂ , U ａ１ , U ａ２ , U ｂ１ , U ｂ２ and U ｂ３ each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, Q ａ and Q ｂ each independently represent a single bond, an ester bond or an amide bond, R ａ and R ｂ each independently represent a linear or branched alkylene group having 1 to 10 carbon atoms which may be substituted with a halogen atom, An － represents an anion selected from the group consisting of halide ions, inorganic acid ions, hydroxide ions and isothiocyanate ions, and m represents an integer from 0 to 6.)

[0078] Therefore, the repeating units derived from the monomers represented by the formulas (A) and (B) are represented by the following formulas (a1) and (b1), respectively. (In the formula, T ａ , T ｂ , U ａ１ , U ａ２ , U ｂ１ , U ｂ２ and U ｂ３ , Q ａ and Q ｂ , R ａ and R ｂ , An － and m have the same meanings as above.)

[0079] In the present invention, "ester bond" means -C(=O)-O- or -O-C(=O)-, and "amide bond" means -NHC(=O)- or -C(=O)NH-.

[0080] In the present invention, the "linear or branched alkylene group having 1 to 10 carbon atoms which may be substituted with a halogen atom" means a linear or branched alkylene group having 1 to 10 carbon atoms, or a linear or branched alkylene group having 1 to 10 carbon atoms substituted with one or more halogen atoms. Here, the "linear or branched alkylene group having 1 to 10 carbon atoms" is a divalent organic group obtained by further removing one hydrogen atom from the above alkyl group. For example, methylene group, ethylene group, propylene group, trimethylene group, tetramethylene group, 1-methylpropylene group, 2-methylpropylene group, dimethylethylene group, ethylethylene group, pentamethylene group, 1-methyl-tetramethylene group, 2-methyl-tetramethylene group, 1,1-dimethyl-trimethylene group, 1,2-dimethyl-trimethylene group, 2,2-dimethyl-trimethylene group, 1-ethyl-trimethylene group, hexamethylene group, octamethylene group, decamethylene group and the like can be mentioned. Among these, an ethylene group, a propylene group, an octamethylene group and a decamethylene group are preferable, a linear or branched alkylene group having 1 to 5 carbon atoms, for example, an ethylene group, a propylene group, a trimethylene group, a tetramethylene group is more preferable, and particularly an ethylene group or a propylene group is preferable. The "linear or branched alkylene group having 1 to 10 carbon atoms substituted with one or more halogen atoms" means that one or more arbitrary hydrogen atoms of such an alkylene group are replaced with halogen atoms. Particularly, those in which a part or all of the hydrogen atoms of an ethylene group or a propylene group are replaced with halogen atoms are preferable.

[0081] In the present invention, the "halogen atom" includes a fluorine atom, a chlorine atom, a bromine atom or an iodine atom. In the present invention, the "halide ion" means an anion of a halogen atom, and includes a fluoride ion, a chloride ion, a bromide ion, an iodide ion, and preferably a chloride ion. In the present invention, the "inorganic acid ion" means a carbonate ion, a sulfate ion, a phosphate ion, a hydrogen phosphate ion, a dihydrogen phosphate ion, a nitrate ion, a perchlorate ion or a borate ion. The above An －Preferred ions include halide ions, sulfate ions, phosphate ions, hydroxide ions, and isothiocyanate ions, with halide ions being particularly preferred.

[0082] In equations (A) and (B), T ａ and T ｂ Preferably, each is independently a hydrogen atom, a methyl group, or an ethyl group, and more preferably, each is independently a hydrogen atom or a methyl group.

[0083] In equations (a), (b), (A), and (B), U ａ１ , U ａ２ , U ｂ１ , U ｂ２ and U ｂ３ Preferably, each is independently a hydrogen atom, a methyl group, or an ethyl group. In formulas (a) and (A), U ａ１ and U ａ２ is more preferably a hydrogen atom. In formulas (b) and (B), U ｂ１ , U ｂ２ (and U ｂ３ ) is more preferably a methyl group or an ethyl group, and particularly preferably a methyl group.

[0084] In equations (A) and (B), Q ａ and Q ｂ Preferably, each is independently an ester bond (-C(=O)-O- or -O-C(=O)-) or an amide bond (-NHC(=O)- or -C(=O)NH-), more preferably independently -C(=O)-O- or -C(=O)NH-, and particularly preferably -C(=O)-O-.

[0085] In equations (A) and (B), R ａ and R ｂ Preferably, each is independently a linear or branched alkylene group having 1 to 3 carbon atoms, which may be substituted with a halogen atom; more preferably, each is independently an ethylene group or a propylene group, or an ethylene group or a propylene group substituted with one chlorine atom; particularly preferably, an ethylene group or a propylene group.

[0086] In formulas (A) and (B), m preferably represents an integer from 0 to 3, more preferably an integer of 1 or 2, and particularly preferably 1.

[0087] Specific examples of the above formula (A) include vinylphosphonic acid, acid phosphooxyethyl (meth)acrylate, 3-chloro-2-acid phosphooxypropyl (meth)acrylate, acid phosphooxypropyl (meth)acrylate, acid phosphooxymethyl (meth)acrylate, acid phosphooxypolyoxyethylene glycol mono(meth)acrylate, acid phosphooxypolyoxypropylene glycol mono(meth)acrylate, etc., but among these, vinylphosphonic acid and acid phosphooxyethyl methacrylate (= 2-(methacryloyloxy)ethyl phosphoric acid) are preferred.

[0088] The structural formulas of vinylphosphonic acid, acid phosphooxyethyl methacrylate (= 2-(methacryloyloxy)ethyl phosphate), and acid phosphooxypolyoxyethylene glycol monomethacrylate are represented by the following formulas (A-1) to (A-3), respectively.

[0089] During synthesis, in addition to these compounds, (meth)acrylate compounds having two functional groups, such as those represented by general formula (C) or (D) described later, may also be used in combination.

[0090] Specific examples of the above formula (B) include dimethylaminoethyl (meth)acrylate, diethylaminoethyl (meth)acrylate, dimethylaminopropyl (meth)acrylate, 2-(t-butylamino)ethyl (meth)acrylate, and methacloylcholinchloride. Among these, dimethylaminoethyl (meth)acrylate, methacloylcholinchloride, or 2-(t-butylamino)ethyl (meth)acrylate are preferred.

[0091] The structural formulas of dimethylaminoethyl acrylate (= 2-(dimethylamino)ethyl acrylate), dimethylaminoethyl methacrylate (= 2-(dimethylamino)ethyl methacrylate), methacloylcholinchloride, and 2-(t-butylamino)ethyl methacrylate (= 2-(t-butylamino)ethyl methacrylate) are represented by the following formulas (B-1) to (B-4).

[0092]

[0093] The proportion of repeating units containing the organic group represented by formula (a) (or the repeating unit represented by formula (a1)) in the copolymer is 20 mol% to 80 mol%, preferably 30 mol% to 70 mol%, and more preferably 40 mol% to 60 mol%. The copolymer may also contain two or more repeating units containing the organic group represented by formula (a) (or the repeating unit represented by formula (a1)).

[0094] The proportion of repeating units containing the organic group represented by formula (b) (or the repeating unit represented by formula (b1)) in the copolymer may be the entire remainder after subtracting the proportion of formula (a) (or formula (a1)) from the total copolymer, or it may be the remainder after subtracting the total proportion of formula (a) (or formula (a1)) and the third component described below. The copolymer may also contain two or more repeating units containing the organic group represented by formula (b) (or the repeating unit represented by formula (b1)).

[0095] Furthermore, the copolymer may also contain copolymerized third components. For example, the third component may be a copolymerized (meth)acrylate compound having two or more functional groups, and a portion of the polymer may be partially three-dimensionally crosslinked. Examples of such third components include bifunctional monomers represented by the following formulas (C) or (D).

[0096] (In the formula, T ｃ , T ｄ and U ｄ Each of these independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, R ｃ and R ｄEach of these independently represents a linear or branched alkylene group having 1 to 10 carbon atoms, which may be substituted with halogen atoms.) In other words, the copolymer preferably includes a crosslinked structure derived from such a bifunctional monomer.

[0097] In equations (C) and (D), T ｃ and T ｄ Preferably, each is independently a hydrogen atom, a methyl group, or an ethyl group, and more preferably, each is independently a hydrogen atom or a methyl group.

[0098] In formulas (C) and (D), U ｄ Preferably, it is a hydrogen atom, a methyl group, or an ethyl group, and more preferably a hydrogen atom.

[0099] In equations (C) and (D), R ｃ and R ｄ Preferably, each is independently a linear or branched alkylene group having 1 to 3 carbon atoms, which may be substituted with a halogen atom; more preferably, each is independently an ethylene group or a propylene group, or an ethylene group or a propylene group substituted with one chlorine atom; particularly preferably, an ethylene group or a propylene group.

[0100] The difunctional monomer represented by formula (C) is preferably ethylene glycol di(meth)acrylate, triethylene glycol di(meth)acrylate, propylene glycol di(meth)acrylate, etc. The difunctional monomer represented by formula (D) is preferably bis(methacryloyloxymethyl) phosphate, bis[2-(methacryloyloxy)ethyl] phosphate, bis[2-(methacryloyloxy)propyl] phosphate, etc.

[0101] Any third component may be a trifunctional monomer. An example of such a trifunctional monomer as a third component is tris(oxy-2,1-ethanediyl) triacrylate.

[0102] Among these, ethylene glycol di(meth)acrylate represented by the following formula (C-1) and bis[2-(methacryloyloxy)ethyl phosphate represented by the following formula (D-1) are particularly preferred.

[0103]

[0104] The copolymer may contain one or more of these third components. Among these, the bifunctional monomer represented by formula (D) is preferred, and the bifunctional monomer represented by formula (D-1) is particularly preferred. The proportion of the third component (for example, a crosslinked structure derived from the bifunctional monomer represented by formula (C) or (D)) in the copolymer is 0 mol% to 50 mol%.

[0105] The method for producing the copolymer is not particularly limited, but examples include the method for producing the copolymer described in pamphlet WO2014 / 196652.

[0106] <<<Coating film of the second embodiment>>> The coating film may also be a coating film obtained from the coating film forming composition described in Brochure WO2021 / 167037. The contents of Brochure WO2021 / 167037 are incorporated into this specification to the same extent as if they were fully expressed. The coating film forming composition described in Brochure WO2021 / 167037 will be described below. In this specification, it will be referred to as the coating film forming composition of the second embodiment.

[0107] The coating film-forming composition of the second embodiment is given by the following formula (1): (In the formula, R １ R represents a hydrogen atom or a methyl group. ２ A compound represented by the following formula (2): (where n represents an alkylene group with 1 to 6 carbon atoms, and n represents an integer from 1 to 30) (In the formula, R ３ R represents a hydrogen atom or a methyl group. ４ Compounds represented by (where represents a monovalent organic group having cationic properties), and the following formula (3): (In the formula, R ５Each of these independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms, R ６ A monomer mixture comprising a compound represented by (where m represents a linear or branched alkylene group having 1 to 10 carbon atoms, which may be substituted with halogen atoms, and m represents an integer from 1 to 30), wherein the ratio of the compound represented by formula (3) to the total mass of the monomer mixture is 2 to 40% by mass, is obtained by polymerizing the monomer mixture, and a solvent.

[0108] In the compound of formula (1), which is a monomer component of the copolymer according to the second embodiment, R １ R represents a hydrogen atom or a methyl group. ２ n represents an alkylene group having 1 to 6 carbon atoms, preferably a linear or branched alkylene group having 1 to 6 carbon atoms, more preferably a linear or branched alkylene group having 2 to 5 carbon atoms, and particularly preferably an ethylene group or a propylene group. n represents an integer from 1 to 30, preferably an integer from 1 to 20, more preferably an integer from 2 to 10, and particularly preferably an integer from 3 to 6.

[0109] Specific examples of the compound of formula (1) above include acid phosphooxyethyl (meth)acrylate, 3-chloro-2-acid phosphooxypropyl (meth)acrylate, acid phosphooxypropyl (meth)acrylate, acid phosphooxymethyl (meth)acrylate, acid phosphooxypolyoxyethylene glycol mono(meth)acrylate, and acid phosphooxypolyoxypropylene glycol mono(meth)acrylate. Among these, acid phosphooxyethyl methacrylate (= 2-(methacryloyloxy)ethyl phosphate), acid phosphooxypolyoxyethylene glycol monomethacrylate, and acid phosphooxypolyoxypropylene glycol monomethacrylate are preferred.

[0110] The structural formulas of acid phosphooxyethyl methacrylate (= 2-(methacryloyloxy)ethyl phosphate), acid phosphooxypolyoxyethylene glycol monomethacrylate, and acid phosphooxypolyoxypropylene glycol monomethacrylate are represented by the following formulas (1-1) to (1-3).

[0111]

[0112] In the compound of formula (2), which is the monomer component of the copolymer according to the second embodiment, R ３ R represents a hydrogen atom or a methyl group. ４ represents a cationic monovalent organic group, typically a monovalent group having a primary, secondary, tertiary, or quaternary ammonium structure. Examples of monovalent groups having a primary, secondary, tertiary, or quaternary ammonium structure are, respectively, represented by the formula: -R ４ａ -NH ２ , -R ４ａ -NHR, -R ４ａ -NRR', -R ４ａ -N ＋ RR'R''[Here, R ４ａ [wherein R is an alkylene group having 1 to 6 carbon atoms, which may be interrupted by an ester bond, amide bond, ether bond, or phosphodiester bond, and R, R', and R'' are independently a linear or branched alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 10 carbon atoms, or an aralkyl group having 7 to 16 carbon atoms]. In the second embodiment, a monovalent group having a primary amine, secondary amine, or tertiary amine structure may be quaternized or chlorinated, and similarly, a monovalent group having a quaternary ammonium structure may be chlorinated.

[0113] Specific examples of the compound of formula (2) above include dimethylaminoethyl (meth)acrylate, diethylaminoethyl (meth)acrylate, dimethylaminopropyl (meth)acrylate, 2-(t-butylamino)ethyl (meth)acrylate, (meth)acryloylcholine chloride, and 2-(meth)acryloyloxyethyl phosphorylcholine (MPC).

[0114] The structural formulas of 2-(dimethylamino)ethyl methacrylate, methacroylcholine chloride, and 2-methacryloyloxyethyl phosphorylcholine (MPC) are represented by the following formulas (2-1) to (2-3).

[0115]

[0116] The compound of formula (3), which is the monomer component of the copolymer according to the second embodiment, is a crosslinkable bifunctional monomer. In the compound of formula (3), R ５ Each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms, preferably a hydrogen atom or a methyl group, R ６ m represents a linear or branched alkylene group having 1 to 10 carbon atoms, which may be substituted with halogen atoms; preferably, a linear or branched alkylene group having 1 to 6 carbon atoms, which may be substituted with halogen atoms; more preferably, a linear or branched alkylene group having 1 to 6 carbon atoms, which may be substituted with chlorine atoms; particularly preferably, an ethylene group, a propylene group, or a trimethylene group; m represents an integer from 1 to 30, preferably from 1 to 20, more preferably from 1 to 10, even more preferably from 1 to 6, and particularly preferably from 1 to 4.

[0117] Specific examples of the compound of formula (3) above include poly(ethylene glycol) di(meth)acrylate, poly(trimethylene glycol) di(meth)acrylate, and poly(propylene glycol) di(meth)acrylate.

[0118] The structural formulas of poly(ethylene glycol) dimethacrylate, poly(trimethylene glycol) dimethacrylate, and poly(propylene glycol) dimethacrylate are represented by the following formulas (3-1) to (3-3).

