JP2018039949A - Stimulus-responsive polymer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a stimulus-responsive polymer which enables production of a culture vessel by a simple method, the culture vessel allowing non-invasive cell peeling.SOLUTION: The invention is related to: a stimulus-responsive polymer which has, as constitutional units, a site X exhibiting temperature-responsiveness and a site Y exhibiting responsiveness to stimulation other than temperature change, in which the site X exhibiting temperature-responsiveness has an ether partial structure or a sulfide partial structure, and the site Y exhibiting responsiveness to stimulation other than temperature change includes a functional group having a pKa of 2 to 12 in water; as well as a culture vessel using the stimulus-responsive polymer; a culture method; and a cell peeling method.SELECTED DRAWING: None

Description

  The present invention relates to a stimulus-responsive polymer and a cell culture container, a cell culture method and a cell detachment method using the same.

  In cell culture, in the step of detaching cells from the cell culture container, a proteolytic enzyme is often used as a detachment solution. However, proteolytic enzymes degrade cell membrane proteins and intercellular adhesion proteins, thus damaging cells.

  Thus, as a technique for non-invasively peeling cells from a cell culture container, a culture container having a stimulus-responsive polymer on its surface has been developed. Non-invasive stimuli include slight temperature changes, slight pH changes, slight salt concentration changes, the addition of some biocompatible substances such as sugars, peptides and weak reducing agents, and light irradiation. In particular, research on the temperature change is progressing, and a temperature-responsive culture vessel using a temperature-responsive polymer is known (Patent Documents 1-4 and Non-Patent Document 1). In such a temperature-responsive culture vessel, cells can be detached by lowering the temperature from the culture temperature.

  However, a conventional temperature-responsive culture vessel needs to immobilize a temperature-responsive polymer such as a poly (N-isopropylacrylamide) (PNIPAM) derivative at a high density on the surface. It could only be manufactured with a low technique.

  Therefore, there is a demand for a temperature-responsive polymer that can sufficiently act on cell detachment even at a low surface density, which enables production of a temperature-responsive culture vessel by a simple technique such as immersion or coating. In addition, a stimulus-responsive polymer that is responsive to a combined stimulus of a temperature change and a non-invasive stimulus other than a temperature change can improve cell detachability.

JP2008-220320 JP 2008-263863 A JP 2010-63439 A JP2012-165730

Polymers 2012, 4, 1478-1498

  As described above, in a conventional temperature-responsive culture vessel, since the temperature-responsive polymer cannot sufficiently act on cell detachment at a low surface density, it must be introduced into the culture vessel at a high density. It was difficult to introduce the polymer into the culture vessel by a simple technique. In addition, a stimulus-responsive polymer that is responsive to temperature changes and stimuli other than temperature changes enables noninvasive cell detachment and can improve cell detachability. Therefore, an object of the present invention is to provide a stimulus-responsive polymer that enables production of a culture vessel capable of non-invasive cell detachment by a simple technique.

  In order to solve the above-mentioned problems, the stimulus-responsive polymer of the present invention has, as structural units, a site X showing temperature responsiveness and a site Y showing responsiveness to stimuli other than temperature changes. The site X exhibiting sex has an ether partial structure or a sulfide partial structure, and the site Y exhibiting responsiveness to stimuli other than temperature changes includes a functional group having a pKa in water of 2 to 12 To do.

  According to the present invention, a culture container capable of non-invasive cell detachment can be produced by a simple technique using a stimulus-responsive polymer.

  Problems, configurations, and effects other than those described above will become apparent from the following description of embodiments.

FIG. 1 shows the temperature responsiveness of polymer 2-2 showing the lower critical solution temperature in the examples. FIG. 2 shows the molecular weight distribution of polymer 2-2 in the examples. FIG. 3 is a photograph showing the progress when the cells were detached in the examples.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. The following description shows specific examples of the contents of the present invention, and the present invention is not limited to these descriptions. Various modifications by those skilled in the art are within the scope of the technical idea disclosed in this specification. Changes and modifications are possible. In all the drawings for explaining the present invention, components having the same function are denoted by the same reference numerals, and repeated description thereof may be omitted.

  The stimulus-responsive polymer of the present invention includes a site X showing temperature responsiveness and a site Y showing responsiveness to stimuli other than temperature changes as structural units. The cell culture container provided with the stimulus-responsive polymer of the present invention on the surface changes the adhesiveness to the surface of the cultured cells by changing the surface state due to temperature changes and stimuli other than temperature changes, and promotes cell detachment. be able to. In particular, when the stimulus-responsive polymer is hydrophobic at the culture temperature and changes to hydrophilicity by the stimulus, the cells generally tend to adhere to the hydrophobic surface, which is advantageous for cell detachment. The stimulus-responsive polymer of the present invention is responsive to both temperature and stimuli other than temperature, and can sufficiently act on cell detachment even at a low surface density. Has cell detachability.

  The site X exhibiting temperature responsiveness is a site that can change properties or structure such as solubility (hydrophilicity or hydrophobicity) of the polymer in accordance with temperature change. For example, if site X changes the solubility of the polymer in response to temperature changes, the polymer may exhibit a lower critical solution temperature and / or an upper critical solution temperature. When the lower critical solution temperature is indicated, the polymer is soluble below the critical temperature, is insoluble above the critical temperature, and when the upper critical solution temperature is indicated, the polymer may be at or above the critical temperature. It is soluble and insoluble below the critical temperature. Here, since the phenomenon essentially required for cell detachment is a change in the surface state felt by the cells, the site X may indicate the lower critical solution temperature and / or the upper critical solution temperature. It does not have to be. This is because the lower critical solution temperature and upper critical solution temperature in a polymer are bulk properties and do not necessarily correlate with the molecular state of the material immobilized on the surface of the culture vessel (changes in structure, mobility, physical properties, etc.). is there. In addition, since the medium contains various organic substances and salts, a compound that does not exhibit the lower critical solution temperature or the upper critical solution temperature in water does not necessarily exhibit the lower critical solution temperature or the upper critical solution temperature in the medium. However, generally, showing the lower critical solution temperature and the upper critical solution temperature in an aqueous solution can be a reference for the solubility of the polymer to be temperature responsive. In one embodiment, the stimulus-responsive polymer of the present invention can exhibit a lower critical solution temperature and / or an upper critical solution temperature in an aqueous solution, and preferably can exhibit a lower critical solution temperature in an aqueous solution. Moreover, when the site | part X isomerizes according to a temperature change, the structure of a polymer changes according to a temperature change.

  There is no particular limitation on the temperature change in which the site X showing temperature responsiveness shows responsiveness as long as it does not adversely affect the cells. For example, the temperature change is not limited, for example, from the culture temperature (eg 37 ° C) to room temperature (eg 25 ° C ), Temperature drop to 20 ° C. or low temperature (eg 4 ° C.).

The site X exhibiting temperature responsiveness has an ether partial structure or a sulfide partial structure. The site X may have an ether partial structure or a sulfide partial structure in either the main chain or the side chain. The site X preferably has two or more ether partial structures or sulfide partial structures. In this case, each ether partial structure or sulfide partial structure may exist continuously or may exist separately. When the moiety X has two or more ether partial structures or sulfide partial structures in succession, this partial structure has the formula: —O— (C _n H _2n ) —O— or —S— (C _n H _2n ) -S- (wherein n is, for example, 1 to 20, preferably 1 to 10, more preferably 1 to 5).