[0119]

[0120] The copolymer according to the second embodiment is obtained by polymerizing a monomer mixture containing the compounds of formulas (1), (2), and (3) above as monomer components. Such a monomer mixture may contain an ethylenically unsaturated monomer, a polysaccharide, or a derivative thereof as an optional fourth component, as long as it does not impair the ability to inhibit the adhesion of biomolecules, etc. Examples of ethylenically unsaturated monomers include one or more ethylenically unsaturated monomers selected from the group consisting of (meth)acrylic acid and its esters; vinyl acetate; vinylpyrrolidone; ethylene; vinyl alcohol; and their hydrophilic functional derivatives. Examples of polysaccharides or derivatives thereof include cellulosic polymers such as hydroxyalkylcellulose (e.g., hydroxyethylcellulose or hydroxypropylcellulose), starch, dextran, and curdlan. Preferably, the copolymer according to the second embodiment is obtained by polymerizing a monomer mixture containing only the compounds of formulas (1), (2), and (3) above as monomer components.

[0121] The copolymer according to the second embodiment is obtained by polymerizing a monomer mixture containing the above-mentioned compounds of formulas (1), (2), and (3) as monomer components, wherein the ratio of the compound of formula (3) to the total mass of the monomer components contained in the monomer mixture is 2 to 40% by mass. By including the compound of formula (3), which is a bifunctional monomer, in an amount of 2 to 40% by mass, preferably 3 to 35% by mass, and more preferably 5 to 30% by mass, relative to the total mass of the monomer components contained in the monomer mixture, the coating film-forming composition according to the second embodiment, which contains the copolymer obtained from the monomer mixture, provides excellent ability to suppress the adhesion of biomolecules. The monomer mixture may contain two or more compounds of formula (3).

[0122] In the monomer mixture, the proportion of the compound of formula (1) to the total mass of the monomer components is 10 to 95% by mass, preferably 10 to 80% by mass, and more preferably 45 to 75% by mass. The monomer mixture may contain two or more compounds of formula (1).

[0123] In the monomer mixture, the proportion of the compound of formula (2) to the total mass of the monomer components is 3 to 90% by mass, preferably 10 to 70% by mass, and more preferably 15 to 35% by mass. The monomer mixture may contain two or more compounds of formula (2).

[0124] The copolymer according to the second embodiment can be synthesized by methods such as radical polymerization, anionic polymerization, and cationic polymerization, which are common methods for synthesizing (meth)acrylic polymers. Various methods are possible for its formation, including solution polymerization, suspension polymerization, emulsion polymerization, and bulk polymerization. In one embodiment, it can be prepared by a manufacturing method that includes a step of reacting (polymerizing) a monomer mixture containing the compounds of formulas (1), (2), and (3) in a solvent at a total monomer component concentration of 0.01 to 20% by mass.

[0125] The solvent used in the polymerization reaction may be water, phosphate buffer, alcohol such as ethanol, or a mixed solvent of these, but it is preferable to include water or ethanol. Furthermore, it is preferable to include 10% to 100% by mass of water or ethanol. Furthermore, it is preferable to include 50% to 100% by mass of water or ethanol. Furthermore, it is preferable to include 80% to 100% by mass of water or ethanol. Furthermore, it is preferable to include 90% to 100% by mass of water or ethanol. Preferably, the total amount of water and ethanol is 100% by mass.

[0126] To efficiently advance polymerization reactions, it is desirable to use polymerization initiators. Examples of polymerization initiators include "thermal radical polymerization initiators" and "photoradical polymerization initiators." Examples of "thermal radical polymerization initiators" include 2,2'-azobis(isobutyronitrile), 2,2'-azobis(2-methylbutyronitrile), 2,2'-azobis(2,4-dimethylvaleronitrile) (Fujifilm Wako Pure Chemical Industries, Ltd. product name; V-65, 10-hour half-life temperature; 51°C), 4,4'-azobis(4-cyanovaleric acid), 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile), 1,1'-azobis(cyclohexane-1-carbonitride), 1-[(1-cyano 2,2'-Azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (Fujifilm Wako Pure Chemical Industries, Ltd.; VA-044, 10-hour half-life temperature; 44°C), 2,2'-Azobis[2-(2-imidazolin-2-yl)propane] (Fujifilm Wako Pure Chemical Industries, Ltd.; VA-061, 10-hour half-life temperature; 61°C), 2,2'-Azobis(2-methylpropionamidine) dihydrochloride, 2,2'-Azo(2-methyl-N-(2-hydroxyethyl) Propionamide (Fujifilm Wako Pure Chemical Industries, Ltd. product name: VA-086, 10-hour half-life temperature: 86°C), benzoyl peroxide (BPO), 2,2'-azobis(N-(2-carboxyethyl)-2-methylpropionamidine) n-hydrate (Fujifilm Wako Pure Chemical Industries, Ltd. product name: VA-057, 10-hour half-life temperature: 57°C), 4,4'-azobis(4-cyanopentanoic acid) (Fujifilm Wako Pure Chemical Industries, Ltd. product name: VA-501), 2,2'-azobis[2-(2-imidazoline] Examples include 2-yl)propane disulfate dihydrate (Fujifilm Wako Pure Chemical Industries, Ltd. product name: VA-046B, 10-hour half-life temperature: 46°C), 2,2'-azobis(2-amidinopropane) dihydrochloride (Fujifilm Wako Pure Chemical Industries, Ltd. product name: V-50, 10-hour half-life temperature: 56°C), peroxodisulfate, t-butyl hydroperoxide, dimethyl 1,1'-azobis(1-cyclohexanecarboxylate) (Fujifilm Wako Pure Chemical Industries, Ltd. product name: VE-073), etc.

[0127] Examples of "photoradical polymerization initiators" include acetophenone, chloroacetophenone, hydroxyacetophenone, 2-aminoacetophenone, dialkylaminoacetophenone, 2,2-diethoxyacetophenone, 2,2-dimethoxy-2'-phenylacetophenone (BASF product name: Irgacure 651), 2-hydroxy-2-methyl-1-phenylpropanone (BASF product name: Irgacure 1173), and 1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxymethylpropanone (B Acetophenones such as 2-hydroxy-1-{4-[4-(2-hydroxy-2-methylpropionyl)benzyl]phenyl}-2-methyl-1-propan-1-one (BASF product name: Irgacure 2959), 2-hydroxy-1-{4-[4-(2-hydroxy-2-methylpropionyl)benzyl]phenyl}-2-methyl-1-propan-1-one (BASF product name: Irgacure 127), 2-methyl-1-[4-(methylthio)phenyl]-2-morpholinopropan-1-one (BASF product name: Irgacure 907), and 2-benzyl-2-(dimethylamino)-4-morpholinobylophenone (BASF product name: Irgacure 369). Benzoin compounds; benzoin, benzoin methyl ether, benzoin ethyl ether, benzoin isopropyl ether, benzoin isobutyl ether, 1-hydroxycyclohexyl phenyl ketone (BASF product name: Irgacure 184), etc.; benzophenone, benzoylbenzoic acid, methyl benzoylbenzoate, methyl-o-benzoyl benzoate, 4-phenylbenzophenone, hydroxybenzophenone, hydroxypropyl benzophenone, acrylbenzophenone, 4,4'-bis(dimethyl benzophenone) Benzophenones such as mino-benzophenone and 4,4'-dichlorobenzophenone; thioxanthones such as thioxanthone, 2-chlorothioxanthone, 2-methylthioxanthone, diethylthioxanthone, and dimethylthioxanthone; acylphosphine oxides such as 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (BASF product name: Irgacure TPO) and bis(2,4,6-trimethylbenzoyl)-phenylphosphine oxide (BASF product name: Irgacure 819);Examples include oxyphenylacetic acid, 2-[2-oxo-2-phenylacetoxyethoxy]ethyl ester and a mixture of oxyphenylacetic acid, 2-(2-hydroxyethoxy)ethyl ester (BASF product name: Irgacure 754), methyl benzoylformate (BASF product name: Irgacure MBF), α-acyl oxime esters, benzyl-(o-ethoxycarbonyl)-α-monoxime, glyoxy esters, 2-ethylanthraquinone, camphorquinone, tetramethylthiuram sulfide, azobisisobutyronitrile, benzoyl peroxide, dialkyl peroxide, tert-butylperoxypivalate, etc.

[0128] Considering solubility in water, ion balance, and interaction with monomers, it is preferable to select from 2,2'-azo(2-methyl-N-(2-hydroxyethyl)propionamide, 2,2'-azobis(N-(2-carboxyethyl)-2-methylpropionamidine) n-hydrate, 4,4'-azobis(4-cyanopentanoic acid), 2,2'-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride, 2,2'-azobis[2-(2-imidazolin-2-yl)propane]disulfate dihydrate, 2,2'-azobis[2-(2-imidazolin-2-yl)propane], 2,2'-azobis(2-amidinopropane)dihydrochloride, and peroxodisulfate. Considering solubility in organic solvents, ion balance, and interactions with monomers, it is preferable to use 2,2'-azobis(2,4-dimethylvaleronitrile) or 2,2'-azobis(isobutyronitrile).

[0129] The amount of polymerization initiator added is 0.05% to 10% by mass relative to the total mass of monomer components used in polymerization.

[0130] The reaction conditions involve heating the reaction vessel to 50-200°C in an oil bath or the like, stirring for 1-48 hours, more preferably 80-150°C for 5-30 hours, to allow the polymerization reaction to proceed and obtain the copolymer of the second embodiment. A nitrogen atmosphere is preferred for the reaction atmosphere.

[0131] The reaction procedure may involve placing all the reactants into a reaction solvent at room temperature and then heating it to the above temperature to polymerize, or by adding all or part of the mixture of reactants dropwise into a preheated solvent.

[0132] The weight-average molecular weight of the copolymer according to the second embodiment may be several thousand to several million, preferably 5,000 to 5,000,000, and more preferably 10,000 to 2,000,000. It may also be a random copolymer, a block copolymer, or a graft copolymer, with a random copolymer being preferred. Furthermore, the copolymer produced in this manner is considered a three-dimensional polymer because it contains a bifunctional monomer, and is dissolved or dispersed in a solution containing water or alcohol.

[0133] The coating film-forming composition according to the second embodiment may be prepared by isolating and purifying the copolymer obtained in this manner, and then diluting it to a predetermined concentration with a desired solvent. Furthermore, the coating film-forming composition according to the second embodiment may be prepared from the reaction solution obtained after the polymerization reaction (i.e., the copolymer-containing varnish).

[0134] The solvents included in the coating film-forming composition of the second embodiment include water, phosphate-buffered saline (PBS), and alcohol. Examples of alcohols include C2-C6 alcohols, such as ethanol, propanol, isopropanol, 1-butanol, 2-butanol, isobutanol, t-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 1-heptanol, 2-heptanol, 2,2-dimethyl-1-propanol (=neopentyl alcohol), 2-methyl-1-propanol, 2-methyl-1-butanol, 2-methyl-2-butanol (=t-amyl alcohol), 3-methyl-1-butanol, 3-methyl-3-pentanol, cyclopentanol, 1-hexanol, 2-hexanol, 3-hexanol, 2,3-dimethyl Examples include ethyl-2-butanol, 3,3-dimethyl-1-butanol, 3,3-dimethyl-2-butanol, 2-ethyl-1-butanol, 2-methyl-1-pentanol, 2-methyl-2-pentanol, 2-methyl-3-pentanol, 3-methyl-1-pentanol, 3-methyl-2-pentanol, 3-methyl-3-pentanol, 4-methyl-1-pentanol, 4-methyl-2-pentanol, 4-methyl-3-pentanol, and cyclohexanol. These can be used individually or as mixed solvents of combinations thereof, but from the viewpoint of dissolving the copolymer, they are preferably selected from water, PBS, ethanol, and propanol.

[0135] In the second embodiment, the concentration of solids in the coating film-forming composition is preferably 0.01 to 50% by mass in order to form a uniform coating film. Furthermore, the concentration of copolymer in the coating film-forming composition is preferably 0.01 to 5% by mass, more preferably 0.01 to 4% by mass, and particularly preferably 0.01 to 3% by mass. If the copolymer concentration is 0.01% by mass or less, the copolymer concentration in the resulting coating film-forming composition is too low to form a coating film of sufficient thickness. If it is 5% by mass or more, the storage stability of the coating film-forming composition deteriorates, and precipitation of dissolved substances or gelation may occur.

[0136] Furthermore, in addition to the copolymer and solvent, the coating film-forming composition of the second embodiment may also contain other substances as needed, provided that they do not impair the performance of the resulting coating film. Examples of other substances include preservatives, surfactants, primers to improve adhesion to the substrate, fungicides, and sugars.

[0137] In order to adjust the ion balance of the copolymer in the coating film-forming composition according to the second embodiment, the process of obtaining the coating film according to the second embodiment may further include a step of pre-adjusting the pH of the coating film-forming composition. pH adjustment may be carried out, for example, by adding a pH adjusting agent to the composition containing the copolymer and a solvent, and setting the pH of the composition to 2 to 13.5, preferably 2 to 8.5, more preferably 3 to 8, or preferably 8.5 to 13.5, even more preferably 10 to 13.5. The type and amount of pH adjusting agent that can be used are appropriately selected according to the concentration of the copolymer and the ratio of its anions to cations. Examples of pH adjusting agents include organic amines such as ammonia, diethanolamine, pyridine, N-methyl-D-glucamine, and tris(hydroxymethyl)aminomethane; alkali metal hydroxides such as potassium hydroxide and sodium hydroxide; alkali metal halides such as potassium chloride and sodium chloride; inorganic acids such as sulfuric acid, phosphoric acid, hydrochloric acid, and carbonic acid or their alkali metal salts; quaternary ammonium cations such as choline, or mixtures thereof (for example, buffer solutions such as phosphate-buffered saline). Among these, ammonia, diethanolamine, sodium hydroxide, choline, N-methyl-D-glucamine, and tris(hydroxymethyl)aminomethane are preferred, with ammonia, diethanolamine, sodium hydroxide, and choline being particularly preferred.

[0138] <<<Coating film of the third embodiment>>> The coating film may also be a coating film obtained from the coating film forming composition described in Brochure WO2022 / 259998. The contents of Brochure WO2022 / 259998 are incorporated into this specification to the same extent as if they were fully expressed. The coating film forming composition described in Brochure WO2022 / 259998 will be described below. In this specification, it will be referred to as the coating film forming composition of the third embodiment.

[0139] The third embodiment of the coating film-forming composition is used to inhibit the adhesion of biological substances. The third embodiment of the coating film-forming composition contains at least a copolymer and, if necessary, other components such as a solvent. The third embodiment of the coating film-forming composition can form a coating film that is poorly soluble in phosphate-buffered saline, but the use of the third embodiment of the coating film-forming composition is not particularly limited as long as it is used to inhibit the adhesion of biological substances, and is not limited to forming a coating film that comes into contact with phosphate-buffered saline.

[0140] The copolymer according to the third embodiment is water-insoluble. Here, "water-soluble" means that 1.0 g or more can be dissolved in 100 g of water at 25°C. "Water-insoluble" means that it does not fall under the category of "water-soluble," that is, its solubility in 100 g of water at 25°C is less than 1.0 g. The copolymer according to the third embodiment has repeating units (A) represented by the following formula (A) and repeating units (B) represented by the following formula (B). The molar ratio (A:B) of repeating units (A) to repeating units (B) in the copolymer according to the third embodiment is 89:11 to 50:50. (In the formula, R １ ~R ３ Each of these independently represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, X １ and X ２ Each of these independently represents an alkylene group having 1 to 5 carbon atoms, which may be interrupted by a single bond, ester bond, ether bond, amide bond, or oxygen atom.