The site X exhibiting temperature responsiveness is preferably an alkylene glycol unit: —O—R ₁ O— (wherein R ₁ O is an oxyalkylene group in which R ₁ is a linear or branched alkylene). Have In the above formula, R ₁ is preferably a linear or branched C _{1 to} C ₂₀ alkylene, more preferably a linear or branched C _{1 to} C ₁₀ alkylene, and particularly preferably a straight chain or branched C _{1 to} C ₂₀ alkylene. a _C 1 -C ₅ alkylene chain or branched. Each alkylene glycol unit may have a linear or branched alkylene moiety. Thus, for example, propylene glycol units are 1,2-propylene glycol units (—O—CH (CH ₃ ) CH ₂ O—) and 1,3-propylene glycol units (—O—CH ₂ CH ₂ CH ₂ O—). ), Butylene glycol units are 1,2-butylene glycol units (—O—C (CH ₂ CH ₃ ) CH ₂ O—), 1,3-butylene glycol units (—O—CH (CH ₃ ) CH ₂ CH ₂ O—) and 1,4-butylene glycol units (—O—CH ₂ CH ₂ CH ₂ CH ₂ O—). The site X particularly preferably has at least one selected from ethylene glycol units (—O—CH ₂ CH ₂ O—), propylene glycol units, butylene glycol units, and pentylene glycol units.

The site X exhibiting temperature responsiveness is preferably a polyalkylene glycol partial structure having two or more oxyalkylene groups: —O— (R ₁ O) _n — (wherein R ₁ is as defined above, n is preferably 1 to 1000). The site X more preferably has at least one selected from a polyethylene glycol partial structure, a polypropylene glycol partial structure, a polybutylene glycol partial structure, and a polypentylene glycol partial structure.

In a preferred embodiment, the temperature-responsive moiety X has the formula: —O— (C _n H _2n ) —O— or —S— (C _n H _2n ) —S—, where n is, for example, 1 to 20, preferably 2 to 10, more preferably 2 to 5), or a polyethylene glycol partial structure, a polypropylene glycol partial structure, or a polybutylene glycol. It has at least one polyalkylene glycol partial structure selected from a partial structure and a polypentylene glycol partial structure.

  Specifically, the site X exhibiting temperature responsiveness includes, for example, alkylene glycol, alkylene dithioether, polyethylene glycol, polypropylene glycol, polybutylene glycol, polypentylene glycol, polyhexylene glycol and other polyalkylene glycols, polyphenylene Glycol, methylcellulose, hydroxypropylcellulose, alkylcelluloses, polyvinyl alcohol methyl ether and polyvinyl alcohol ether as partial structures, preferably alkylene glycol partial structure, alkylene dithioether partial structure, polyethylene glycol partial structure, polypropylene glycol moiety Structure, polybutylene glycol partial structure and polypentylene glycol partial structure. The site | part X may have the combination of these partial structures.

  As the site X exhibiting temperature responsiveness, a polymer having an ether partial structure or a sulfide partial structure in the side chain can also be used. Examples of the polymer capable of introducing an ether partial structure or a sulfide partial structure into the side chain include poly (N-isopropylacrylamide), poly (N-diisopropylacrylamide), poly (N-ethylacrylamide), and poly (N- Diethyl acrylamide), poly (N-methyl acrylamide), poly (N-dimethyl acrylamide), polyacrylamide, poly (butyl methacrylate), poly (propyl methacrylate), poly (ethyl methacrylate), poly (methyl methacrylate) , Polymethacrylic acid, poly (butyl acrylate), poly (propyl acrylate), poly (ethyl acrylate), poly (methyl acrylate), polyacrylic acid, poly (N-vinylpyrrolidone), poly (N-vinyl) Caprolactam), polylactic acid, N Acylation-.epsilon.-polylysine,-.epsilon.-N-alkylated polylysine, can be used γ- polyglutamic acid amide derivatives and δ- polyaspartic acid amide derivatives.

  In one embodiment, the site | part X which shows temperature responsiveness has the group derived from a raw material in both ends. The group derived from the raw material is not particularly limited as long as it is a polymerizable group, and examples thereof include a group derived from an epoxy group, a group derived from a carboxylic acid group, and a group derived from an amine group. That is, the site X preferably has an ether partial structure or a sulfide partial structure and groups derived from raw materials at both ends. In one embodiment, the site X consists of an ether partial structure or a sulfide partial structure, and groups derived from raw materials at both ends.

In a preferred embodiment, the site X exhibiting temperature responsiveness is represented by the following formula:
Formula: L-O- (R _{< 1 >} O) _n- L '(I)
(Wherein R ₁ is a linear or branched alkylene group, L and L ′ are groups derived from raw materials, and n is 1 to 1000),
_{Formula: L-O- (C n H} 2n) -O-L '(II)
(N is, for example, 1 to 20, preferably 2 to 10, more preferably 2 to 5), or the formula: LS— (C _n H _2n ) —SL ′ (III)
(N is, for example, 1 to 20, preferably 2 to 10, more preferably 2 to 5)
It is represented by In the formula (I), R ₁ is preferably linear or branched C ₁ -C ₂₀ alkylene, more preferably linear or branched C ₁ -C ₁₀ alkylene, and particularly preferably Is a linear or branched C ₁ -C ₅ alkylene. In the above formulas (I) to (III), L and L ′ are, for example, an epoxy group-derived group, a carboxylic acid group-derived group, and an amine group-derived group. Specifically, for example, —CH ( _OH) CH 2 -, - CO- or - _(CH 2) a NH-.

  Alternatively, the site X may be an amino acid-derived moiety such as lysine or glutamic acid substituted with an ether partial structure or a sulfide partial structure. Moreover, the alkylene part which has an ether partial structure or sulfide partial structure in a side chain formed when the monomer which has a double bond as a raw material is used may be sufficient.

（式中、ａは１〜１０００であり、ｂは１〜５００であり、ｃは１〜４００であり、ｄは１〜５００であり、ｅは１〜５００であり、ｇは１〜５００である。） Part X showing temperature responsiveness is particularly preferably at least one selected from the following.

(In the formula, a is 1-1000, b is 1-500, c is 1-400, d is 1-500, e is 1-500, and g is 1-500. is there.)

  The site X exhibiting temperature responsiveness may be either a small molecule or a polymer, but an oligomer or a polymer is preferable because it is desirable that the cell has a molecular weight of a certain level or more in order to feel a change in the surface state. When the site | part X is an oligomer or a polymer, the number average molecular weight of the site | part X is about 300-10000, for example.

  The part X exhibiting temperature responsiveness may be responsive to stimuli other than temperature. If site X is responsive to stimuli other than temperature, cell detachment can be promoted without damaging the cells, and site Y showing responsiveness to stimuli other than temperature changes. The effect of can be enhanced. Examples of stimuli other than temperature include non-invasive changes in external environment that are not invasive to cells, and are not particularly limited. For example, pH changes, salt concentration changes, composition changes, sugar / peptide / Examples include addition of a biocompatible substance such as a weak reducing agent and light irradiation. Site X is preferably responsive to changes in pH and / or salt concentration in addition to temperature changes. These stimuli are preferably weak so as not to adversely affect the cells. For example, when the site X is responsive to a pH change, the pH change is, for example, from pH 7.4 to pH 6.5, pH 7 .4 to pH 7.0 or a slight change from pH 7.4 to pH 8.0, and when site X is responsive to changes in salt concentration or composition, for example, Dulbecco's Modified Eagle Medium (DMEM) Change from serum-free medium for stem cells (TeSR-E8) to PBS buffer or HEPES buffer.