[0141] The copolymer according to the third embodiment may have two or more repeating units (A). The copolymer according to the third embodiment may have two or more repeating units (B). Preferably, the copolymer according to the third embodiment has one repeating unit (A) and one repeating unit (B).

[0142] Examples of alkyl groups having 1 to 5 carbon atoms include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, s-butyl group, t-butyl group, n-pentyl group, 1-methylbutyl group, 2-methylbutyl group, 3-methylbutyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, and 1-ethylpropyl group. １ ~R ３ Each of these is preferably a hydrogen atom, a methyl group, or an ethyl group.

[0143] The above terms "ester bond" means -C(=O)-O- or -O-C(=O)-, "ether bond" means -O-, and "amide bond" means -NHC(=O)- or -C(=O)NH-.

[0144] The alkylene group having 1 to 5 carbon atoms may be interrupted by an oxygen atom. Examples of alkylene groups having 1 to 5 carbon atoms include the methylene group, ethylene group, propylene group, trimethylene group, tetramethylene group, 1-methylpropylene group, 2-methylpropylene group, dimethylethylene group, ethylethylene group, pentamethylene group, 1-methyltetramethylene group, 2-methyltetramethylene group, 1,1-dimethyl-trimethylene group, 1,2-dimethyl-trimethylene group, 2,2-dimethyl-trimethylene group, and 1-ethyl-trimethylene group. １ and X ２ The group is preferably a methylene group, an ethylene group, or a propylene group. "May be interrupted by an oxygen atom" means that one or more carbon-carbon bonds in an alkylene group having 1 to 5 carbon atoms are bonded via an ether bond.

[0145] The copolymer according to the third embodiment is R １ and R ２is a hydrogen atom, R ３ is a methyl group, X １ and X ２ A copolymer in which the bonds are single bonds is preferred.

[0146] The molar ratio (A:B) of repeating unit (A) to repeating unit (B) is 89:11 to 50:50. In the copolymer according to the third embodiment, if the total number of moles of repeating unit (A) and repeating unit (B) is 100, the molar ratio (A:B) of repeating unit (A) to repeating unit (B) can be expressed as (100-m):m. In this case, the range of m is 11 to 50. The lower limit of m may be 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. The upper limit of m may be 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 38, 37, 36, or 35. Examples of the range of m include 12-49, 12-48, 15-48, 20-49, 20-45, 22-49, or 22-45.

[0147] In the copolymer according to the third embodiment, the total mol% of repeating units (A) and repeating units (B) in the total repeating units is not particularly limited, but is preferably 90 mol% or more, more preferably 95 mol% or more, even more preferably 99.5 mol% or more, and particularly preferably 100%.

[0148] In the third embodiment, in order to obtain a coating film that is poorly soluble in phosphate-buffered saline, the molar ratio of repeating units (A) to repeating units (B) in the copolymer is set to a specific range. Therefore, in the third embodiment, a coating film that is poorly soluble in phosphate-buffered saline can be obtained without crosslinking the copolymer. Thus, the copolymer does not need to have photosensitive groups for crosslinking. That is, it is preferable that the copolymer does not have photosensitive groups. Examples of photosensitive groups include azide groups. In the third embodiment, the copolymer does not need to have photosensitive groups for crosslinking. Therefore, when forming the coating film, it is not necessary to irradiate the copolymer with light to crosslink it. Thus, the process for forming the coating film can be simplified.

[0149] The viscosity-average degree of polymerization (hereinafter sometimes simply referred to as "degree of polymerization") of the copolymer according to the third embodiment is not particularly limited, but from the viewpoint of suitably obtaining the effects of the third embodiment, it is preferably 200 to 3,000, more preferably 200 to 2,500, and particularly preferably 200 to 2,000. The viscosity-average degree of polymerization is measured when the copolymer is fully saponified. The viscosity-average degree of polymerization of polyvinyl alcohol obtained by fully saponification is a value calculated from the intrinsic viscosity [η] (g / dL) measured at 30°C using an Ostwald viscometer with deionized water as the solvent, by the following formula: log(P) = 1.613 × log([η] × 10) ４ (8.29) Here, P represents the viscosity-average degree of polymerization. The viscosity-average degree of polymerization can be determined according to JIS K 6726.

[0150] The method for producing the copolymer according to the third embodiment is not particularly limited, but for example, one method is to polymerize a compound represented by the following formula (C) to produce a homopolymer, and then partially hydrolyze the obtained homopolymer by a known saponification reaction to obtain a copolymer. (In the formula, R １ , R ３ , and X １ (This is synonymous with the above.)

[0151] Furthermore, as a method for producing a copolymer according to the third embodiment, for example, a method of copolymerizing a compound represented by the following formula (C) with a compound represented by the following formula (D) to obtain a copolymer is mentioned. (In the formula, R １ ~R ３ , X １ , and X ２ (This is synonymous with the above.)

[0152] The copolymer according to the third embodiment may be a random copolymer or a block copolymer. A commercially available product may be used as the copolymer according to the third embodiment. A specific example of a commercially available copolymer is polyvinyl acetate (manufactured by Nippon Vinyl Acetate & Polyvinyl Alcohol, trade name JMR-10L®).

[0153] The content of the copolymer in the film-forming component of the coating film-forming composition of the third embodiment is not particularly limited, but is preferably 80% by mass or more, more preferably 90% by mass or more, and particularly preferably 95% by mass or more. The film-forming component refers to the components of the composition excluding the solvent component from all components.

[0154] The copolymer content in the third embodiment of the coating film-forming composition is not particularly limited, but from the viewpoint of easily forming a coating film of a desired thickness, it is preferably 0.1 to 10% by mass, more preferably 0.3 to 8% by mass, and particularly preferably 0.5 to 5% by mass. The copolymer content in the coating film-forming composition may also be 0.02 to 2% by mass or 0.05 to 1% by mass.

[0155] Examples of solvents in the third embodiment include water, phosphate-buffered saline (PBS), alcohol, and water-soluble organic solvents (excluding alcohol).

[0156] Examples of alcohols include those with 2 to 6 carbon atoms. Examples of alcohols include ethanol, propanol, isopropanol, 1-butanol, 2-butanol, isobutanol, t-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 1-heptanol, 2-heptanol, 2,2-dimethyl-1-propanol (neopentyl alcohol), 2-methyl-1-propanol, 2-methyl-1-butanol, 2-methyl-2-butanol (t-amyl alcohol), 3-methyl-1-butanol, 3-methyl-3-pentanol, cyclopentanol, 1- Examples include xanol, 2-hexanol, 3-hexanol, 2,3-dimethyl-2-butanol, 3,3-dimethyl-1-butanol, 3,3-dimethyl-2-butanol, 2-ethyl-1-butanol, 2-methyl-1-pentanol, 2-methyl-2-pentanol, 2-methyl-3-pentanol, 3-methyl-1-pentanol, 3-methyl-2-pentanol, 3-methyl-3-pentanol, 4-methyl-1-pentanol, 4-methyl-2-pentanol, 4-methyl-3-pentanol, and cyclohexanol. These can be used individually or in combination of two or more.

[0157] Water-soluble organic solvents are organic solvents that can be mixed with water and alcohol in any proportion and do not separate after mixing. Examples of water-soluble organic solvents include ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, methyl cellosolve acetate, ethyl cellosolve acetate, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, propylene glycol, propylene glycol monomethyl ether, propylene glycol monoethyl ether, propylene glycol monomethyl ether acetate, and propylene glycol propyl ether acetate. These can be used individually or in combination of two or more.

[0158] In the third embodiment of the coating film-forming composition, water, phosphate-buffered saline (PBS), alcohol, or a water-soluble organic solvent may be used alone as the solvent. In the third embodiment of the coating film-forming composition, two or more of water, phosphate-buffered saline (PBS), alcohol, and a water-soluble organic solvent may be used in combination as the solvent. From the viewpoint of copolymer solubility, the solvent is preferably selected from water, alcohol, a water-soluble organic solvent, and a combination of two or more thereof, and more preferably selected from water, ethanol, a water-soluble organic solvent, and a combination of two or more thereof.

[0159] The following solvent combinations are preferred: • Water and alcohol • Water, alcohol, and water-soluble organic solvent • Alcohol and water-soluble organic solvent

[0160] The following solvent combinations are more preferable: • Water and ethanol • Water, ethanol, and propylene glycol monomethyl ether • Ethanol and propylene glycol monomethyl ether

[0161] In the third embodiment of the coating film-forming composition, the mass ratio of water to alcohol is, for example, 1:99 to 70:30 and 1:99 to 50:50. In the third embodiment of the coating film-forming composition, the mass ratio of water:alcohol:water-soluble organic solvent (A:B:C) is, for example, 5 to 30:65 to 92:1 to 30 (where A+B+C is 100). In the third embodiment of the coating film-forming composition, the mixing ratio (mass ratio) of alcohol to water-soluble organic solvent is, for example, 30:70 to 97:3.

[0162] The solvent content in the coating film-forming composition of the third embodiment is not particularly limited, but from the viewpoint of easily forming a coating film of a desired thickness, it is preferably 90% by mass or more, more preferably 92% by mass or more, and particularly preferably 95% by mass or more.

[0163] The third embodiment of the coating film-forming composition may optionally contain other components. Examples of other components include pH adjusters, preservatives, surfactants, fungicides, sugars, and the like.

[0164] An example of a cell holder before cells are placed is described below. As shown in Figure 1, the cell holder before cells are placed has a main body and a cell-holding underlayment that is a spot. It is also preferable to have a cell holder with a cell-holding underlayment covering the entire surface of the inner bottom surface instead of the cell-holding underlayment covering that is a spot as shown in Figure 1. The main body may have an outer wall, an inner bottom surface, and an opening. The opening is formed by the outer wall, and the inner bottom surface may be located below the opening. Multiple cell-holding underlayment coverings are arranged at intervals on the inner bottom surface. The main body may not have an opening. In the case of a main body without an opening, the cell-holding underlayment covering may be arranged on the inner bottom surface and on the upper surface inside the main body.

[0165] The cell retainer before cells are placed has a main body, a cell adhesion substance, and a cell-retaining base membrane which acts as a spot. The main body may have an outer wall, an inner bottom surface, and an opening. The opening is formed by the outer wall, and the inner bottom surface may be located below the opening. Multiple cell-retaining base membranes are arranged at intervals on the inner bottom surface. The cell adhesion substance is placed on the cell-retaining base membranes.

[0166] The cell retainer before cells are placed comprises a main body, a cell adhesion substance, a cell-retaining underlayer film (which acts as a spot), and a coating film. The main body may have an outer wall, an inner bottom surface, and an opening. The opening is formed by the outer wall, and the inner bottom surface is located below the opening. The surface of the inner bottom surface has a coating film that inhibits cell adhesion. Multiple cell-retaining underlayer films are arranged at intervals on the coating film. The cell adhesion substance is placed on the cell-retaining underlayer films.

[0167] (Method for producing a cell retainer) The method for producing a cell retainer of the present invention includes the following step (I): Step (I): A step of applying droplets of a cell retaining base film forming agent to a substrate to form a cell retaining base film.

[0168] The method for producing a cell retainer may further include any of the following steps (II) to (III): Step (II): A step of applying a coating agent containing a polymer having cell adhesion inhibitory ability onto a substrate to form a coating film. Step (III): A step of applying droplets containing a cell adhesion substance to a cell retaining base film to distribute the cell adhesion substance onto the cell retaining base film. Step (II) is performed before step (I). Step (III) is performed after step (I) and after step (II).

[0169] <Step (I)> Step (I) is a step in which droplets of a cell-retaining base film forming agent are applied to the substrate to form a cell-retaining base film.

[0170] Examples of substrates include those mentioned in the description of the cell retainer of the present invention.

[0171] The cell-retaining underlayer film-forming agent (hereinafter sometimes referred to as the "underlayer film-forming agent") contains a polymer and a solvent, and may also contain other components. Examples of polymers include those listed in the description of the cell retainer of the present invention. The solvent is not particularly limited, but is preferably an aqueous solution containing water, phosphate-buffered saline (PBS), alcohol, or water-soluble organic solvent (excluding alcohol). Examples of aqueous solutions include water, saline or salt-containing aqueous solutions such as phosphate buffer solution, or mixed solvents combining water or a salt-containing aqueous solution with alcohol. Alcohols include those with 2 to 6 carbon atoms, such as ethanol, propanol, isopropanol, 1-butanol, 2-butanol, isobutanol, t-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 1-heptanol, 2-heptanol, 2,2-dimethyl-1-propanol (= neopentyl alcohol), 2-methyl-1-propanol, 2-methyl-1-butanol, 2-methyl-2-butanol (= t-amyl alcohol), 3-methyl-1-butanol, 3-methyl-3-pentanol, cyclopentanol, and 1-hexanol. Examples of alcohols include 2-hexanol, 3-hexanol, 2,3-dimethyl-2-butanol, 3,3-dimethyl-1-butanol, 3,3-dimethyl-2-butanol, 2-ethyl-1-butanol, 2-methyl-1-pentanol, 2-methyl-2-pentanol, 2-methyl-3-pentanol, 3-methyl-1-pentanol, 3-methyl-2-pentanol, 3-methyl-3-pentanol, 4-methyl-1-pentanol, 4-methyl-2-pentanol, 4-methyl-3-pentanol, and cyclohexanol, which may be used individually or as mixed solvents of these. The water content in the aqueous solution is, for example, 50% to 100% by mass, 80% to 100% by mass, or 90% to 100% by mass. Examples of alcohols include alcohols having 2 to 6 carbon atoms.Examples of alcohols include ethanol, propanol, isopropanol, 1-butanol, 2-butanol, isobutanol, t-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 1-heptanol, 2-heptanol, 2,2-dimethyl-1-propanol (neopentyl alcohol), 2-methyl-1-propanol, 2-methyl-1-butanol, 2-methyl-2-butanol (t-amyl alcohol), 3-methyl-1-butanol, 3-methyl-3-pentanol, cyclopentanol, 1- Examples include xanol, 2-hexanol, 3-hexanol, 2,3-dimethyl-2-butanol, 3,3-dimethyl-1-butanol, 3,3-dimethyl-2-butanol, 2-ethyl-1-butanol, 2-methyl-1-pentanol, 2-methyl-2-pentanol, 2-methyl-3-pentanol, 3-methyl-1-pentanol, 3-methyl-2-pentanol, 3-methyl-3-pentanol, 4-methyl-1-pentanol, 4-methyl-2-pentanol, 4-methyl-3-pentanol, and cyclohexanol. These can be used individually or in combination of two or more. Furthermore, a water-soluble organic solvent refers to an organic solvent that can be mixed with water and alcohol in any proportion and does not separate after mixing. Examples of water-soluble organic solvents include ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, methyl cellosolve acetate, ethyl cellosolve acetate, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, propylene glycol, propylene glycol monomethyl ether, propylene glycol monoethyl ether, propylene glycol monomethyl ether acetate, and propylene glycol propyl ether acetate. These can be used individually or in combination of two or more. In addition to the polymer, cell adhesion substance, and solvent mentioned above, other substances may be added to the primer-forming agent as needed, provided that they do not impair the performance of the resulting primer. Examples of other substances include pH adjusters, crosslinking agents, preservatives, surfactants, primers to improve adhesion to containers or substrates, antifungal agents, and sugars.

[0172] There are no particular limitations on the method for applying droplets of the undercoat-forming agent to the substrate, but inkjet printing, screen printing, spray printing, slit coating, gravure printing, bar coating, roll-to-roll, etc. can be used, but preferably inkjet printing, gravure printing, bar coating, or other printing technology is used. As for other coating methods, for example, a method in which a substrate with areas where a cell-retaining undercoat-forming agent is not to be formed is immersed in the undercoat-forming agent, or a method in which the undercoat-forming agent is added to a substrate (container) container with areas where a cell-retaining undercoat-forming agent is not to be formed is immersed in the undercoat-forming agent, or a method in which the undercoat-forming agent is added to a container with areas where a cell-retaining undercoat-forming agent is immersed in the undercoat-forming agent, or a method in which the undercoat-forming agent is added to a container with areas where a cell-retaining undercoat-forming agent is immersed in the undercoat-forming agent, or a method in which the undercoat-forming agent is immersed in a container The standing period is carried out by appropriately selecting the time and temperature depending on the material of the container or substrate and the type of cell culture substrate forming agent, but for example, it is carried out from 1 minute to 24 hours, preferably from 5 minutes to 3 hours, at 10 to 80°C.

[0173] Furthermore, after forming a cell-retaining underlayer on the substrate surface by this method, subsequent steps may be carried out either directly without a drying process, or after washing with water or the medium used for cell culture (e.g., water, buffer solution, culture medium, etc.).

[0174] <Step (II)> Step (II) is a step of applying a coating agent containing a polymer having cell adhesion inhibitory ability onto a substrate to form a coating film. The coating agent is not particularly limited as long as it contains a polymer having cell adhesion inhibitory ability, but for example, it contains a polymer having cell adhesion inhibitory ability and a solvent. Examples of the coating film and coating agent include the coating film and coating agent mentioned in the description of the cell holder of the present invention. The method of applying the coating agent onto the substrate is not particularly limited, but inkjet printing, screen printing, spray printing, slit coating, gravure printing, bar coating, roll-to-roll, etc. can be used, but preferably it is done using printing techniques such as inkjet printing, gravure printing, or bar coating.

[0175] <Step (III)> Step (III) is a step in which droplets containing cell adhesion material are applied to the cell-retaining base film, thereby distributing the cell adhesion material on the cell-retaining base film.

[0176] Examples of cell adhesion substances include the cell adhesion substances mentioned in the description of the cell holder of the present invention.

[0177] Droplets containing cell adhesion substances are formed, for example, from a liquid containing cell adhesion substances. The liquid containing cell adhesion substances contains, for example, cell adhesion substances and a solvent. Examples of solvents include those listed in the description of cell-retaining underlayer-forming agents.

[0178] The method for applying droplets containing cell adhesion material is not particularly limited, but inkjet methods, screen printing methods, slit coating methods, roll-to-roll methods, etc., can be used, but preferably inkjet methods or printing techniques such as screen printing are used.

[0179] (Retention and retrieval of suspended cells) The cell retainer described above is suitable for retaining suspended cells on a cell retaining substrate. Furthermore, a cell retainer having suspended cells retained on a cell retaining substrate is suitable for retrieving the suspended cells.

[0180] <Maintaining Suspended Cells> Suspended cells can be maintained by seeding culture cells onto a cell-retaining substrate. One method of seeding cells is to add a culture medium containing dispersed suspended cells to a substrate on which a cell-retaining substrate has been formed. For example, the following total number of cells are dispersed in the culture medium, and the medium is added to the cell retainer. The lower limit of the total number of cells should be, for example, equal to or greater than the number of spots (n) present in the cell retainer. The culture medium is prepared according to the type of suspended cells. One type of suspended cell may be used, or two or more types of suspended cells may be used.

[0181] There are no particular restrictions on the cell concentration in the culture medium in which the suspension cells are dispersed.

[0182] The culture medium can be appropriately selected depending on the type of cells to be used. For example, when culturing mammalian cells, a culture medium commonly used for mammalian cell culture can be used. Examples of culture media for mammalian cells include Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM / F12, McCoy's 5A Medium, Eagle's Minimum Essential Medium (EMEM), αMEM (alpha Modified Eagle's Minimum Essential Medium), and MEM (Minimum Essential Medium). Medium), RPMI 1640 medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB 131 medium, William's Medium E, IPL 41 medium, Fischer's medium, StemPro 34 (Invitrogen), X-VIVO 10 (Kembrex), X-VIVO 15 (Kembrex), HPGM (Kembrex), StemSpan Examples include H3000 (Stem Cell Technology), StemSpanSFEM (Stem Cell Technology), Stemline II (Sigma-Aldrich), QBSF-60 (Quality Biological), StemProhESCSFM (Invitrogen), mTeSR1 or SR2 medium (Stem Cell Technology), Sf-900II (Invitrogen), Opti-Pro (Invitrogen), and HuMedia-KG2 (Kurabo Industries Ltd.).

[0183] Those skilled in the art may freely add sodium, potassium, calcium, magnesium, phosphorus, chlorine, various amino acids, various vitamins, antibiotics, serum, fatty acids, sugars, etc. to the above culture medium as needed. When culturing mammalian cells, those skilled in the art may also add one or more other chemical or biological components as needed.

[0184] Components that can be added to culture media for mammalian cells include fetal bovine serum, human serum, horse serum, insulin, transferrin, lactoferrin, cholesterol, ethanolamine, sodium selenite, monothioglycerol, 2-mercaptoethanol, bovine serum albumin, sodium pyruvate, polyethylene glycol, various vitamins, various amino acids, agar, agarose, collagen, methylcellulose, various cytokines, various hormones, various growth factors, various extracellular matrix components, and various cell adhesion molecules.

[0185] Cytokines that can be added to the culture medium include, for example, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-14 (IL-14), and interleukin-14. Examples include, but are not limited to, IL-15, IL-18, IL-21, IFN-α, IFN-β, IFN-γ, granulocyte colony-stimulating factor (G-CSF), monocyte colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), flk2 / flt3 ligand (FL), leukemia cell inhibitor (LIF), oncostatin M (OM), erythropoietin (EPO), and thrombopoietin (TPO).

[0186] Hormones that can be added to the culture medium include melatonin, serotonin, thyroxine, triiodothyronine, epinephrine, norepinephrine, dopamine, anti-Müllerian hormone, adiponectin, adrenocorticotropic hormone, angiotensinogen and angiotensin, antidiuretic hormone, atrial natriuretic peptide, calcitonin, cholecystokinin, corticotropin-releasing hormone, erythropoietin, follicle-stimulating hormone, gastrin, ghrelin, glucagon, gonadotropin-releasing hormone, growth hormone-releasing hormone, human chorionic gonadotropin, human placental lactogen, growth hormone, inhibin, insulin, insulin-like growth factor, leptin, luteinizing hormone, and melanocyte-stimulating hormone. Examples include, but are not limited to, oxytocin, parathyroid hormone, prolactin, secretin, somatostatin, thrombopoietin, thyroid-stimulating hormone, thyrotropin-releasing hormone, cortisol, aldosterone, testosterone, dehydroepiandrosterone, androstenedione, dihydrotestosterone, estradiol, estrone, estriol, progesterone, calcitriol, calcidiol, prostaglandins, leukotrienes, prostacyclins, thromboxanes, prolactin-releasing hormone, lipotropin, brain natriuretic peptide, neuropeptide Y, histamine, endothelin, pancreatic polypeptides, renin, and enkephalins.

[0187] Growth factors that can be added to the culture medium include transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), macrophage inflammatory protein-1α (MIP-1α), epidermal growth factor (EGF), fibroblast growth factor-1, 2, 3, 4, 5, 6, 7, 8, or 9 (FGF-1, 2, 3, 4, 5, 6, 7, 8, 9), neuronal growth factor (NGF), hepatocyte growth factor (HGF), leukemia inhibitor (LIF), and protease. Examples include, but are not limited to, Syn I, protease nexin II, platelet-derived growth factor (PDGF), cholinergic differentiation factor (CDF), chemokines, Notch ligands (such as Delta1), Wnt protein, angiopoietin-like proteins 2, 3, 5, or 7 (Angpt2, 3, 5, 7), insulin-like growth factor (IGF), insulin-like growth factor-binding protein (IGFB), and pleiotrophin.

[0188] Furthermore, genetically modified cytokines and growth factors with altered amino acid sequences can also be added. Examples include the IL-6 / soluble IL-6 receptor complex or Hyper IL-6 (a fusion protein of IL-6 and the soluble IL-6 receptor).

[0189] Examples of various extracellular matrix and cell adhesion molecules include collagen I to XIX, fibronectin, laminin-1 to 12, nitrogen, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, various elastins, various proteoglycans, various cadherins, desmocolin, desmoglein, various integrins, E-selectin, P-selectin, L-selectin, immunoglobulin superfamily, Matrigel, poly-D-lysine, poly-L-lysine, chitin, chitosan, Sepharose, hyaluronic acid, alginate gel, various hydrogels, and their cleavage fragments.

[0190] Examples of antibiotics that can be added to the culture medium include sulfonamides, penicillin, pheneticillin, methicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, nafcillin, ampicillin, penicillin, amoxicillin, cyclacillin, carbenicillin, ticarcillin, piperacillin, azurocillin, mexdulocillin, mesilinum, andinocillin, cephalosporins and their derivatives, oxolinic acid, amifloxacin, temafloxacin, nalidixic acid, pyromidic acid, ciprofloxan, cinoxacin, norfloxacin, perfloxacin, rozaxacin, and ofloxacin. Examples include xacin, enoxacin, pipemidic acid, sulbactam, crab phosphate, β-bromopenicillaneic acid, β-chloropenicillaneic acid, 6-acetylmethylene-penicillaneic acid, cefoxazole, sultanpicillin, adinosillin, and formaldehyde foudrate ester of sulbactam, tazobactam, aztreonam, sulfazetine, isosulfazetine, nocardisine, methyl phenylacetamidephosphonate, chlortetracycline, oxytetracycline, tetracycline, demeclocycline, doxycycline, metacycline, and minocycline.

[0191] As mentioned above, serum and / or serum substitutes may be added to the culture medium. The concentration of serum added to the culture medium should be set appropriately depending on the cell type, culture conditions, and purpose of culture, but in one embodiment, the concentration of serum (and / or serum substitute) in the culture medium can be 15% by weight or less, 10% by weight or less, 9% by weight or less, 8% by weight or less, 7% by weight or less, 6% by weight or less, 5% by weight or less, 4% by weight or less, 3% by weight or less, 2% by weight or less, 1% by weight or less, 0.9% by weight or less, 0.8% by weight or less, 0.7% by weight or less, 0.6% by weight or less, 0.5% by weight or less, 0.4% by weight or less, or 0.3% by weight or less. The serum concentration may be kept constant throughout the culture period, or it may be increased or decreased as needed when changing the culture medium, etc.

[0192] <Recovery of Suspended Cells> The cell retainer having suspended cells held in the cell-retaining substrate as described above is suitably used for recovering the suspended cells.

[0193] The method for recovering suspended cells may be any method that can recover suspended cells. Preferred recovery methods include, for example, suspension by pipetting of the culture medium, cooling, and shaking, more preferably by pipetting or shaking, and particularly preferably by pipetting. Physical impact makes it easier to detach the suspended cells. Pipetting also makes it easier to apply a liquid flow to the cell-holding membrane to be recovered, thus making it easier to detach the suspended cells. For cooling before shaking, it is preferable to cool the cell holder containing the suspended cells at 0°C to 10°C for 10 to 60 minutes.

[0194] In the cell holder that holds the suspended cells, it is preferable to detach the suspended cells from the cell holder by applying a shear stress of 0.8 Pa to 15 Pa. Suspended cells held in the cell holder of the present invention are difficult to detach with a weak force of 0.8 Pa or less, but are easily detached from the cell holder by applying a shear stress of a certain level or higher (greater than 0.8 Pa). Here, the shear stress for detaching the suspended cells is preferably 15 Pa or less from the viewpoint of suppressing damage to the cells and foaming of the culture medium, and from the viewpoint of stable operation of the device, etc.

[0195] Shear stress is the maximum stress applied to suspended cells when they are detached. It can be calculated using spreadsheet software such as Excel or fluid analysis software, by inputting appropriate values ​​for variables such as culture medium density, culture medium viscosity, culture vessel shape (aspect ratio), fluid flow velocity and kinematic viscosity coefficient caused by shaking of the vessel and fluid flow.

[0196] The present invention will be described in more detail below based on synthesis examples, examples, test examples, etc., but the present invention is not limited to these.

[0197] <Method for Measuring Molecular Weight> The weight-average molecular weight shown in the synthesis example below is the result obtained by Gel Filtration Chromatography (hereinafter abbreviated as GFC). (Measurement conditions) ・Apparatus: HLC-8320GPC (manufactured by Tosoh Corporation) ・GFC column: TSKgel G 6000 + 3000 PWXL-CP ・Flow rate: 1.0 mL / min ・Eluent: Salt-containing water / organic mixed solvent ・Column temperature: 40°C ・Detector: RI ・Injection concentration: Polymer solids 0.05% by mass ・Injection volume: 100 μL ・Calibration curve: Cubic approximation curve ・Standard samples: Polyethylene oxide (manufactured by Agilent) x 10 types

[0198] <Synthesis Example 1> 24.00 g of 2-(dimethylamino)ethyl methacrylate (manufactured by Tokyo Chemical Industry Co., Ltd.), 1.46 g of methacrylic acid (manufactured by Tokyo Chemical Industry Co., Ltd.), 5.09 g of ethylene glycol dimethacrylate (manufactured by Tokyo Chemical Industry Co., Ltd.), 0.31 g of dimethyl 1,1'-azobis(1-cyclohexanecarboxylate) (VE-073, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and 111.09 g of 2-propanol were mixed and polymerized dropwise into 166.62 2-propanol at the reflux temperature to synthesize a polymer. The reaction product was reprecipitation with hexane, a poor solvent, and the precipitate was recovered by filtration and dried under reduced pressure. The weight-average molecular weight of this polymer according to GFC was 228,000.

[0199] <Comparative Synthesis Example 1> 18.01 g of acid phosphooxypolypropylene glycol monomethacrylate (average number of moles of propylene oxide added: 5; molecular weight per phosphate group calculated by titration: 618) (product name: PPM-5P, anionic homopolymer, manufactured by Toho Chemical Industries, Ltd.), 0.18 g of dimethyl 1,1'-azobis(1-cyclohexanecarboxylate) (VE-073, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and 72.99 g of 2-propanol were mixed. Polymers were synthesized by polymerization at a reflux temperature while stirring.

[0200] <Comparative Synthesis Example 2> PMB30 (ion complex polymer (neutral)) was synthesized according to S. H. Ye et al. / Journal of Membrane Science 210 (2002) 411-421. The reaction product was reprecipitation with a poor solvent, diethyl ether / chloroform = 8 / 2 (volume ratio), and the precipitate was recovered by filtration and dried under reduced pressure.

[0201] <Preparation Example 1> Polyvinyl acetate (JMR-150L®, manufactured by Nippon Vinyl Acetate & Polamine Co., Ltd. (degree of polymerization 1480, degree of saponification 22.7%)) was dissolved in ethanol / 1-methoxy-2-propanol (7 / 3 mass ratio) to a concentration of 3.0 mg / g to prepare a coating film-forming composition 1 having cell adhesion inhibitory ability. The obtained composition was transparent and homogeneous.

[0202] <Preparation Example 2> 0.05 g of the polymer obtained in Synthesis Example 1 was added to 49.95 g of sterile water and thoroughly stirred. 3.00 g of the resulting solution was then added to 7.00 g of sterile water and thoroughly stirred to prepare primer-forming agent 1.

[0203] <Preparation Example 3> 0.05 g of the polymer obtained in Synthesis Example 1 was added to 49.95 g of sterile water and thoroughly stirred. 1.00 g of the resulting solution was added to 9.00 g of sterile water and thoroughly stirred to prepare primer-forming agent 2.