  The site X showing temperature responsiveness includes a structure showing responsiveness to stimuli other than temperature change, or the ether partial structure or sulfide partial structure contained in the site X is sensitive to stimuli other than temperature change. However, by showing responsiveness, it is possible to show responsiveness to these stimuli in addition to temperature responsiveness. For example, the ether moiety or sulfide moiety contained in site X can be responsive to changes in pH and / or salt concentration. In addition to the ether partial structure or the sulfide partial structure, the site X includes a coordination site capable of interacting with a salt, a proton, or the like, so that it can respond to changes in pH and / or salt concentration. it can. The coordination site is not particularly limited, and examples thereof include another ether, another thioether, a carboxylic acid group, a nitrogen-containing aromatic group, an amine group, a carboxylic acid group, and a phosphate group. From the viewpoint of preventing non-specific adsorption to the container, a structure such as polyether is preferred.

  The site Y exhibiting responsiveness to stimuli other than temperature changes is not particularly limited. For example, pH changes, salt concentration changes, composition changes, addition of biocompatible substances such as sugars / peptides / weak reducing agents And responsiveness to non-invasive stimulation to cells such as light irradiation. The site Y can act on cell adhesion or exfoliation, in particular cell exfoliation, by changing properties such as polymer solubility by these stimuli. These stimuli are preferably stimuli that are weak enough not to adversely affect the cells. For example, when the site Y is responsive to a pH change, the pH change is usually from pH 7.4 to pH 6.5, pH 7 .4 to pH 7.0, or a slight change from pH 7.4 to pH 8.0, and if site Y is responsive to changes in salt concentration or composition, for example from DMEM or TeSR-E8 to PBS buffer Change to liquid or HEPES buffer. Site Y is preferably responsive to pH, salt concentration and / or sugar addition, more preferably responsive to pH and / or salt concentration, and particularly responsive to pH.

  The site Y exhibiting responsiveness to stimuli other than temperature changes has a different structure depending on the stimulus used, but the site Y contains a functional group having a pKa in water of 2 to 12. This is preferred when the stimulus is a pH change and / or a salt concentration change. When the pKa in water of the functional group at the site Y is within the above range, the pKa of the water is 15.7 or less and the pKa of the oxonium ion in water is -1.7 or more, Responsiveness in the vicinity of pH 7 where culture is possible can be realized. The site Y more preferably contains a functional group having a pKa in water of 3 or more and 11 or less. Here, in the present invention, the functional group of a predetermined pKa includes a functional group in which the pKa of the conjugate acid or conjugate base is within the range. In addition, when the same functional group exists in one molecule, each pKa may be different. Even if the pKa in water when present alone does not fall within the above range, the pKa in water only needs to fall within the above range when present in plural.

  Examples of the functional group possessed by the site Y that exhibits responsiveness to stimuli other than temperature changes include carboxylic acid groups (aliphatic carboxylic acids and aromatic carboxylic acids), amine groups (aliphatic primary amine groups, aliphatic groups). Secondary amine group, aliphatic tertiary amine group, aromatic primary amine group, aromatic secondary amine group, aromatic tertiary amine group), imine group, thioether group, boronic acid group (aliphatic boronic acid group, Aromatic boronic acid group), phosphoric acid group (aliphatic phosphoric acid group, aromatic phosphoric acid group), nitrogen-containing aromatic group (pyrrole group, imidazolyl group, pyridyl group, pyrimidyl group, oxazolyl group, thiazolyl group and triazolyl group) Etc.) and phenolic hydroxyl groups and the like. The functional group possessed by the site Y is preferably a carboxylic acid group, an amine group, a thioether group, a boronic acid group, and a nitrogen-containing aromatic group (particularly an imidazole group). These groups usually have a pKa in water of 3 or more and 11 or less. The functional group possessed by the site Y may be a combination of the above. Preferred combinations of the functional groups possessed by the site Y include combinations of amine groups and carboxylic acid groups, combinations of amine groups and thioether groups, combinations of amine groups, thioether groups, and carboxylic acid groups, and amine groups and nitrogen-containing aromatic groups ( In particular, a combination of imidazole groups).

  The site Y exhibiting responsiveness to stimuli other than temperature changes may have at least one of the above functional groups, but preferably the site Y has two or more of the above functional groups, More preferably, it has two or three functional groups.

  The portion Y that exhibits responsiveness to stimuli other than temperature change preferably has an alkylenediamine partial structure or an alkylaminothioether partial structure, and these may further have a carboxylic acid group or an amine group. A moiety derived from an amino acid (eg, lysine, cysteine, glutamic acid, etc.) may be used.

  In one embodiment, the site Y that exhibits responsiveness to stimuli other than temperature changes preferably has a group derived from a raw material at both ends. The group derived from the raw material is not particularly limited as long as it is a polymerizable group, and examples thereof include a group derived from an amine group, a group derived from a thiol group, and a group derived from a carboxylic acid group. In the site Y, the group derived from the raw material can be a functional group having a pKa in water of 2 or more and 12 or less.

In preferred embodiments, moiety Y is of the formula: —R ₂ — (C _n H _2n ) —R ₃ —, wherein R ₂ and R ₃ are each NH or S, and n is, for example, 1 to 1 20 and preferably 1 to 10 and more preferably 1 to 5), and the alkylene moiety may be substituted with a carboxylic acid group or an amine group. Also, site Y has the formula: -NH- in _{(C n} H 2n) -CO- _(wherein, n, for example, from 1 to 20, preferably 1 to 10, more preferably, 1 to The alkylene moiety may be substituted with a carboxylic acid group or an amine group. Moreover, the site | part Y may be amino acid origin parts, such as a lysine, a cysteine, and glutamic acid. The site Y is represented by the formula: —CH ₂ CHR ₄ — (wherein R ₄ is selected from a carboxylic acid group, an amine group, a boronic acid group, and a nitrogen-containing aromatic group, and preferably a boronic acid group. And particularly preferably a phenylboronic acid group).

  The site Y includes a coordination site selected from, for example, an ether, a thioether, a carbonyl group, a nitrogen-containing aromatic group, an amine group, a carboxylic acid group, and a phosphate group when the stimulus is a change in salt concentration. In addition, when the stimulus is sugar addition, the site Y preferably includes a site capable of reacting with a sugar such as a boronic acid group. Where the stimulus is the addition of a reducing agent, site Y preferably includes a reducible site such as disulfide. Where the stimulus is light irradiation, site Y preferably includes photoresponsive sites such as azobenzene, spiropyran, diarylethene and o-nitrobenzyl groups. In addition, for example, when using a functional group that is cleaved by reduction, such as disulfide, or a functional group that is cleaved by light, such as o-nitrobenzyl group, the components generated by the cleavage of the bond dissolve in the medium. It is necessary to pay attention to the effects on cells.

Part Y that exhibits responsiveness to stimuli other than temperature changes is particularly preferably at least one selected from the following.

  The site Y exhibiting responsiveness to stimuli other than temperature changes may be either a low molecule or a polymer, but is preferably a low molecule.

  The molar ratio of the site X showing temperature responsiveness in the stimulus-responsive polymer and the site Y showing responsiveness to stimuli other than temperature change is, for example, 10: 1 to 1:10, preferably 5: It is 1-1: 5, More preferably, it is 3: 1 to 1: 3.

  The stimulus-responsive polymer of the present invention has a site X ′ or Y ′ that regulates physical properties in addition to a site X showing temperature responsiveness and a site Y showing responsiveness to stimuli other than temperature changes. Also good. Having a site X ′ or Y ′ that regulates physical properties allows the polymer to be tuned to the desired physical properties, such as increasing responsiveness to temperature or non-temperature stimuli, changing solubility, charge It is possible to change the density, impart another responsiveness, adjust cell adhesion, impart sustained drug release, and the like. Here, increasing the responsiveness to temperature or a stimulus other than temperature means increasing the degree of responsiveness, or widening the range of stimuli other than the temperature or temperature that can be responded.