[0204] <Preparation Example 4> 0.05 g of the polymer obtained in Synthesis Example 1 was added to 49.95 g of sterile water and thoroughly mixed. 2.20 g of the resulting solution was then mixed thoroughly with 26.24 g of sterile water and 0.88 mL of 0.5 mg / mL of Vitronectin VTN-N (Gibco) to prepare primer-forming agent 3.

[0205] <Preparation Example 5> 0.05 g of the polymer obtained in Synthesis Example 1 was added to 49.95 g of sterile water and thoroughly mixed. 2.20 g of the resulting solution was then mixed thoroughly with 26.24 g of sterile water and 0.88 mL of 0.5 mg / mL of Vitronectin VTN-N (Gibco) to prepare the primer-forming agent 4.

[0206] <Preparation Example 6> 0.08 g of the polymer obtained in Synthesis Example 1 was mixed with 39.92 g of 2-propanol and stirred thoroughly to prepare the primer-forming agent 5.

[0207] <Preparation Example 7> 0.20 g of the polymer obtained in Synthesis Example 1 was mixed with 39.80 g of 2-propanol and stirred thoroughly to prepare the primer-forming agent 6.

[0208] <Preparation Example 8> To prepare primer film-forming agent 7, 0.07 g of Epomin SP-200 (Nippon Shokubai Co., Ltd.) was mixed with 13.93 g of 2-propanol and 0.70 g of a 50% aqueous solution of glutaraldehyde (Tokyo Chemical Industry Co., Ltd.) and stirred thoroughly.

[0209] <Preparation Example 9> 0.10 g of the polymer obtained in Comparative Synthesis Example 2 above was mixed with 19.90 g of ethanol and stirred thoroughly to prepare the primer-forming agent 8.

[0210] <Preparation Example 10> 0.17 g of poly(2-hydroxyethyl methacrylate) (Sigma-Aldrich) was mixed with 8.23 ​​g of ethanol and 0.28 g of 1 mol / L hydrochloric acid (Kanto Chemical Co.) and stirred thoroughly to prepare primer-forming agent 9.

[0211] <Preparation Example 11> 0.68 g of polyacrylic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) was mixed with 22.11 g of ethanol and 1.4 g of 1 mol / L hydrochloric acid (Kanto Chemical Co., Ltd.) and stirred thoroughly to prepare the primer-forming agent 10.

[0212] <Preparation Example 12> 0.19 g of the polymer obtained in Comparative Synthesis Example 1 was added to 38.2 g of ethanol and thoroughly stirred to prepare the primer-forming agent 11.

[0213] <Preparation Example 13> 0.30 g of ε-poly-L-lysine 25% solution (JNC Co.), 7.24 g of Otsuka distilled water, and 7.46 g of ethanol were added and thoroughly stirred to prepare the primer-forming agent 12.

[0214] <Preparation Example 14> 0.02 g of the polymer obtained in Synthesis Example 1 was mixed with 19.98 g of 2-propanol and stirred thoroughly to prepare the primer-forming agent 13.

[0215] <Preparation Example 15> Polyvinyl acetate (JMR-150L®, manufactured by Nippon Vinyl Acetate & Polamine Co., Ltd. (degree of polymerization 1480, degree of saponification 22.7%)) was dissolved in ethanol / 1-methoxy-2-propanol (85 / 15 mass ratio) to a concentration of 20 mg / g to prepare a coating film-forming composition 2 having cell adhesion inhibitory ability. The obtained composition was transparent and homogeneous.

[0216] <Production Example 1> (Production of a substrate with cell adhesion inhibitory ability by inkjet) Using an inkjet device (Seiko Epson Corporation, R&D inkjet device) and an inkjet head (Seiko Epson Corporation, Precision Core head S800-A1), an appropriate amount of the coating film forming composition 1 prepared in Production Example 1 was applied to a polystyrene substrate measuring 79 mm x 121 mm in a circular shape with a diameter of 18 mm. The coated polystyrene substrate was dried in an oven at 70°C for 24 hours to produce a substrate 1 with cell adhesion inhibitory ability.

[0217] <Preparation Example 2> In each well of a substrate having cell adhesion inhibitory ability (Sumitomo Bakelite Co., Ltd., PrimeSurface® Plate 24F, Model No.: MS-90240), the undercoat-forming agents 1 and 2 obtained in Preparation Examples 2 and 3 were applied using a micropipette at a rate of 0.5 μL / spot to create 10 spots / well. Then, the substrate was dried in an oven at 70°C for 24 hours to prepare a cell temporary adsorption substrate (cell holder) 1.

[0218] <Preparation Example 3> Using an inkjet device (Seiko Epson Corporation, R&D inkjet device) and an inkjet head (Seiko Epson Corporation, Precision Core head S800-A1), an appropriate amount of the undercoat-forming agent 3 prepared in Preparation Example 4 was applied to the culture surface of a substrate with cell adhesion-inhibiting ability (Sumitomo Bakelite Co., Ltd., PrimeSurface® plate 24F, model number: MS-90240), forming numerous spots with a diameter of 400 μm. After drying at room temperature, the substrate was dried in a constant temperature dryer at 70°C for one day to prepare a cell temporary adsorption substrate 2. Sterilization was performed by irradiation with gamma rays at 25 kGy.

[0219] <Preparation Example 4> Using an inkjet device (Seiko Epson Corporation, R&D inkjet device) and an inkjet head (Seiko Epson Corporation, Precision Core head S800-A1), an appropriate amount of the undercoat-forming agent 4 prepared in Preparation Example 5 was applied to the culture surface of the substrate 1 having cell adhesion-inhibiting ability prepared in Preparation Example 1, and numerous spots with a diameter of 400 μm were formed. After drying the substrate with numerous spots at room temperature, it was dried in a constant temperature dryer at 70°C for one day and attached to a bottomless 24-well plate (C-STECH Co., Ltd.) to prepare a cell temporary adsorption substrate 3. Sterilization was performed by irradiation with gamma rays at 25 kGy.

[0220] <Fabrication Example 5> (Fabrication of a substrate with cell adhesion inhibitory ability using a bar coater) Using a small automatic film applicator (Allgood Co., Ltd.) and a bar coater (wet film thickness 10 μm), the coating film-forming composition 2 prepared in Preparation Example 15 was applied to a polystyrene substrate measuring 79 mm x 121 mm. The coated polystyrene substrate was dried in a 70°C oven for 24 hours and attached to a bottomless 24-well plate (C-Stec Co., Ltd.) to produce a substrate 2 with cell adhesion inhibitory ability. Sterilization was performed by irradiation with gamma rays at 25 kGy.

[0221] <Preparation Example 6> Using a small automatic film applicator (Allgood Co., Ltd.) and a bar coater (wet film thickness 10 μm), the undercoat-forming agent 5 prepared in Preparation Example 6 was applied to a polystyrene substrate measuring 79 mm x 121 mm. The coated polystyrene substrate was dried in a 60°C oven for 6 hours. The film thickness of the coating film was confirmed to be 200 Å using a spectroscopic ellipsometer on the above polystyrene substrate. The substrate was attached to a bottomless 24-well plate (C-Stec Co., Ltd.) to prepare a cell temporary adsorption substrate 4. Sterilization was performed by irradiating with gamma rays at 25 kGy.

[0222] <Preparation Example 7> Using a small automatic film applicator (Allgood Co., Ltd.) and a bar coater (wet film thickness 10 μm), the undercoat-forming agent 6 prepared in Preparation Example 7 was applied to a polystyrene substrate measuring 79 mm x 121 mm. The coated polystyrene substrate was dried in a 60°C oven for 6 hours. The film thickness of the coating film was confirmed to be 420 Å using a spectroscopic ellipsometer on the above polystyrene substrate. It was attached to a bottomless 24-well plate (C-STEC Co., Ltd.) to prepare a cell temporary adsorption substrate 5. Sterilization was performed by irradiating with gamma rays at 25 kGy.

[0223] <Preparation Example 8> Using a small automatic film applicator (Allgood Co., Ltd.) and a bar coater (wet film thickness 10 μm), the undercoat-forming agent 6 prepared in Preparation Example 7 was applied to an ethylene vinyl acetate copolymer substrate (EVA, Achilles Co., Ltd.) measuring 79 mm x 121 mm. The coated EVA substrate was dried in a 60°C oven for 6 hours and attached to a bottomless 24-well plate (C-Stec Co., Ltd.) to prepare a cell temporary adsorption substrate 6. Sterilization was performed by irradiating with gamma rays at 25 kGy.

[0224] <Preparation Example 9> Using a small automatic film applicator (Allgood Co., Ltd.) and a bar coater (wet film thickness 10 μm), the undercoat film forming agent 5 prepared in Preparation Example 6 was applied to a polyethylene substrate (Hosokawa Yoko Co., Ltd.) measuring 79 mm x 121 mm. The coated polyethylene substrate was dried in a 60°C oven for 6 hours and attached to a bottomless 24-well plate (C-Stec Co., Ltd.) to prepare a cell temporary adsorption substrate 7. Sterilization was performed by irradiating with gamma rays at 25 kGy.

[0225] <Preparation Example 10> Using a gravure printing apparatus (Nissho Gravure Co., Ltd., Gravoproof CM and 150-line plates), the undercoat-forming agent 6 prepared in Preparation Example 7 was applied to a polyethylene substrate (Hosokawa Yoko Co., Ltd.) measuring 79 mm x 121 mm. The coated polyethylene substrate was dried in a 60°C oven for 6 hours and attached to a bottomless 24-well plate (C-Stec Co., Ltd.) to prepare a cell temporary adsorption substrate 8. Sterilization was performed by irradiating with gamma rays at 25 kGy.

[0226] <Preparation Example 11> Using a spin coater, an appropriate amount of the undercoat-forming agent 7 prepared in Preparation Example 8 was dropped onto a polystyrene substrate measuring 25 mm x 25 mm and coated at a rotation speed of 1500 rpm. The coated polystyrene substrate was dried in a 60°C oven for 6 hours and attached to a bottomless 24-well plate (manufactured by CS Tech Co., Ltd.) to prepare a cell temporary adsorption substrate 9.

[0227] <Preparation Example 12> Using a small automatic film applicator (Allgood Co., Ltd.) and a bar coater (wet film thickness 10 μm), the undercoat-forming agent 13 prepared in Preparation Example 14 was applied to a polystyrene substrate measuring 79 mm x 121 mm. The applied polystyrene substrate was dried in a 60°C oven for 6 hours and then attached to a bottomless 24-well plate (C-Stec Co., Ltd.) to prepare a cell temporary adsorption substrate 10. Sterilization was performed by irradiating with gamma rays at 25 kGy.

[0228] <Preparation Example 13> Using a small automatic film applicator (Allgood Co., Ltd.) and a bar coater (wet film thickness 10 μm), the undercoat-forming agent 13 prepared in Preparation Example 14 was applied to an ethylene vinyl acetate copolymer substrate (EVA) measuring 79 mm x 121 mm (Achilles Corporation). The coated EVA substrate was dried in a 60°C oven for 6 hours and attached to a bottomless 24-well plate (C-Stec Co., Ltd.) to prepare a cell temporary adsorption substrate 11. Sterilization was performed by irradiating with gamma rays at 25 kGy.

[0229] <Preparation Example 14> Using a small automatic film applicator (Allgood Co., Ltd.) and a bar coater (wet film thickness 10 μm), the undercoat-forming agent 13 prepared in Preparation Example 14 was applied to a polyethylene substrate (Sumibe Co., Ltd., Sumilight CEL) measuring 79 mm x 121 mm. The coated polyethylene substrate was dried in a 60°C oven for 6 hours and attached to a bottomless 24-well plate (C-Stec Co., Ltd.) to prepare a cell temporary adsorption substrate 12. Sterilization was performed by irradiating with gamma rays at 25 kGy.

[0230] <Preparation Example 15> Using a small automatic film applicator (Allgood Co., Ltd.) and a bar coater (wet film thickness 10 μm), the undercoat forming agent 5 prepared in Preparation Example 6 was applied to the culture surface of the cell adhesion-inhibiting substrate 2 prepared in Preparation Example 5. The coated polystyrene substrate was dried in a 60°C oven for 6 hours and attached to a bottomless 24-well plate (C-Stec Co., Ltd.) to prepare a cell temporary adsorption substrate 13. Sterilization was performed by irradiating with gamma rays at 25 kGy.

[0231] <Preparation Example 16> Using a small automatic film applicator (Allgood Co., Ltd.) and a bar coater (wet film thickness 10 μm), the undercoat-forming agent 5 prepared in Preparation Example 6 was applied to a polyethylene substrate (Achilles Co., Ltd.) measuring 79 mm x 121 mm. The coated polyethylene substrate was dried in a 60°C oven for 6 hours and attached to a bottomless 24-well plate (C-Stec Co., Ltd.) to prepare a cell temporary adsorption substrate 14. Sterilization was performed by irradiating with gamma rays at 25 kGy.

[0232] <Preparation Example 17> Using a small automatic film applicator (Allgood Co., Ltd.) and a bar coater (wet film thickness 10 μm), the undercoat-forming agent 5 prepared in Preparation Example 6 was applied to an ethylene vinyl acetate copolymer substrate (EVA, Achilles Co., Ltd.) measuring 79 mm x 121 mm. The coated EVA substrate was dried in a 60°C oven for 6 hours. A cell temporary adsorption substrate 15 was prepared by heat sealing using a heat sealer to form a culture bag shape. Sterilization was performed by irradiating with gamma rays at 25 kGy.

[0233] <Example 18> A commercially available poly-D-lysine dish, BioCoat Poly-D-Lysine 60 mm Dish (Corning), was used as the cell temporary adsorption substrate 16.

[0234] <Comparative Fabrication Example 1> Using a small automatic film applicator (Allgood Co., Ltd.) and a bar coater (wet film thickness 10 μm), the undercoat-forming agent 8 prepared in Preparation Example 9 was applied to a polystyrene substrate measuring 79 mm x 121 mm. After application, the polystyrene substrate was dried in a 70°C oven for 24 hours and attached to a bottomless 24-well plate (C-Stec Co., Ltd.) to prepare Comparative Substrate 1. Sterilization was performed by irradiating with gamma rays at 25 kGy.

[0235] <Comparative Fabrication Example 2> Using a small automatic film applicator (Allgood Co., Ltd.) and a bar coater (wet film thickness 10 μm), the undercoat-forming agent 9 prepared in Preparation Example 10 was applied to a polystyrene substrate measuring 79 mm x 121 mm. After application, the polystyrene substrate was dried in a 70°C oven for 24 hours and attached to a bottomless 24-well plate (C-Stec Co., Ltd.) to prepare Comparative Substrate 2. Sterilization was performed by irradiating with gamma rays at 25 kGy.

[0236] <Comparative Fabrication Example 3> Using a small automatic film applicator (Allgood Co., Ltd.) and a bar coater (wet film thickness 10 μm), the undercoat forming agent 10 prepared in Preparation Example 11 was applied to a polystyrene substrate measuring 79 mm x 121 mm. After application, the polystyrene substrate was dried in a 70°C oven for 24 hours and attached to a bottomless 24-well plate (C-Stec Co., Ltd.) to prepare comparative substrate 3. Sterilization was performed by irradiating with gamma rays at 25 kGy.

[0237] <Comparative Fabrication Example 4> Using a small automatic film applicator (Allgood Co., Ltd.) and a bar coater (wet film thickness 10 μm), the undercoat forming agent 11 prepared in Preparation Example 12 was applied to a polystyrene substrate measuring 79 mm x 121 mm. The applied polystyrene substrate was dried in a 70°C oven for 24 hours and attached to a bottomless 24-well plate (C-Stec Co., Ltd.) to prepare comparative substrate 4. Sterilization was performed by irradiating with gamma rays at 25 kGy.