  The site X ′ or Y ′ that regulates physical properties is not particularly limited as long as it includes a physical property regulating site that can change the physical properties of the stimulus-responsive polymer. In the present invention, the site X ′ has a structure similar to the site X, and the site Y ′ has a structure similar to the site Y. The site X ′ may be one that can be used as the site X, but in one polymer, the site X ′ is different from the site X. Similarly, the site Y ′ may be one that can be used as the site Y, but the site Y ′ is different from the site Y in one polymer.

  Specifically, for example, the site X ′ or Y ′ is a carboxylic acid group, boronic acid group, phosphoric acid group, sulfonic acid, amine group, imine group, imidazolyl group, pyridyl group, guanidyl group, quaternary ammonium group, etc. In the case of having an amino acid-derived moiety such as lysine, for example, the responsiveness to the pH change of the stimulus-responsive polymer can be changed. When the site X 'or Y' contains photoresponsive moieties such as azobenzene, spiropyran, diarylethene and o-nitrobenzyl groups, the photoresponsiveness of the stimulus responsive polymer can be increased. Site X ′ or Y ′ is gelatin, collagen, polylysine, RDG peptide, laminin, fibronectin, vitronectin, chitin, chitosan, integrin, cadherin, albumin, globulin, heparin, heparan sulfate, dextran sulfate, polyglutamic acid, polyaspartic acid, When an adhesive moiety such as elastin is included, the cell adhesiveness of the stimulus-responsive polymer can be adjusted. When the site X ′ or Y ′ includes a structure in which a physiologically active substance is introduced through a hydrolyzable bond such as an ester bond, sustained drug release can be imparted.

  The site X ′ has a structure similar to that of the site X. The site X ′ preferably has a group derived from a raw material at both ends. The group derived from the raw material is the same as the raw material of the site X described above.

  The site Y ′ has a structure similar to that of the site Y. The site Y ′ preferably has a group derived from a raw material at both ends. The group derived from the raw material is the same as the raw material of the site Y.

Part X ′ or Y ′ that regulates physical properties is particularly preferably at least one selected from the following.

  The molar ratio of the site X exhibiting temperature responsiveness in the stimuli-responsive polymer to the site X ′ that regulates physical properties is, for example, 100: 1 to 1:10, preferably 20: 1 to 1: 5. The molar ratio of the site Y that exhibits responsiveness to stimuli other than temperature changes and the site Y ′ that regulates physical properties is, for example, 100: 1 to 1:10, preferably 20: 1 to 1: 5.

  The stimulus-responsive polymer of the present invention is preferably water-soluble. The number average molecular weight of the stimulus-responsive polymer is preferably 500 to 500,000, more preferably 500 to 200,000, and particularly preferably 500 to 100,000. Preferred stimuli-responsive polymers are the polymers 1-1 to 1-4, 2-1 to 2-7, 3-1 to 3-4, 4-1 to 4-4, and 5-1 to 5-4 of the examples. The polymer.

  The stimuli-responsive polymer of the present invention includes, for example, a raw material of site X that exhibits temperature responsiveness, a raw material of site Y that exhibits responsiveness to stimuli other than changes in temperature, and a site X ′ that adjusts physical properties as the case may be. It can be produced by copolymerizing the raw material of Y ′. A monomer or a macromonomer can be used as a raw material for each part. In addition to these raw materials, copolymerization may be performed in the state where another monomer or macromonomer coexists. As the polymerization mode, for example, radical polymerization, cationic polymerization, anionic polymerization, coordination polymerization, polycondensation, polyaddition, addition condensation, ring-opening polymerization, metathesis polymerization, etc. can be used, and it is appropriately selected according to the desired structure. it can. The polymerization mode is preferably radical polymerization, polycondensation, polyaddition, addition condensation and ring-opening polymerization. The functional group in the raw material may be protected as necessary. In this case, it is preferable to purify after deprotection after polymerization.

  In one embodiment, when a stimuli-responsive polymer is produced by polycondensation or ring-opening polymerization, the starting materials for the site X and the site Y may be those having groups capable of polycondensation or ring-opening polymerization at both ends. it can. For example, in the case of polycondensation, a material having a carboxylic acid group and / or an amine group at both ends can be used as a raw material for the site X and the site Y. Specifically, as a material for the site X and the site Y, A material having a carboxylic acid group at one end and an amine group at the other end can be used, and a material having a carboxylic acid group at both ends is used as a raw material for part X. Those having amine groups at both ends can also be used. It is preferable that the raw materials of the site X ′ and the site Y ′ have the same end groups as the site X and the site Y, respectively. In the case of ring-opening polymerization, for example, a material having an epoxy group at both ends can be used as the raw material for the site X, and a nucleophilic group (for example, an amine group or a thiol group) can be used as the material for the site Y. What has both ends can be used. The polymerization conditions can be appropriately selected depending on the raw materials used and the polymerization method.

  In one embodiment, when producing a stimuli-responsive polymer by radical addition polymerization, the raw material of the site X and the site Y may be any material having a radical addition-polymerizable group, for example, a double bond. The raw material which has is used. In this case, preferably, the raw material of the site X has an ether partial structure or a sulfide partial structure in the side chain, and the raw material of the site Y has a functional group in the side chain. In this polymerization method, a polymer in which the sites X and Y are combined in any order is obtained. The polymerization conditions can be appropriately selected depending on the raw materials used and the polymerization method.

  Alternatively, the stimuli-responsive polymer of the present invention uses a polymer as a raw material, introduces site X or site Y into the side chain of the polymer, and introduces site X or site Y into all or part of the side chain. It can also be manufactured. For example, a side chain of a polymer having a site X as a structural unit by reacting a polymer having a site X exhibiting temperature responsiveness as a structural unit with a raw material of the site Y exhibiting responsiveness to a stimulus other than a temperature change. A site Y may be introduced into the polymer, or a polymer having a site Y that exhibits responsiveness to a stimulus other than a temperature change as a constituent unit is reacted with a raw material of the site X that exhibits temperature responsiveness to react with the site Y. May be introduced into the side chain of the polymer having as a structural unit. In one embodiment, for example, polylysine or polyglutamic acid is used as a polymer having the site Y as a structural unit, and the site X exhibiting temperature responsiveness can be introduced into these side chains. In this case, site Y is lysine or glutamic acid, which is a structural unit of polylysine or polyglutamic acid, respectively.

  In general, the response of a stimulus-responsive polymer to a temperature change or stimulus other than the temperature change is affected by the introduction amount of a site exhibiting the corresponding response and the molecular weight of the polymer. For example, the monomer concentration, the monomer ratio, the solvent, By controlling the polymerization conditions such as reaction temperature and atmosphere, the responsiveness range can be controlled.

  When a raw material having a plurality of reaction sites is used, the polymer obtained is not necessarily linear, but may be branched or networked, but may be within a range that does not affect the function. In addition, the polymer tacticity, polymerization regioselectivity, and terminal functional groups may be a mixture of multiple structures within a range that does not affect the function, and the bonding mode and terminal structure in the polymer are not limited. .

  The present invention also includes a stimulus-responsive polymer produced by the production method described above. Therefore, the present invention also includes a stimulus-responsive polymer obtained by copolymerizing the raw material of the site X, the raw material of the site Y, and the raw material of the site X ′ or Y ′ that optionally controls physical properties.

  The stimulus-responsive polymer of the present invention is represented by the following formula (1-1), formula (1-2), formula (1-3) or formula (2-1) depending on the structural unit and structure. .

（式中、Ｘは温度応答性を示す部位であり、Ｙは温度変化以外の刺激に対して応答性を示す部位であり、Ｘはエーテル部分構造またはスルフィド部分構造を有し、Ｙは水中でのｐＫａが２以上１２以下の官能基を含み、ｎは、好ましくは、２〜１０００である）。 Stimulus responsive polymer of formula (1-1)

(In the formula, X is a site showing temperature responsiveness, Y is a site showing responsiveness to stimuli other than temperature change, X has an ether partial structure or a sulfide partial structure, and Y is in water. PKa includes a functional group of 2 or more and 12 or less, and n is preferably 2 to 1000).

  The stimulus-responsive polymer of the formula (1-1) has a site X showing temperature responsiveness and a site Y showing responsiveness to stimuli other than temperature change as structural units. The site X and site Y and preferred ones thereof are as described above. The stimulus-responsive polymer of Formula (1-1) has a structure in which the sites X and Y are alternately bonded. By selecting an appropriate combination of each raw material and an appropriate molecular weight, the desired cell detachability can be obtained. The stimuli-responsive polymer of formula (1-1) has a site X that exhibits temperature responsiveness and a site Y that exhibits responsiveness to stimuli other than temperature changes. Can be peeled off and can sufficiently act on cell detachment even at a low density.

  The stimuli-responsive polymer of the formula (1-1) is, for example, polycondensation of a raw material of the site X having an epoxy group or a carboxylic acid group at both ends and a raw material of the site Y having a nucleophilic group at both ends. It can be produced by ring-opening polymerization.

（式中、Ｘは温度応答性を示す部位であり、Ｙは温度変化以外の刺激に対して応答性を示す部位であり、Ｙ’は物性を調節する部位であり、Ｘはエーテル部分構造またはスルフィド部分構造を有し、Ｙは水中でのｐＫａが２以上１２以下の官能基を含み、Ｙ’は物性調節部位を含み、Ｙと同一ではなく、ｎは、好ましくは、１〜１０００であり、ｎ’は、好ましくは、１〜５００である）。 Stimulus responsive polymer of formula (1-2)

(In the formula, X is a site showing temperature responsiveness, Y is a site showing responsiveness to stimuli other than temperature change, Y ′ is a site that regulates physical properties, and X is an ether partial structure or It has a sulfide partial structure, Y contains a functional group having a pKa in water of 2 or more and 12 or less, Y 'contains a physical property controlling site, is not the same as Y, and n is preferably 1-1000 , N ′ is preferably 1 to 500).

  The polymer of the formula (1-2) has a site X that exhibits temperature responsiveness, a site Y that exhibits responsiveness to stimuli other than temperature changes, and a site Y ′ that regulates physical properties as structural units. The site X, the site Y and the site Y ′ and preferred ones thereof are as described above.

  In the polymer of formula (1-2), a part of the part Y is a physical property of the structure in which the part X showing temperature responsiveness and the part Y showing responsiveness to stimuli other than temperature change are alternately bonded. It has a structure substituted by a site Y ′ that regulates. By having the site Y ′ for controlling the physical properties, it is possible to obtain physical properties that are difficult to obtain with the polymer of the formula (1-1) having only the site X and the site Y as structural units.

  The stimulus-responsive polymer of the formula (1-2) includes, for example, a raw material of the site X having an epoxy group or a carboxylic acid group at both ends, and a raw material of the site Y and the site Y ′ having a nucleophilic group at both ends. Can be produced by polycondensation or ring-opening polymerization.

（式中、Ｘは温度応答性を示す部位であり、Ｙは温度変化以外の刺激に対して応答性を示す部位であり、Ｘ’は物性を調節する部位であり、Ｘはエーテル部分構造またはスルフィド部分構造を有し、Ｙは水中でのｐＫａが２以上１２以下の官能基を含み、Ｘ’は物性調節部位を含み、Ｘと同一ではなく、ｎは、好ましくは、１〜１０００であり、ｎ’は、好ましくは、１〜５００である。） Stimulus responsive polymer of formula (1-3)

(In the formula, X is a site showing temperature responsiveness, Y is a site showing responsiveness to stimuli other than temperature change, X ′ is a site that regulates physical properties, and X is an ether partial structure or It has a sulfide partial structure, Y contains a functional group having a pKa in water of 2 or more and 12 or less, X ′ contains a physical property regulating site, is not identical to X, and n is preferably 1 to 1000 , N ′ is preferably 1 to 500.)

  The polymer of the formula (1-3) has, as structural units, a site X that exhibits temperature responsiveness, a site Y that exhibits responsiveness to stimuli other than temperature changes, and a site X ′ that regulates physical properties. The site X, the site X ′ and the site Y and preferred ones thereof are as described above.

  The polymer of the formula (1-3) is composed of the structure in which the site X showing temperature responsiveness and the site Y showing responsiveness to stimuli other than temperature change are alternately bonded. A part of the structure is replaced by a site X ′ that regulates physical properties. By having the site X ′ for controlling the physical properties, it is possible to obtain physical properties that are difficult to obtain with the polymer of the formula (1-1) having only the site X and the site Y as structural units.

  The stimuli-responsive polymer of the formula (1-3) includes, for example, a raw material of the site X and the site X ′ having an epoxy group or a carboxylic acid group at both ends, and a raw material of the site Y having a nucleophilic group at both ends. Can be produced by polycondensation or ring-opening polymerization.

（式中、Ｘは温度応答性を示す部位であり、Ｙは温度変化以外の刺激に対して応答性を示す部位であり、Ｘはエーテル部分構造またはスルフィド部分構造を有し、Ｙは水中でのｐＫａが２以上１２以下の官能基を含み、ｎは、好ましくは、１〜１０００であり、ｎ’は、好ましくは、１〜５００である） Stimulus responsive polymer of formula (2-1)

(In the formula, X is a site showing temperature responsiveness, Y is a site showing responsiveness to stimuli other than temperature change, X has an ether partial structure or a sulfide partial structure, and Y is in water. PKa includes a functional group of 2 or more and 12 or less, n is preferably 1 to 1000, and n ′ is preferably 1 to 500)

  The polymer of the formula (2-1) has a site X showing temperature responsiveness and a site Y showing responsiveness to stimuli other than temperature change as structural units. Unlike the polymer of the formula (1-1), the polymer of the formula (2-1) has a structure in which the site X and the site Y are combined in any order, such as a random copolymer and a block copolymer. It has a structure. Such a structure can also be used when the binding order of the part X showing temperature responsiveness and the part Y showing responsiveness to stimuli other than temperature changes is not important.

  The polymer of the formula (2-1) is, for example, radical polymerization, cationic polymerization, anionic polymerization, coordination polymerization, polycondensation, polyaddition, addition condensation, ring-opening polymerization, using each raw material of the site X and the site Y. It can be produced by metathesis polymerization or the like. Alternatively, the polymer of the formula (2-1) can be obtained by introducing the site Y into the side chain of the polymer having the site X as a structural unit or the site X into the side chain of the polymer having the site Y as a structural unit. Can be manufactured.

  The present invention also includes a culture vessel provided with the stimulus-responsive polymer on the surface. In the present invention, since the stimulus-responsive polymer functions sufficiently even at a low density, the polymer can be introduced into the culture vessel by a simple method, and the stimulus-responsive polymer is responsive to stimuli other than temperature. Therefore, it has excellent cell detachability.

  The immobilization of the stimulus-responsive polymer on the surface of the culture vessel is not particularly limited, and can be performed by, for example, covalent bond, charge interaction, physical adsorption and the like. Considering productivity and durability, the stimulus-responsive polymer can be immobilized on the surface of the culture vessel by forming a covalent bond between the functional group of the stimulus-responsive polymer and the functional group on the surface of the culture vessel, which can be easily implemented. preferable.