[0238] <Comparative Fabrication Example 5> Using a spin coater, an appropriate amount of the undercoat-forming agent 12 prepared in Preparation Example 13 was dropped onto a polystyrene substrate measuring 25 mm x 25 mm and coated at a rotation speed of 1500 rpm. The coated polystyrene substrate was dried in a 60°C oven for 6 hours and attached to a bottomless 24-well plate (manufactured by CS Tech Co., Ltd.) to prepare comparative substrate 5.

[0239] <Test Example 1> Contact angle was measured using a cell temporary adsorption substrate 4 and an untreated polystyrene substrate without coating. The contact angle of 2.0 μL of bubbles in water was measured using a contact angle meter DMCMC3C (Kyowa Interface Chemical Co., Ltd.) with a U-shaped needle. The results are shown in Table 1.

[0240]

[0241] As shown in Table 1, the untreated polystyrene substrate exhibited hydrophobicity, while the surface of the cell temporary adsorption substrate 4 showed relatively high hydrophilicity.

[0242] <Example 1> Cell adsorption and recovery test in serum medium using immortalized human T lymphocyte cell lines when the charge of the material is changed (Cell preparation) Jurkat cells (manufactured by ATCC), which are suspension cells, were used. For cell culture, a medium (hereinafter referred to as RPMI serum-containing medium) was used, which is RPMI-1640 (manufactured by Fujifilm Wako Pure Chemical Corporation) with fetal bovine serum (FBS) added at a concentration of 10% (v / v). The cells were cultured at 37°C / CO2 ２ Cells were cultured statically for at least two days in an incubator under a 5% carbon dioxide concentration using a 100 mm cell culture surface-untreated dish (10 mL of culture medium) manufactured by AGC Technoglass. After centrifuging the culture medium (Tommy Seiko Co., Ltd., model EIX-136, 300 × g / 3 min, room temperature), the supernatant was removed, and RPMI serum-containing medium was added to form a culture medium of 1.6 × 10⁶. ６ A cell suspension was prepared at cells / mL.

[0243] (Test Example 2: Suspended Cell Adsorption (Retention) Experiment) Add 500 μL of the cell suspension prepared above to cell temporary adsorption substrates 4, 5, 9, 16, comparison substrates 1-4, and an untreated polystyrene substrate, and collect 8 × 10⁶ cells per well. ５Cells were seeded to become cells. Then, at a maintained carbon dioxide concentration of 5%, the temperature was 37°C / CO2. ２ The cells were left to stand in an incubator for 3 hours. After 3 hours, the culture medium was collected from the wells (A: cells not adsorbed to the substrate), 500 μL of new culture medium was gently added through the wall of the well, and the culture medium was collected again from the well (B: cells detached from the substrate during the culture medium change). Furthermore, 500 μL of new culture medium was gently added through the wall of the well, and the cells were suspended by pipetting so that the culture medium touched the bottom of the well. After that, the culture medium was collected from the wells (C: cells that maintained adsorption to the substrate during the culture medium change and detached from the substrate by pipetting). 500 μL of new culture medium was added to the wells (D: cells that did not detach from the substrate even after pipetting). Equal volumes of CellTiter-Glo (manufactured by Promega Co., Ltd.) were added to the cell suspensions (A) to (D), and after suspension, the cells were left to stand for 10 minutes or more to elute ATP. Subsequently, 100 μL each of the ATP-eluted solution was dispensed into a 96-well luminescence measurement white plate (Sumitomo Bakelite Co., Ltd., MS-8496W), and the amount of ATP was quantified by measuring the luminescence using an Enspire (PerkinElmer). Based on this luminescence value, the percentage of viable cells adsorbed (retained) and recovered from the substrate (cell provisional adsorption rate) was calculated using the following formula: (C) / ((A) + (B) + (C) + (D)) × 100 (%). The results are shown in Table 2.

[0244] (Test Example 3: Suspended Cell Adsorption (Retention) Experiment 2) Add 500 μL of the cell suspension prepared above to the comparison substrate 5, and add 8 × 10⁶ cells per well. ５ Cells were seeded to become cells. Then, at a maintained carbon dioxide concentration of 5%, the temperature was 37°C / CO2. ２The culture medium was left to stand in an incubator for 3 hours. After 3 hours, the medium was collected from the wells, 500 μL of new medium was gently added through the wall of the well, and the medium was collected again (washing step). Furthermore, 500 μL of new medium was gently added through the wall of the well, and the medium was suspended by pipetting so that it touched the bottom of the well. After that, the medium was collected from the wells. The number of collected cells was measured using a fully automated cell counter TC20 (BioRAD), and was found to be 5.15 × 10⁶. ４ It was confirmed that the cells were cells and that the recovery rate of suspended cells relative to the number of seeds (cell provisional adsorption rate) was approximately 7%.

[0245] <Test Example 4> Surface Zeta Potential Measurement As an important parameter for measuring the attractive behavior of cells, the surface zeta potential of the substrate was measured. Measurements were performed on representative substrates: cell temporary adsorption substrates 4, 5, 9, and 16, comparison substrates 1 to 5, and an untreated polystyrene substrate. Each coated substrate was cut to a size of 4 mm x 10 mm and set in a surface potential measurement unit (Malvern). As standard monitor particles, micromerNH3+ (micromod) diluted 25 times with ultrapure water was used for substrates coated with cationic materials under room temperature and pH 7 conditions. ZTS1240 (Malvern) diluted 1000 times with ultrapure water was used for substrates coated with neutral and anionic materials. Of the above measurement targets, cell temporary adsorption substrates 4, 5, 9, and 16, and comparison substrate 5 are substrates coated with cationic materials, while comparison substrates 1 to 4 and the untreated polystyrene substrate are substrates coated with neutral and anionic materials. A surface potential measurement unit with a substrate was placed in a polystyrene measurement cell into which each monitor particle solution had been introduced, and the surface zeta potential was measured using a Zetasizer Nano (Malvern). The results are shown in Table 2.

[0246]

[0247] Table 2 shows that the comparative substrates and untreated polystyrene substrates with a cell pre-adsorption rate of less than 50% had negative or weakly cationic zeta potentials (less than 25 mV), while the cell pre-adsorption substrates with a cell pre-adsorption rate of 50% or more had surface zeta potentials of 25 mV or higher. This indicates that the surface potential of the substrate is a parameter closely related to the cell pre-adsorption rate, and further demonstrates that exhibiting cationic properties with a surface potential of 25 mV or higher is important for cell pre-adsorption.

[0248] <Test Example 5> Quantitative Test of Cell Detachment Force A test of cell detachment force was conducted using cell temporary adsorption substrates 4 and 5 and an untreated polystyrene substrate.

[0249] (Fabrication of substrate-mounted microchannels) Each substrate was attached to an open-type rectangular microchannel (channel width 0.5 mm, height 2.2 mm, length 50 mm) (manufactured by CS Tech Co., Ltd.) (this is called a substrate-mounted microwell plate).

[0250] (Cell preparation) Jurkat cells (manufactured by ATCC), which are suspension cells, were used. 3.0 × 10⁶ cells were prepared using the same method as in Example 1. ６ A cell suspension was prepared at cells / mL.

[0251] (Quantitative test of cell seeding and cell detachment force) 500 μL of the cell suspension prepared above was added to each of the substrate-mounted microwell plates prepared above. Then, under a 5% carbon dioxide concentration, the plates were incubated at 37°C for 2 hours. ２ The culture medium was left standing in an incubator. After 2 hours, a 50 mL syringe (Terumo Corporation) was filled with the culture medium and set in a syringe pump (company name). Flow rates of 0, 5, 10, 25, and 50 mL / min (equivalent to shear stresses of 0, 0.86, 1.7, 4.3, and 8.6 Pa) were applied every 10 seconds. Microscope images were taken after flowing at each flow rate to confirm the cell adsorption and desorption behavior. The shear stress was calculated by determining the wall shear stress τ in a rectangular channel n. Specifically, the following formula, which represents the wall shear stress in the flow velocity region where the Reynolds number represents laminar flow, was used. Here, C is a constant that depends on the cross-sectional shape of the channel, and in the rectangular parallelepiped microchannel with an aspect ratio of 4.4 used above, C can be approximated as 74.4. μ is the kinematic viscosity, which was approximated as 0.001 (Pa·s) for water at 20°C in the culture medium. u is the average flow velocity (m / s), and Dh is the hydraulic diameter, which was set to 0.00081 (m) for the channel with dimensions of 2.2 mm vertically and 0.5 mm horizontally. The wall shear stress (Pa) acting on the cells was calculated using this formula at each flow velocity.

[0252] As shown in Figure 4, cells detached from untreated polystyrene with a weak shear stress of 0.86 Pa. On the other hand, with cell temporary adsorption substrates 4 and 5, cells did not detach with a weak shear stress of 0.86 Pa, but most cells detached at shear stresses of 4.3 Pa and 8.6 Pa, respectively. From these results, it was shown that by using cell temporary adsorption substrates, cell detachment can be prevented up to a certain level of shear stress, and cells can be detached and recovered when a shear stress above a certain level is applied.

[0253] <Example 2: Cell provisional adsorption and recovery test in serum medium using human T cells> (Cell preparation) The cells used were human buffy coat-derived T cells (StemBioSys), which are suspension cells. RPMI serum-containing medium was used for thawing and culturing the cells. Frozen human T cells (5 × 10) ６ The cells (1 mL) were thawed in a 37°C water bath for 2 minutes. After thawing, the cells were suspended in 19 mL of the above culture medium (hereinafter referred to as "culture medium"). The suspension was centrifuged (Tommy Seiko Co., Ltd., model number EIX-136, 300 × g / 10 min, room temperature), the supernatant was removed, and the culture medium was added to form a 3.7 × 10⁶ solution. ６ A cell suspension was prepared at cells / mL.

[0254] (Test Example 6: Suspended Cell Adsorption (Retention) Experiment) Add 500 μL of the cell suspension prepared above to the wells of the plate (cell temporary adsorption substrate 5) prepared in Preparation Example 7, and add 1.85 × 10⁶ per well. ６ Cells were seeded to become cells. Then, under a 5% carbon dioxide concentration, the cells were incubated at 37°C for 3 hours. ２The culture medium was left standing in an incubator. After 3 hours, the medium was removed and 500 μL of new medium was added through the wall of the well. The medium was removed again and 500 μL of new medium was added through the wall of the well. The presence or absence of cell adsorption in the well was checked using Cell. ３ The comparison was based on observations made using iMager duos (manufactured by SCREEN Holdings Co., Ltd.).

[0255] The observation results were evaluated according to the following criteria to confirm the adsorption effect of suspended cells. [Evaluation Criteria] ○: Seeded suspended cells were observed at the bottom of the well even after the culture medium was changed (adsorption). ×: Seeded suspended cells were not observed at the bottom of the well even after the culture medium was changed.

[0256] Table 3 shows the results of the suspension cell adsorption (retention) experiment. The plate with the cell temporary adsorption substrate 5 prepared in example 7 showed adsorption of human T cells, and cells were observed at the bottom of the wells even after the culture medium was changed.

[0257]

[0258] (Test Example 7: Cell Recovery Experiment by Liquid Flow) Suspension was performed by pipetting so that the culture medium touched the bottom surface of the wells of the cell temporary adsorption substrate 5 from Test Example 6, on which suspended cells had been adsorbed. Then, the culture medium was collected from the wells, 500 μL of new culture medium was added, and the culture medium was collected again. 500 μL of new culture medium was added to each well. The presence or absence of cell adsorption in each well was checked by Cell. ３ The comparison was based on observations made using iMager duos (manufactured by SCREEN Holdings Co., Ltd.).

[0259] The observation results were evaluated according to the following criteria to confirm the effectiveness of adsorbed (retained) cell recovery by liquid flow. [Evaluation Criteria] ○: Almost no adsorbed floating cells were observed after suspension by pipetting. ×: Even after suspension by pipetting, floating cells were observed at the bottom of the well (adsorbed).

[0260] Figures 5A to 5C show the same portion of cells in the wells 3 hours after cell seeding, after medium change, pipetting, and collection. ３ The image shows the results of observations made using iMagazine Duos.

[0261] Table 4 shows the results of the cell retrieval experiment using liquid flow to remove adsorbed (retained) cells. After suspension by pipetting, almost no suspended cells that had been adsorbed (retained) were observed.

[0262]

[0263] (Test Example 8: Calculation of Adsorbed (Retained) Cell Recovery Rate by Cell Count) The number of cells recovered from the well after pipetting in Test Example 7 above was measured using a NucleoCounter NC-200 (ChemoMetec), and the result was 1.83 × 10⁻⁶. ６ It was confirmed that the cells were cells and that the cell recovery rate relative to the number of seeds was approximately 99%.

[0264] <Example 3: Cell pre-adsorption and recovery test in serum medium using immortalized human T lymphocyte cell lines> (Cell preparation) Jurkat cells (manufactured by ATCC), which are suspension cells, were used. 1.0 × 10⁶ cells were prepared using the same method as in Example 1. ６ A cell suspension was prepared at cells / mL.

[0265] (Test Example 9: Suspended Cell Adsorption (Retention) Experiment) 500 μL of the cell suspension prepared above was added to each well of the cell temporary adsorption substrate 1 prepared in Preparation Example 2, at each concentration. Then, under a 5% carbon dioxide concentration, it was incubated at 37°C for 2 hours. ２ The plates were left standing in an incubator. After 2 hours, the plates prepared above were shaken for 5-10 seconds, the culture medium was removed, and 500 μL of new culture medium was added through the walls of the wells. After shaking for 5-10 seconds, the culture medium was removed again, and 500 μL of new culture medium was added through the walls of the wells. The presence or absence of cell adsorption in each well was checked using the Cell method. ３ The comparison was based on observations made using iMager duos (manufactured by SCREEN Holdings Co., Ltd.).

[0266] The observation results were evaluated according to the following criteria to confirm the adsorption effect of suspended cells. [Evaluation Criteria] ○: Seeded suspended cells were observed on the spot even after medium change and shaking (adsorption). ×: Seeded suspended cells were not observed on the spot even after medium change and shaking. Figure 6 shows the Cell of the suspended cell adsorption experiment in Test Example 9. ３ The image shows the results of observations made using iMagazine Duos.

[0267] Table 5 shows the results of the suspension cell adsorption experiment. Adsorption of suspension cells was observed on the varnish-coated plates with base film-forming agents 1 and 2, and the cells were observed as spots even after medium changes and shaking. Adsorption (retention) of suspension cells was not observed in the uncoated samples, and the cells were not observed as spots even after medium changes and shaking.

[0268]

[0269] (Test Example 10: Cell Recovery Experiment by Liquid Flow) Suspension was performed by pipetting into each well of a varnish-coated plate with base film-forming agent 1 and base film-forming agent 2, on which suspended cells had been adsorbed, so that the culture medium touched the bottom surface of the well. Then, the culture medium was collected from the well, 500 μL of new culture medium was added, and the culture medium was collected again. 500 μL of new culture medium was added to each well. The presence or absence of cell adsorption in each well was checked by Cell. ３ The comparison was based on observations made using iMager duos (manufactured by SCREEN Holdings Co., Ltd.).

[0270] The observation results were evaluated according to the following criteria to confirm the effect of adsorbed cell recovery by liquid flow. [Evaluation Criteria] ○: After suspension by pipetting, almost no adsorbed floating cells were observed on the spot. Figure 7 shows the cells of the same spot before and after pipetting in the floating cell adsorption experiment of Test Example 10. ３ The image shows the results of observations made using iMagazine Duos.

[0271] Table 6 shows the results of the experiment to recover adsorbed cells using liquid flow. After suspension by pipetting, almost no adsorbed floating cells were observed on any of the spots.