  The method for forming a covalent bond used for immobilizing the stimulus-responsive polymer on the surface of the culture vessel is not particularly limited. For example, amide formation, epoxy ring opening, imine formation, Michael type addition, ene reaction, thioene reaction, Diels -Alder reaction, Husgen reaction, native chemical ligation, nucleophilic addition reaction, electrophilic addition reaction, sigmatropic rearrangement, etc. are considered. Reactions that can utilize functional groups present in are preferred. In general, the culture vessel base material is a resin such as polystyrene. Oxidation or introduction of a cationic polymer may be performed as a hydrophilic treatment on the surface, and products with carboxylic acid groups or amine groups as surface functional groups are available. Easy. From the above, as a method for forming a covalent bond used for immobilizing a stimulus-responsive polymer on the surface of a culture vessel, amide formation, epoxy ring opening, imine formation, Michael type addition, and the like are preferable.

  The present invention also includes a method for culturing cells using a culture vessel having the stimulus-responsive polymer on its surface.

  In the culture method of the present invention, the cells are adhered and cultured by bringing the cell suspension into contact with the cell suspension. Prior to contacting the cell suspension, a component that promotes cell adhesion may be coated or immobilized on the culture vessel. Components that promote cell adhesion include, for example, gelatin, collagen, poly-L-lysine, RDG peptide, laminin, fibronectin, vitronectin, integrin, cadherin, an antibody specific for a membrane protein, or a molecule containing a partial structure thereof, or Although it is a molecule | numerator etc. containing them, it is not limited to them.

  The surface density of the stimuli-responsive polymer and the component that promotes cell adhesion on the surface of the cell culture container has an effect on cell culture properties and cell detachability. For example, if the surface density of the stimulus-responsive polymer is too high, the cells cannot be cultured because there are few components that promote cell adhesion, and if the surface density of the stimulus-responsive polymer is too low, the cells cannot be detached. It is possible. However, since these depend on the cells to be cultured, it is necessary to select a material and surface density suitable for the target cells when carrying out.

  The cells to be cultured are preferably adherent cells, such as fibroblasts, mesenchymal stem cells, ES cells, iPS cells, and dedifferentiated cells, but are not limited thereto. In addition, it is necessary to select appropriately the said component which promotes cell adhesion by the cell to culture.

  The cells can be detached by applying an appropriate stimulus to the surface of the culture container after the cells are adhered and cultured in the culture container having the stimulus-responsive polymer of the present invention on the surface. Appropriate stimuli are changes in the external environment that are non-invasive to the cell, such as medium temperature, pH, salt concentration, chemical potential, redox potential or the light intensity to which the cell is exposed. Change. Therefore, the present invention relates to a cell comprising a process for changing the temperature, pH, salt concentration, chemical potential, redox potential or light intensity to which the cell is exposed, in a state where the cell is adhered to the culture vessel. A peeling method is also included. When using a temperature or liquid factor (pH, substance addition, etc.) as a stimulus, cells can be easily detached by adding a stripping solution containing them. Before adding the stripping solution, the medium may or may not be removed. Although appropriate conditions vary depending on the cells to be cultured, general culture conditions can be used. Since the cell detachment method of the present invention can detach cells without damaging the cells, an excellent cell survival rate is achieved.

  The temperature change in the cell detachment method may be a temperature change within a culturable temperature range, for example, a temperature change from 37 ° C. to 4 ° C., 37 ° C. to 20 ° C., 37 ° C. to 25 ° C., etc. However, it is not limited to these. The pH change in the cell detachment method may be a pH change within a range that does not adversely affect the cells in the short term, for example, pH 7.4 to pH 6.5, pH 7.4 to pH 7.0, pH 7.4. The pH change from pH to 8.0 is not limited to these. The salt concentration change in the cell peeling method is a salt concentration change within a range that does not adversely affect the cells in the short term. This salt concentration may be the salt concentration of the whole solution or the salt concentration of a specific substance, for example, change from medium to PBS buffer, change from medium to HEPES buffer, change from medium to another medium. Although it is a change etc., it is not limited to these. However, it is desirable that the osmotic pressure does not change greatly from the viewpoint of preventing damage to the cells in these cases.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated concretely, the technical scope of this invention is not limited to this.

A stimulus-responsive polymer of the present invention was prepared. The molar equivalent described in the following polymer preparation is the molar equivalent at the time of preparation, and is the molar equivalent of each raw material relative to the raw material corresponding to the site X. Moreover, what was protected as needed was used for the functional group in a raw material, In this case, it refine | purified after implementing deprotection after superposition | polymerization.

<Preparation of polymers 1-1 to 1-4>
Polymers 1-1 to 1-1 represented by formula (1-1) using a raw material having an epoxy group at both ends corresponding to part X and a raw material having two or more nucleophilic parts corresponding to part Y for polymerization 1-4 was prepared.

  The polymer 1-1 was polymerized by heating a water-ethanol (1: 1) solution of PEG2000-diglycidyl ether and 1,4-butanediamine (1.2 molar equivalent) at 60 ° C. for 2 hours. The obtained polymer was purified by reprecipitation or dialysis (MWCO1000), and pulverized by lyophilization to obtain polymer 1-1. The number average molecular weight of the polymer 1-1 was about 40,000.

  Polymer 1-2 was prepared in the same manner as Polymer 1-1 from PPG640-diglycidyl ether and ethylenediamine (1.2 molar equivalent). The number average molecular weight of the polymer 1-2 was about 30,000. It was confirmed that polymer 1-2 exhibited a lower limit dissolution critical temperature in Dulbecco's modified Eagle medium (DMEM).

  Polymer 1-3 was prepared from PBG400-diglycidyl ether and ethylenediamine (1.4 molar equivalent) in the same manner as Polymer 1-1. The number average molecular weight of the polymer 1-3 was about 20,000.

  Polymer 1-4 was prepared in the same manner as polymer 1-1 from rand-PEG / PPG1000-diglycidyl ether and lysine (1.4 molar equivalent). The number average molecular weight of the polymer 1-4 was about 10,000.

（式中、ａは１〜１０００であり、ｂは１〜５００であり、ｃは１〜４００であり、ｄは１〜５００であり、ｅは１〜５００である。） Table 1 shows the structures of the polymers 1-1 to 1-4.

(In the formula, a is 1-1000, b is 1-500, c is 1-400, d is 1-500, and e is 1-500.)

<Preparation of polymers 2-1 to 2-7>
A polymer represented by the formula (1-2) using a raw material having an epoxy group at both ends corresponding to the site X and a raw material having two or more nucleophilic sites corresponding to the site Y or the site Y ′ for polymerization. 2-1 to 2-7 were prepared.

  Polymer 2-1 was prepared in the same manner as polymer 1-1 from PEG2000-diglycidyl ether, 1,4-butanediamine (0.8 molar equivalent) and cysteine (0.8 molar equivalent). The number average molecular weight of the polymer 2-1 was about 40,000.

  Polymer 2-2 was prepared in the same manner as Polymer 1-1 from PPG640-diglycidyl ether, ethylenediamine (1.2 molar equivalent) and lysine (0.4 molar equivalent). It was confirmed that polymer 2-2 exhibited a lower limit dissolution critical temperature in DMEM. In FIG. 1, the result of having measured the temperature responsiveness in the aqueous solution of the polymer 2-2 by the temperature variable light transmittance measurement is shown. From FIG. 1, it was confirmed that the polymer 2-2 exhibits a lower critical solution temperature in an aqueous solution. The number average molecular weight of the polymer 2-2 was about 30,000. FIG. 2 shows the molecular weight distribution of the polymer 2-2.