[0272]

[0273] (Test Example 11: Cell Recovery Experiment by Cooling and Shaking) Suspended cells were adsorbed and 500 μL of medium at 4°C was added to each well of a varnish-coated plate with base film-forming agent 1 and base film-forming agent 2, via the wall of the well. Then, it was left to stand at 4°C for 25 minutes. After shaking the plate prepared above for about 5 to 10 seconds, the presence or absence of cell adsorption in each well was checked using Cell. ３ Comparisons were made based on observations using iMager duos (manufactured by SCREEN Holdings Co., Ltd.). Subsequently, the culture medium was collected from the wells and 500 μL of new culture medium was added through the well walls. After shaking for 5 to 10 seconds, the culture medium was collected again and 500 μL of new culture medium was added through the well walls.

[0274] The observation results were evaluated according to the following criteria to confirm the effect of adsorbed cell recovery. [Evaluation Criteria] ○: After cooling and shaking, the adsorbed floating cells detached from the spot. Figure 8 shows the same spot before and after cooling and shaking in the floating cell adsorption experiment of Test Example 11. ３ The image shows the results of observations made using iMagazine Duos.

[0275] Table 7 shows the results of the cell recovery experiment using cooling and shaking. When culture medium at 4°C was added, the mixture was left to stand at 4°C for 25 minutes and then shaken, the adsorbed suspension cells were partially no longer observed on the spots.

[0276]

[0277] (Test Example 12: Calculation of Adsorbed Cell Recovery Rate by ATPassay) After pipetting and cooling in Test Examples 10-11 above, the culture medium collected from the wells was centrifuged (Tommy Seiko Co., Ltd., model MX-307, 300 x g / 3 min, room temperature), the supernatant was removed, and 500 μL of fresh culture medium was added and the cells were suspended. 500 μL of CellTiter-Glo (Promega Co., Ltd.) was added to this cell suspension, and after suspension, it was allowed to stand for 10 minutes or more to elute ATP (A). Similarly, 500 μL of CellTiter-Glo (Promega Co., Ltd.) was added to the 500 μL of culture medium remaining in the wells, and after suspension, it was allowed to stand for 10 minutes or more to elute ATP (B). Subsequently, 100 μL each of solutions (A) and (B) were dispensed into a 96-well luminescence measurement white plate (Sumitomo Bakelite Co., Ltd., MS-8496W), and the amount of ATP was quantified by measuring the luminescence using an Enspire (PerkinElmer). Based on these luminescence values, the recovery rate of suspension cells in test examples 10-11 was calculated using the following formula, and it was confirmed that the recovery rate in all cases was 50% or higher: (A) / ((A) + (B)) × 100 (%)

[0278] <Example 4: Cell aspiration test using human erythroleukemia cells in serum medium>

[0279] (Cell Preparation) Human erythroleukemia cell line (K562: ATCC Corporation) was used as the cell culture medium. Bovine fetal serum (FBS) was added to RPMI-1640 (Fujifilm Wako Pure Chemical Corporation) at a concentration of 10% (v / v) for cell culture. The cells were cultured at 37°C / CO2. ２ Cells were cultured statically for at least two days in a Corning 100 mm cell culture surface-treated dish (10 mL of culture medium) while maintaining a 5% carbon dioxide concentration in an incubator. The cell suspension was collected and centrifuged (Tommy Seiko Co., Ltd., model EIX-136, 200 × g / 3 min, room temperature). The supernatant was removed, and the above culture medium was added to form a 1.0 × 10⁶ cell suspension. ６ A cell suspension was prepared at cells / mL.

[0280] (Cell seeding, medium change, observation) 0.35 mL of the cell suspension prepared above was added to the cell temporary adsorption substrate 2 prepared in Preparation Example 3. Then, at a maintained carbon dioxide concentration of 5%, the substrate was heated at 37°C / CO2. ２The culture medium was left standing in an incubator. After 2 hours, the entire volume of the medium was aspirated, washed by adding 1 mL of medium and aspirating again, and then 1 mL of medium was added again. After that, Cell ３ Observation was performed using iMager duos (manufactured by SCREEN Holdings Co., Ltd.) to evaluate the presence or absence of cell adsorption. The results are shown in Figure 9.

[0281] Before the culture medium was changed, many cells were concentrated on the underlayer forming agent on the temporary adsorption substrate. After the culture medium was changed, no cells were present on the cell adhesion inhibition pattern, and cells were adsorbed only on the underlayer forming agent. These results demonstrate that cells can be adsorbed onto the temporary adsorption substrate in serum culture medium.

[0282] <Example 5: Cell aspiration test in serum-free medium using human erythroleukemia cells> (Cell preparation) The same method was used as in Example 4, except that RPMI-1640 was used as the culture medium after cell detachment, to prepare 1.0 × 10⁶ cells. ６ A cell suspension was prepared at cells / mL.

[0283] (Cell seeding, medium exchange, observation) The cell temporary adsorption substrate 2 prepared in Example 3 was cultured and evaluated in the same manner as in Example 4. The results are shown in Figure 10.

[0284] Similar to Example 4, it was shown that before the medium change, many cells were accumulated on the underlayer forming agent on the temporary adsorption substrate, and after the medium change, no cells were present on the cell adhesion inhibition pattern, and cells were adsorbed only on the underlayer forming agent. From the above, it was shown that cells can be adsorbed onto the temporary adsorption substrate even in serum-free medium.

[0285] <Example 6: Cell aspiration test using human erythroleukemia cells in serum-free medium>

[0286] (Cell preparation) 1.0 × 10⁶ cells were prepared using the same method as in Example 5. ６ A cell suspension was prepared at cells / mL.

[0287] (Cell seeding, medium change, observation) 0.35 mL of the cell suspension prepared above was added to the cell temporary adsorption substrate 3 prepared in Example 4. Then, culture was carried out in the same manner as in Example 4, and observation was performed after the medium change. The results are shown in Figure 11.

[0288] After the culture medium was changed, it was shown that cells were adsorbed only on the underlying film-forming agent. Therefore, it was demonstrated that even when the material for the cell adhesion inhibition pattern was changed in serum culture medium, cells could still be adsorbed onto the temporary adsorption substrate.

[0289] <Example 7: Evaluation of the time required for temporary cell adsorption (retention) to the substrate by a cell temporary adsorption and recovery test in serum medium using an immortalized human T lymphocyte cell line> (Cell preparation) The cells used were Jurkat cells (manufactured by ATCC), which are suspension cells. 1.6 × 10⁶ cells were prepared using the same method as in Example 1. ６ A cell suspension was prepared at cells / mL.

[0290] (Test Example 13: Suspended Cell Adsorption (Retention) Experiment) Add 500 μL of the cell suspension prepared above to the wells of the plate (cell temporary adsorption substrate 5) prepared in Preparation Example 7, and add 8 × 10 units per well. ５ Cells were seeded to become cells. Then, at a maintained carbon dioxide concentration of 5%, the temperature was 37°C / CO2. ２ After standing in the incubator for 0, 1, 2, or 3 hours, the culture medium was collected from the wells (A: cells not adsorbed to the substrate), 500 μL of new culture medium was gently added through the well wall, and the culture medium was collected again from the wells (B: cells detached from the substrate during the culture medium change). Furthermore, 500 μL of new culture medium was gently added through the well wall. The presence or absence of cell adsorption in the wells was compared based on observation using a microscope (Olympus Corporation).

[0291] The observation results were evaluated using the same evaluation criteria as in Test Example 6 of Example 2, and the adsorption effect on suspended cells was confirmed.

[0292] (Test Example 14: Calculation of the percentage of viable cells adsorbed (retained) and recovered on the substrate using ATPassay) Suspension was performed by pipetting into the wells of the cell temporary adsorption substrate 5 plate from Test Example 13, where the suspended cells were adsorbed, so that the culture medium touched the bottom of the wells. Then, the culture medium was collected from the wells, 500 μL of new culture medium was added, and the culture medium was collected again (C: Cells that maintained adsorption to the substrate during the culture medium exchange and detached from the substrate by pipetting). 500 μL of new culture medium was added to the wells (D: Cells that did not detach from the substrate even after pipetting).

[0293] Equal volumes of CellTiter-Glo (Promega Co., Ltd.) were added to cell suspensions (A) to (D), and after suspension, the cells were allowed to stand for at least 10 minutes to elute ATP. Subsequently, 100 μL of each ATP-eluted solution was dispensed into a 96-well luminescence measurement white plate (Sumitomo Bakelite Co., Ltd., MS-8496W), and the amount of ATP was quantified by measuring the luminescence using an Enspire (PerkinElmer). Based on these luminescence values, the percentage of viable cells adsorbed (retained) and recovered from the substrate was calculated using the following formula. Note that (C) has been washed and is now 1000 μL, so this was reflected in the formula below. ((C) × 2) / ((A) + (B) + (C) + (D)) × 100 (%)

[0294] (Test Example 15: Measurement of the viability of cells adsorbed (retained) and recovered from the substrate film) The viability of cells contained in the suspension of Test Example 14 (C) was measured using a cell counter TC-20 (manufactured by BIO-RAD).

[0295] Table 8 shows the evaluation results of the suspension cell adsorption (retention) experiment in Test Example 13, the percentage of viable cells adsorbed (retained) and recovered on the substrate in Test Example 14, and the viability of cells adsorbed (retained) and recovered on the substrate as measured in Test Example 15. In plates left standing for 0 hours after cell seeding, no adsorption of suspension cells was observed, and no cells were observed at the bottom of the wells after medium exchange. On the other hand, in plates left standing for 1 hour, 2 hours, or 3 hours, adsorption of suspension cells was observed in all cases, and cells were observed at the bottom of the wells even after medium exchange. Furthermore, the percentage of viable cells adsorbed (retained) and recovered on the substrate was 90% or more, and the viability of those cells was also 90% or more. From the above, it was shown that the cell temporary adsorption substrate 5 plate prepared in Preparation Example 7 can exhibit a suspension cell adsorption (retention) effect at least 1 hour after cell seeding.

[0296]

[0297] <Example 8: Verification of the effect of low-adhesion substrate material by cell temporary adsorption and recovery test in serum medium using human T cells> (Cell preparation) The cells used were suspension cells derived from human buffy coat (StemBioSys). 8 × 10⁻⁶ cells were prepared using the same method as in Example 2. ５ A cell suspension was prepared at cells / mL.

[0298] (Test Example 16: Suspended Cell Adsorption (Retention) Experiment) Add 500 μL of the cell suspension prepared above to the wells of the plates (cell temporary adsorption substrates 4 and 13) prepared in Preparation Examples 6 and 15, and add 4 × 10 units per well. ５ Cells were seeded to become cells. Then, at a maintained carbon dioxide concentration of 5%, the temperature was 37°C / CO2. ２ The cells were left undisturbed in an incubator for 21 hours. After 21 hours, the cells were left undisturbed. ３ After observation with iMager duos (manufactured by SCREEN Holdings Co., Ltd.), the culture medium was removed and 500 μL of new medium was gently added through the well wall. The culture medium was removed again and 500 μL of new medium was gently added through the well wall. The presence or absence of cell adsorption in the well was again checked using Cell ３The comparison was based on observations made using iMager duos.

[0299] The observation results were evaluated using the same evaluation criteria as in Test Example 6 of Example 2, and the adsorption effect on suspended cells was confirmed.

[0300] (Test Example 17: Cell Recovery Experiment by Liquid Flow) Suspension was performed by pipetting into the wells of the cell temporary adsorption substrates 4 and 13 plates from Test Example 16, on which suspended cells had been adsorbed, so that the culture medium touched the bottom of the wells. Then, the culture medium was collected from the wells, 500 μL of new culture medium was added, and the culture medium was collected again. 500 μL of new culture medium was added to each well. The presence or absence of cell adsorption in each well was checked by Cell. ３ The comparison was based on observations made using iMager duos (manufactured by SCREEN Holdings Co., Ltd.).

[0301] The observation results were evaluated using the same evaluation criteria as in Test Example 7 of Example 2, and the effect of retrieving adsorbed (retained) cells by liquid flow was confirmed.

[0302] Figure 12 shows the same portion of cells in the wells of test examples 16-17, 21 hours after cell seeding, after medium change, pipetting, and collection. ３ The image shows the results of observations made using iMagazine Duos.

[0303] (Test Example 18: Calculation of the percentage of viable cells adsorbed (retained) and recovered from the substrate film, and measurement of cell viability) The number of viable cells and cell viability recovered from the wells after pipetting in Test Example 17 above were measured using a Nucleo Counter NC-200 (ChemoMetec). Based on the value of the number of viable cells (*), the percentage of viable cells adsorbed (retained) and recovered from the substrate film was calculated using the following formula: (*) / 4 × 10 ５ ) × 100 (%)

[0304] Table 9 shows the evaluation results of the suspension cell adsorption (retention) experiment in Test Example 16, the evaluation results of the adsorbed cell recovery experiment by liquid flow in Test Example 17, and the calculation of the percentage of viable cells adsorbed (retained) and recovered on the base film in Test Example 18, as well as the results of viability measurement. The plates of cell temporary adsorption substrates 4 and 13 prepared in Preparation Examples 6 and 15 showed adsorption of human T cells, and cells were observed at the bottom of the wells even after medium exchange. Furthermore, after suspension by pipetting, almost no adsorbed (retained) cells were observed. Moreover, the percentage of viable cells adsorbed (retained) and recovered on the base film was 75% or more, and the viability of those cells was 90% or more. This demonstrates that the base film of the present invention can exhibit a suspension cell adsorption (retention) effect even with human T cells, regardless of whether or not a low-adhesion material is coated on the underlying layer.

[0305]

[0306] <Example 9: Evaluation of the versatility of a base coat coating substrate by cell provisional adsorption and recovery test in serum medium using human T cells> (Cell preparation) The cells used were human buffy coat-derived T cells (StemBioSys) activated with CD3 / CD28 antibody (STEMCELL Technologies, #10971) and cultured on a large scale. For expansion culture, ImmunoCult-XF T Cell Expansion Medium (STEMCELL Technologies, #ST-10981) was supplemented with Human Recombinant IL-2 (hereinafter, rhIL-2) (STEMCELL Technologies, #78036.3) at a concentration of 10 ng / mL (hereinafter, XF serum-free medium). After activation, the cells were kept at 37°C / CO2. ２ The cultures were incubated in an incubator at a 5% carbon dioxide concentration for at least 3 days. After centrifugation of the culture medium (300 × g / 10 min, room temperature), the supernatant was removed, and RPMI serum-containing medium was added, resulting in a 2 × 10⁶ culture. ６ A cell suspension was prepared at cells / mL.

[0307] (Test Example 19: Calculation of the percentage of live cells adsorbed (retained) and recovered on the substrate using ATPassay) Add 500 μL of the cell suspension prepared above to the wells of the plates (cell temporary adsorption substrates 10-12) prepared in Preparation Examples 12-14, and calculate 1 × 10⁶ cells per well. ６ Cells were seeded to become cells. Then, at 37°C, CO2 was released while maintaining a 5% carbon dioxide concentration. ２ After standing in the incubator for 3 hours, the percentage of live cells adsorbed (retained) and recovered from the substrate was calculated in the same manner as in Test Examples 13 and 14 of Example 7.

[0308] (Test Example 20: Measurement of cell viability after adsorption (retention) and recovery from the substrate film) The viability of cells contained in the suspension from Test Example 19 (cells that maintained adsorption to the substrate film during culture medium exchange and were detached from the substrate film by pipetting) was measured using a Nucleo Counter NC-200 (ChemoMetec).

[0309] Table 10 shows the percentage of viable cells adsorbed (retained) and recovered from the substrate film calculated in Test Example 19, and the viability of cells adsorbed (retained) and recovered from the substrate film measured in Test Example 20. In all of the cell temporary adsorption substrates 10 to 12 prepared in Preparation Examples 12 to 14, more than 75% of the cells were adsorbed (retained) and recovered from the substrate film, and the viability of those cells was more than 90%. From the above, it has been shown that the substrate film of the present invention can be applied to various substrates and exhibit a floating cell adsorption (retention) effect.

[0310]

[0311] <Example 10: Cell provisional adsorption and recovery test in serum-free medium using human T cells> (Cell preparation) As in Example 9, human T cells were activated with CD3 / CD28 antibody and cultured in XF serum-free medium. After centrifuging the cell culture medium (300 × g / 10 min, room temperature), the supernatant was removed and XF serum-free medium was added to form 2 × 10⁶ cells. ６ A cell suspension was prepared at cells / mL.

[0312] 500 μL of the cell suspension prepared above was added to the wells of the plate (cell temporary adsorption substrate 10) prepared in Preparation Example 12. The percentage of viable cells adsorbed (retained) and recovered from the substrate was calculated using ATPassay, and the viability of the cells adsorbed (retained) and recovered from the substrate was measured, similar to Test Examples 19 and 20 of Example 9. The results are shown in Table 11. Even when using serum-free medium, more than 75% of the cells could be adsorbed (retained) and recovered from the substrate, and their cell viability was more than 90%. Therefore, it was demonstrated that the substrate of the present invention can exhibit a suspension cell adsorption (retention) effect even when using serum-free medium.

[0313]

[0314] <Example 11: Evaluation of the properties of human T cells collected after adsorption (retention) to a substrate film> (Cell preparation) The cells used were suspension cells derived from human buffy coat (StemBioSys). Similar to the cell preparation in Example 2, thawing, centrifugation, and supernatant removal were performed, and RPMI serum-containing medium was added to form 1.6 × 10⁶ cells. ６ A cell suspension was prepared at cells / mL.

[0315] (Adsorption (retention) and retrieval of human T cells to the substrate) Add 500 μL of the cell suspension prepared above to the wells of the plates (cell temporary adsorption substrates 5 and 6) prepared in examples 7 and 8, and collect 8 × 10⁶ cells per well. ５ Cells were seeded to become cells, and the temperature was maintained at 37°C / CO2 while keeping the carbon dioxide concentration at 5%. ２ The cells were left standing in an incubator for 22 hours. The same procedure as in Test Examples 13 and 14 of Example 7 was performed, and the cells that were adsorbed (retained) on the substrate were collected (cells that maintained adsorption to the substrate during medium exchange and were detached from the substrate by pipetting). For comparison, 500 μL of the cell suspension prepared above was added to the wells of an untreated plate (AGC Technoglass Co., Ltd., #1820-024-MYP), and the plate was incubated at 37°C / CO2. ２ The cells were left to stand in an incubator for 22 hours. After that, the entire volume was collected and the cells were recovered as cells that had not undergone adsorption to the substrate membrane.

[0316] (Test Example 21: Cell Proliferation Test) - Activation of T cells (Day 0 to Day 3) After centrifuging (300 x g / 10 min, room temperature) the cells recovered from the wells of each plate as above and removing the supernatant, a medium supplemented with CD3 / CD28 antibody (25 μL / mL) in XF serum-free medium was added to prepare a cell suspension with a viable cell number concentration of 1 x 10 ６ cells / mL. The cell suspension was seeded onto an untreated plate (manufactured by AGC Techno Glass Co., Ltd., #1820-024-MYP), and left standing in an incubator at 37°C / CO ２ for 3 days while maintaining a 5% carbon dioxide concentration to activate the cells.

[0317] - Expansion culture (Day 3 to Day 10) The activated T cells were recovered, and XF serum-free medium was added to prepare a cell suspension with a viable cell number concentration of 1 x 10 ５ cells / mL. The cell suspension was seeded onto an untreated plate and left standing in an incubator at 37°C / CO ２ for 4 days. At the time of seeding and 4 days later, an equal volume of CellTiter-Glo (manufactured by Promega Corporation) was added to the cell suspension to elute ATP. The ATP eluate was dispensed into a 96-well white luminescence measurement plate, and the amount of ATP was quantified by measuring luminescence with Enspire (ATP assay). Based on this luminescence value, the cell proliferation rate for the 4 days from Day 3 to Day 7 was calculated. The T cells 4 days after the above expansion culture were recovered, and the cell seeding and ATP assay were performed in the same manner to calculate the cell proliferation rate for the 3 days from Day 7 to Day 10.

[0318] The calculation results of the cell proliferation rate in the expansion culture (Day 3 to Day 10) of Test Example 21 are shown in Fig. 13. It was shown that there was almost no difference in the proliferative ability of the cells between those that had not undergone adsorption to the basement membrane and those that had been adsorbed (retained) on the basement membrane and then recovered.

[0319] (Test Example 22: Population Evaluation by Flow Cytometry) The T cells expanded in the above Test Example 21 were collected on Day 10. After centrifugation, the supernatant was removed and replaced with a solution prepared by adding fetal bovine serum (FBS) at a concentration of 2% (v / v) to PBS (manufactured by FUJIFILM Wako Pure Chemical Corporation). CD3 staining (manufactured by BD, #555339), CD4 staining (manufactured by BD, #566913), and CD8 staining (manufactured by BD, #555367) were performed, and CD3-positive cells (i.e., T cells) were analyzed by measuring the fluorescence intensity using flow cytometry (BD FACS Fortessa X20). For only CD3-positive cells, the expression of CD4 and CD8 was analyzed. Then, the ratios of CD4-positive / CD8-negative cells (i.e., helper T cells) and CD4-negative / CD8-positive cells (i.e., killer T cells) were calculated, and the populations of each cell were compared.

[0320] The detection results of CD4 and CD8 positive cells by flow cytometry are shown in Fig. 14. It was shown that there was almost no difference in the population (CD4 / CD8 ratio) during cell growth between the cells that had not undergone adsorption to the basement membrane and the cells recovered after adsorption (retention) to the basement membrane.

[0321] <Example 12: Cell Pretention and Recovery Test in Serum Medium Using an Immortalized Strain of Human T Lymphocyte Cells in a Cell Culture Bag> (Cell Preparation) As the cells, Jurkat cells (manufactured by ATCC), which are floating cells, were used. Similar to the cell preparation in Example 1, culturing, centrifugation, and removal of the supernatant were performed, and RPMI serum-containing medium was added to prepare a cell suspension of 1.54×10 ６ cells / mL.

[0322] (Adsorption (Retention) and Recovery of Human T Cells to the Basement Membrane) To the cell culture bag prepared in Production Example 17, 32 mL of the cell suspension prepared above was added, and the cells were seeded to obtain 4.93×10 ７ cells per bag. With the side of the cell pretention substrate 15 facing down, while maintaining a carbon dioxide concentration of 5%, at 37°C / CO ２The bag was left standing in an incubator for 3 hours. After that, 17 mL of culture medium was collected from the bag (a: cells not adsorbed to the substrate), 17 mL of new culture medium was gently added, and another 17 mL of culture medium was collected from the bag (b: cells detached from the substrate during the culture medium change). The bag with the adsorbed suspension cells was kneaded by hand for 1 minute, and then 30 mL of culture medium was collected from the bag (c: cells that maintained adsorption to the substrate during the culture medium change and detached from the substrate by kneading the bag by hand).

[0323] The number of viable cells and their viability in culture media (a) to (c) were measured using a cell counter TC-20 (manufactured by BIO-RAD). The ratio of viable cells in each culture medium to the number of seeded cells was calculated using the following formula: ((a) or (b) or (c) / 4.93 × 10⁻¹⁰ ７ ) × 100 (%) The results are shown in Table 12. The percentage of viable cells adsorbed (retained) and recovered on the substrate was over 90%, and the viability of those cells was also over 90%. From the above, it was shown that the cell culture bag prepared in example 17 can exhibit a suspension cell adsorption (retention) effect, and that the adsorbed cells can be detached by physical stimulation.

[0324]

[0325] <Example 13: Cell provisional adsorption and recovery test in serum medium using immortalized human T lymphocyte cell lines depending on differences in the application method of the base material type and base film forming agent> (Cell preparation) Jurkat cells (manufactured by ATCC), which are suspension cells, were used. In the same manner as in Example 1, culture, centrifugation, and supernatant removal were performed, and RPMI serum-containing medium was added to form 1.6 × 10⁶ cells. ６ A cell suspension was prepared at cells / mL.

[0326] (Test Example 23: Suspended Cell Adsorption (Retention) Experiment) Add 500 μL of the cell suspension prepared above to the wells of the plates (cell temporary adsorption substrates 6, 7, 14) prepared in Preparation Examples 8, 9, and 16, and add 8 × 10 units per well. ５ Cells were seeded to become cells. Then, at a maintained carbon dioxide concentration of 5%, the temperature was 37°C / CO2. ２After standing in an incubator for 3 hours, the culture medium was collected from the wells (A: cells not adsorbed to the substrate), 500 μL of new culture medium was gently added through the well wall, and the culture medium was collected again from the wells (B: cells detached from the substrate during the culture medium change). Furthermore, 500 μL of new culture medium was gently added through the well wall. The presence or absence of cell adsorption in the wells was compared based on observation using a microscope (manufactured by Olympus Corporation).

[0327] The observation results were evaluated using the same evaluation criteria as in Test Example 6 of Example 2, and the adsorption effect on suspended cells was confirmed.

[0328] (Test Example 24: Calculation of the percentage of live cells adsorbed (retained) and recovered on the substrate by cell counting) Suspension was performed by pipetting on the wells of plates 6, 7, and 14 of the cell temporary adsorption substrate from Test Example 23, to which suspended cells had adsorbed, so that the culture medium touched the bottom of the wells. Then, the culture medium was collected from the wells, 500 μL of new culture medium was added, and the culture medium was collected again (C: Cells that maintained adsorption to the substrate during the culture medium exchange and detached from the substrate by pipetting). The number of cells in the cell suspension recovered by pipetting in (C) was measured using a fully automated cell counter TC20 (BioRAD), and the recovery rate relative to the seeding number was calculated. The results are shown in Table 13.

[0329] (Test Example 25: Measurement of the viability of cells adsorbed (retained) and recovered from the substrate film) The viability of cells contained in the suspension of Test Example 24 (C) was measured using a cell counter TC-20 (manufactured by BIO-RAD).

[0330] Table 13 shows the evaluation results of the suspension cell adsorption (retention) experiment in Test Example 23, the percentage of viable cells adsorbed (retained) and recovered on the substrate film calculated in Test Example 24, and the viability of cells adsorbed (retained) and recovered on the substrate film measured in Test Example 25. Regardless of the type of substrate material or the application method of the substrate film-forming agent, adsorption of suspension cells was observed in all cases, and cells were observed at the bottom of the wells even after the culture medium was changed. Furthermore, the percentage of viable cells adsorbed (retained) and recovered on the substrate film was 85% or more.

[0331]

[0332] The present invention allows suspension cells to be held in a cell holder. Therefore, in a closed-system cell manufacturing apparatus, by connecting a closed-system container having a cell holder, observation and monitoring of suspension cells becomes possible. Furthermore, when changing the culture medium for suspension cells, the culture medium can be easily changed by holding the suspension cells in the cell holder without centrifugation. By using suspension cells held in the cell holder, it is easier to perform treatments such as gene modification and drug infiltration compared to treating them in a suspension state, resulting in more uniform treatment.

[0333] Furthermore, this invention allows for the recovery of suspended cells held in the cell holder. This enables the recovery of suspended cells that have been observed and quality-evaluated, thereby improving the yield of cell culture. When the cell holder is installed in a flow channel of a closed-system cell manufacturing apparatus, it becomes possible to reuse the suspended cells after they have been held in place. Additionally, since suspended cells can be recovered without enzymatic treatment, damage to adherent cells during recovery is easily reduced.

[0334] 1 Substrate 1A Coating film 2 Cell adhesive substance 3 Cell retention base film 100 Cell holder

Claims

1. A cell retainer comprising a substrate and a cell-retaining underlayer film disposed on the surface of the substrate, wherein the cell-retaining underlayer film contains a polymer, and is used to retain suspended cells on the cell-retaining underlayer film.

2. The cell retainer according to claim 1, wherein the surface of the cell-retaining underlayer has an opposite charge to that of the suspended cells.

3. The cell retainer according to claim 1, wherein the zeta potential of the surface of the cell retaining underlayer is +25 mV to +80 mV.

4. The cell holder according to claim 2, wherein the suspended cells can be held in place on the cell-holding base membrane by electrical attraction, and the suspended cells can be detached from the cell-holding base membrane when a force greater than the attractive force is applied to the suspended cells.

5. The cell holder according to claim 4, further comprising suspended cells held in a cell-holding base membrane, wherein the suspended cells are held in place without detaching from the cell-holding base membrane when the shear stress on the suspended cells is 0.8 Pa or less.

6. The cell holder according to claim 4, further comprising suspended cells held in a cell-holding base membrane, wherein the suspended cells detach from the cell-holding base membrane when the shear stress on the suspended cells is 15 Pa or less.

7. The cell holder according to claim 6, wherein when the shear stress on the suspended cells is 0.8 Pa or less, the suspended cells are held without detaching from the cell-holding substrate.

8. The cell retainer according to claim 1, wherein the polymer contains at least one of a polymer (A) containing a repeating unit represented by the following formula (a-1), a polymer (B) containing a repeating unit represented by the following formula (b-1), and a polymer (C) containing a repeating unit represented by the following formula (c-1). (In formula (a-1), R ａ１ represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, and Z ａ represents the following formula (Z1), the following formula (Z2), or the following formula (Z3). In formula (b-1), X ｂ represents a linear alkylene group having 1 to 3 carbon atoms, and Y ｂ represents a guanidino group or an imidazolyl group.) (In formula (Z1), Z ｂ represents the following formula (Ia) or the following formula (Ia-2). In formula (Z2), R ａ２１ represents a linear or branched alkyl group having 1 to 5 carbon atoms. In formula (Z3), R ａ３１ and R ａ３２ each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms. In formulas (Z1) to (Z3), * represents a bond.) (In formula (Ia) and formula (Ia-2), R ａ２ represents a linear or branched alkylene group having 1 to 5 carbon atoms, U ａ１ and U ａ２ each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, U ａ３ represents a hydrogen atom, a linear or branched alkyl group having 1 to 5 carbon atoms, or an aralkyl group, X － represents a salt-forming anion, and * represents a bond.) 9. The cell retainer according to claim 8, wherein the polymer (A) includes a crosslinked structure.

10. The cell retainer according to claim 8, wherein the polymer (A) comprises at least one of the following structures: the structure represented by formula (IIIa), the structure represented by formula (IVa), and the structure represented by formula (Va). (In the formula, R ｃ and R ｄ Each of these independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, and R ｅ (where n represents a linear or branched alkylene group with 1 to 5 carbon atoms, and n represents a number from 1 to 50.) 11. The cell holder according to claim 1, wherein the surface of the substrate has a coating film having the ability to inhibit cell adhesion, and the cell-holding underlayer film is disposed on the coating film.

12. The cell retainer according to claim 1, comprising a cell adhesion substance disposed on the cell retaining base film.

13. A method for holding suspended cells in the cell-holding base membrane of a cell holder according to any one of claims 1 to 12.

14. The method for holding suspended cells according to claim 13, wherein the cell holder holding the suspended cells is detached from the cell holder by applying a shear stress of 0.8 Pa to 15 Pa.

15. A method for recovering suspended cells as described in claim 13, comprising recovering the suspended cells from the cell-retaining substrate.

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