  Polymer 2-3 was prepared in the same manner as Polymer 1-1 from PPG640-diglycidyl ether, ethylenediamine (1.25 molar equivalent) and 4,4'-diaminoazobenzene (0.25 molar equivalent). The number average molecular weight of the polymer 2-3 was about 20,000. It was confirmed that the polymer 2-3 exhibits a lower limit melting critical temperature in DMEM.

  Polymer 2-4 was prepared in the same manner as polymer 1-1 from rand-PEG / PPG1000-diglycidyl ether, ethylenediamine (1.2 molar equivalent) and lysine (0.4 molar equivalent). The number average molecular weight of the polymer 2-4 was about 40,000.

  Polymer 2-5 was prepared in the same manner as Polymer 1-1 from butylene glycol diglycidyl ether, cysteine (1.5 molar equivalent) and ω, ω'-dithiol-PNIPAM (0.3 molar equivalent). The number average molecular weight of the polymer 2-5 was about 50,000. It was confirmed that the polymer 2-5 exhibited a lower limit dissolution critical temperature in DMEM.

  Polymer 2-6 was prepared from PEG2000-diglycidyl ether, 1,4-butanediamine (1.6 molar equivalent) and ε-polylysine (0.16 molar equivalent) in the same manner as Polymer 1-1. The number average molecular weight of the polymer 2-6 was about 100,000.

  Polymer 2-7 was prepared from PPG640-diglycidyl ether, ethylenediamine (1.6 molar equivalent) and gelatin (0.16 molar equivalent) in the same manner as Polymer 1-1. The number average molecular weight of the polymer 2-7 was about 100,000. It was confirmed that the polymer 2-7 exhibits a lower limit melting critical temperature in DMEM.

（式中、ａは１〜１０００であり、ｂは１〜５００であり、ｃは１〜４００であり、ｄは１〜５００であり、ｅは１〜５００である。） Table 2 shows the structures of the polymers 2-1 to 2-7. In Table 2, [Y]: [Y ′] indicates the charged molar ratio during synthesis.

(In the formula, a is 1-1000, b is 1-500, c is 1-400, d is 1-500, and e is 1-500.)

<Preparation of polymers 3-1 to 3-4>
A polymer represented by the formula (1-3) using a raw material having an epoxy group at both ends corresponding to the site X or the site X ′ and a raw material having two or more nucleophilic sites corresponding to the site Y for polymerization. 3-1 to 3-4 were prepared.

  Polymer 3-1 was prepared in the same manner as polymer 1-1 from PEG2000-diglycidyl ether, butylene glycol diglycidyl ether (0.5 molar equivalent) and cysteine (1.5 molar equivalent). The number average molecular weight of the polymer 3-1 was about 40,000.

  Polymer 3-2 was prepared from PEG2000-diglycidyl ether, octafluorodecane bisoxide (0.05 molar equivalent) and 1,4-butanediamine (1.5 molar equivalent) in the same manner as Polymer 1-1. . The number average molecular weight of the polymer 3-2 was about 40,000.

  Polymer 3-3 was prepared in the same manner as Polymer 1-1 from PEG2000-diglycidyl ether, PBG1000-diglycidyl ether (2 molar equivalent) and ethylenediamine (4.8 molar equivalent). The number average molecular weight of the polymer 3-3 was about 40,000.

  Polymer 3-4 was prepared in the same manner as Polymer 1-1 from PPG640-diglycidyl ether, butylene glycol diglycidyl ether (0.33 molar equivalent) and ethylenediamine (2 molar equivalent). The number average molecular weight of the polymer 3-4 was about 30,000.

（式中、ａは１〜１０００であり、ｂは１〜５００であり、ｃは１〜４００である。） Table 3 shows the structures of the polymers 3-1 to 3-4. In Table 3, [X]: [X ′] represents the charged molar ratio at the time of synthesis.

(In the formula, a is 1-1000, b is 1-500, and c is 1-400.)

<Preparation of polymers 4-1 to 4-4>
Polymerization by dehydration condensation reaction using a raw material having a carboxylic acid at both ends corresponding to moiety X or optionally X ′ and a raw material having an amine at both ends corresponding to moiety Y or optionally Y ′, by the polymerization by dehydration condensation reaction. A polymer represented by formula (1-1), formula (1-2), or formula (1-3) was prepared.

  Polymerization was performed by stirring ω, ω′-dicarboxyl PEG 1000 and spermidine (1.5 molar equivalent) with 1.2 equivalents of condensing agent DMT-MM in water-ethanol (1: 1) solvent. Obtained. The obtained polymer was purified by reprecipitation or dialysis (MWCO1000) and pulverized by lyophilization to obtain polymer 4-1. The number average molecular weight of the polymer 4-1 was about 20,000.

  Polymer 4-2 was prepared from 3,5-dithiaoctane-1,8-dicarboxylic acid, spermidine (1.5 molar equivalent) and 1,4-butanediamine (0.3 molar equivalent) in the same manner as polymer 4-1. Prepared. The number average molecular weight of the polymer 4-2 was about 5000.

  Polymer 4-3 was prepared from ω, ω′-dicarboxyl PEG 1000, ω, ω′-dicarboxyl PPG640 (2 molar equivalent) and lysine protected with a carboxyl group (4.8 molar equivalent) from polymer 4-1 Prepared in the same manner. The number average molecular weight of the polymer 4-3 was about 10,000.

  Polymer 4-4 was prepared in the same manner as Polymer 4-1 from ω, ω'-diaminoPPG640 and amino-protected glutamic acid (1.5 molar equivalents). The number average molecular weight of the polymer 4-4 was about 10,000.

Table 4 shows the structures of the polymers 4-1 to 4-4. In Table 4, [X]: [X ′] and [Y]: [Y ′] indicate the charged molar ratio at the time of synthesis.

<Preparation of polymers 5-1 to 5-4>
A polymer represented by the formula (2-1) was prepared by using dehydration condensation reaction or radical addition reaction for polymerization.

  Butoxyethoxyacetic acid and ε-polylysine were stirred with 1.2 equivalents of DMT-MM in water to introduce a butoxyethoxyacetamide moiety into the side chain of ε-polylysine. The obtained polymer was purified by reprecipitation or dialysis, and powdered by lyophilization to obtain polymer 5-1. The number average molecular weight of the polymer 5-1 was about 6000.

  By stirring N, N-bis (ethoxyethyl) amine and poly-γ-glutamic acid with 1.2 equivalents of DMT-MM in water, N, N-bis (ethoxyethyl) is added to the side chain of poly-γ-glutamic acid. ) An amide moiety was introduced. The obtained polymer was purified by reprecipitation or dialysis, and pulverized by lyophilization to obtain polymer 5-2. The number average molecular weight of the polymer 5-2 was about 10,000.

  Polymerization was carried out by stirring a PPG640 derivative having a carboxylic acid group and an amine group at the end and histidine with 1.2 equivalents of DMT-MM in a water-ethanol (1: 1) solvent. The obtained polymer was purified by reprecipitation or dialysis, and pulverized by lyophilization to obtain polymer 5-3. The number average molecular weight of the polymer 5-3 was about 20,000.

  Polymerization was carried out by heating a mixture of two types of monomers having a carbon-carbon double bond, vinyl (diethylene glycol) ether and styrene boronic acid and AIBN (0.01 equivalents) to 60 ° C. in acetone. The obtained polymer was purified by reprecipitation or dialysis (MWCO1000), and pulverized by lyophilization to obtain polymer 5-4. The number average molecular weight of the polymer 5-4 was about 5000.

Table 5 shows the structures of the polymers 5-1 to 5-4. In Table 5, [X]: [Y] indicates the charged molar ratio at the time of synthesis.

<Production of culture vessel>
Culture container of Examples 1-7 About the typical example of the polymer obtained above, the polymer was introduce | transduced into the culture container surface and the culture container provided with this polymer on the surface was produced.

  A 1 wt% EDC / HCl solution (pH 5.8) was allowed to act at 40 ° C. for 2 hours on a polystyrene 6-well dish having carboxylic acid groups as surface functional groups. Thereafter, the 6-well dish was washed with a pH 5.8 buffer solution, and 0.1 wt% of the polymer having primary or secondary amine groups (Polymer 1-1, 2-2, 3-3, 4-4) The solution (pH 5.8) was allowed to act at 40 ° C. for 12 hours. Thereafter, the 6-well dish was washed with PBS of pH 7.4, then washed with pure water, dried, and sterilized with ultraviolet light to produce the culture containers of Examples 1 to 4, respectively.

  Further, in a polystyrene 6-well dish having amine groups as surface functional groups, 0.1% by weight of a polymer having a carboxylic acid group (polymers 1-4, 2-4, 5-2) and 1% by weight of DMT-MM. % 5.8 buffer solution was allowed to act at 40 ° C. for 2 hours. Thereafter, the 6-well dish was washed with PBS of pH 7.4, then washed with pure water, dried, and sterilized with ultraviolet light to produce the culture containers of Examples 5-7.

Culture Containers of Comparative Examples 1 to 9 The culture containers of Comparative Examples 1 to 5 are ε-polylysine, poly-γ-glutamic acid, polyvinylamine, amine-terminated poly (N- Isopropyl acrylamide) or gelatin (polymers 6-1, 6-2, 6-3, 6-4, or 6-5, respectively) is introduced into the surface of the culture container in the same manner as in the culture containers of Examples 1-4. Produced.

  In the culture containers of Comparative Examples 6 and 7, polyethylene glycol or polypropylene glycol (referred to as polymers 6-6 or 6-7, respectively) is used as a compound that does not include a site Y that exhibits responsiveness to stimuli other than temperature changes. In the same manner as in the culture container of the above-mentioned example, it was introduced into the surface of the culture container.

Table 6 shows the compound names or structures of the polymers 6-1 to 6-7.

  As a culture container of Comparative Example 8, an untreated culture container (culture container used in Examples 1 to 4 and Comparative Examples 1 to 5) having a carboxylic acid group as a surface functional group, the surface of which is not polymer-modified. Using. Further, as the culture vessel of Comparative Example 9, an untreated culture vessel (culture vessel used in Examples 5 to 7) having an amine group as a surface functional group, the surface of which was not polymer-modified, was used.

<Culture / Peel test>
Cell culture and peeling tests were performed using the culture containers of Examples 1 to 7 and the culture containers of Comparative Examples 1 to 9.

Cell culture and detachment tests were performed as follows:
The surface of the culture vessel was coated with laminin according to a general protocol, iPS cells were seeded in the culture vessel, and cultured in a CO ₂ incubator for 7 days. The medium and the like were in accordance with general feeder-less conditions. Thereafter, the culture vessel was taken out from the CO ₂ incubator, the medium was replaced with a predetermined stripping solution, and left at room temperature. After a certain time, the cells were detached when the culture vessel was lightly pipetted and stirred. Cells that did not detach were completely detached by enzyme treatment, counted, and added to the number of cells detached by the stimulus response to obtain the total cell count, and the detachment rate (number of cells detached by the stimulus response to the total number of cells) The ratio was calculated. The results are shown in Table 7. FIG. 3 is a photograph showing the progress of the cell detachment test for one of the culture containers of the examples (during culturing → after 5 minutes from the addition of the detachment solution (PBS) → after pipetting). In addition, when the undifferentiated state of the iPS cells cultured in the culture vessels 1 to 7 was confirmed by ALP staining, all the colonies were stained blue, and it was confirmed by visual observation that the undifferentiation rate was at least 90% or more. It was.

  From Table 7, in the culture containers of Examples 1 to 7, the survival rate of the detached cells was 85% or more. On the other hand, in the culture containers of Comparative Examples 1 to 9, the viability of the detached cells was very low. In the culture container using the polymer of the present invention, the survival rate of the detached cells was remarkably improved, and the polymer of the present invention was found to be very useful in the cell culture method and the cell detachment method.

<Cytotoxicity evaluation>
In order to evaluate the cytotoxicity of the polymer 2-2, a study was conducted to add a polymer to the medium when culturing iPS cells using a general protocol (Example 8). When all the polymers reach from the surface of the culture vessel, the polymer concentration in the medium is considered to be about 1 μg / well at the maximum. Therefore, in this evaluation, the culture was performed in a state where the polymer 2-2 was added to the medium at a concentration of 2 μg / well which is twice the estimated maximum value. As a result, in the normal medium containing no polymer, the growth rate was 68 times and survival rate was 91% on the 6th day, whereas in the medium added with 2 μg / well of the polymer, the growth rate was 63 on the 6th day. The survival rate was 95%, which was the same result as that of a normal medium. From this, it was found that there is no cytotoxicity derived from the polymer of the present invention in the use for immobilization in a culture vessel.

<Examination of polymer immobilization conditions and laminin treatment conditions>
In the polymer 2-2, the robustness of the polymer immobilization and laminin treatment conditions was examined (Example 9).

  Change the concentration of the polymer added to the 6-well dish after EDC / HCl activation within the range of 0.1 to 0.01% by weight, and change the laminin addition condition within the range of the normal concentration to 1/10 of the normal concentration. The same examination as Example 2 was implemented using this. As a result, within the examined range, the cell proliferation rate, the cell detachment rate due to the stimulus response, and the survival rate were almost the same, and the robustness was confirmed.

Claims (10)

  A stimulus-responsive polymer having as constituent units a site X exhibiting temperature responsiveness and a site Y exhibiting responsiveness to stimuli other than temperature changes, wherein the site X exhibiting temperature responsiveness is an ether partial structure or sulfide The stimulus-responsive polymer, wherein the site Y having a partial structure and exhibiting responsiveness to a stimulus other than a temperature change includes a functional group having a pKa in water of 2 or more and 12 or less.   The stimuli-responsive polymer according to claim 1, wherein the site X exhibiting temperature responsiveness has two or more ether partial structures or sulfide partial structures.   The stimuli-responsive polymer according to claim 2, wherein the temperature-responsive portion X has at least one selected from a polyethylene glycol partial structure, a polypropylene glycol partial structure, a polybutylene glycol partial structure, and a polypentylene glycol partial structure. .   The stimulus-responsive polymer according to claim 1, wherein the site Y exhibiting responsiveness to a stimulus other than a temperature change includes a functional group having a pKa in water of 3 to 11.   The site Y showing responsiveness to stimuli other than temperature change is at least selected from carboxylic acid groups, amine groups, imine groups, thioether groups, boronic acid groups, phosphoric acid groups, nitrogen-containing aromatic groups, and phenolic hydroxyl groups. 5. The stimulus responsive polymer of claim 4, having one.   The stimulus-responsive polymer according to claim 1, further comprising a site X ′ or Y ′ that regulates physical properties.   The stimuli-responsive polymer of claim 1, which can exhibit a lower critical solution temperature in an aqueous solution.   A culture vessel provided with the stimulus-responsive polymer according to any one of claims 1 to 7 on a surface thereof.   A culture method in which the cells are adhered and cultured by bringing the cell suspension into contact with the culture container according to claim 8. A cell comprising a process of changing the temperature, pH, salt concentration, chemical potential, redox potential or light intensity to which the cell is exposed, in a state where the cell is adhered to the culture container according to claim 8. Peeling method.

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