JP2017012019A - Cell structure manufacturing method, cell structure - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce a cell structure including a lumen-shaped cell structure that extends inside a lump-state cell structure in a mesh state.SOLUTION: A method for producing a cell structure comprises: a preparation process of preparing a temperature-responsive polymer composition; a providing process of coating a first culture surface and a second culture surface of a cell culture vessel with a temperature-responsive polymer composition to provide a first coated region and a second coated region respectively having surface zeta potential at 0 to 50 mV; an inoculation process which comprises a first inoculation process of inoculating a first cell on the first coated region and a second inoculation process of inoculating a second cell on the second coated region; a pre-culture process which comprises a first culture process of culturing the inoculated first cell to prepare a lumen-shaped cell structure and a second culture process of culturing the inoculated second cell to prepare a sheet-shaped cell structure; a sticking process of sticking the lumen-shaped cell structure onto the sheet-shaped cell structure to prepare a composite cell structure; and a post-culture process of culturing the composite cell structure. The present invention also provides the cell structure produced by the production method.SELECTED DRAWING: Figure 1

Description

  The present invention particularly relates to a method for producing a cell structure including a tubular cell structure extending like a mesh inside a massive cell structure, and a cell structure produced by the production method.

  In recent years, it has been desired to realize tailor-made medical care for the purpose of improving a patient's QOL. In tailor-made medical care, regenerative medicine plays a major role in regenerating tissues and organs that have fallen into dysfunction or deficiency using the patient's own cells.

  Regenerative medicine requires an operation in which cells collected from a patient's tissue are cultured in a cell incubator to form the tissue, and then the tissue is transplanted into the patient. Therefore, a technique for culturing cells to form a cell structure such as a tissue and a technique for recovering the cell structure as it is are desired.

  As a method of forming a cell structure having a three-dimensional structure that mimics the structure of a tissue, for example, the cell culture vessel is coated with a specific cell culture composition and the cell culture vessel is coated in a lump shape. A method of obtaining a cell structure in the form of (pellet) or lumen (tube) is known (see Patent Document 1).

JP 2014-027919 A

  Although the method described in Patent Document 1 is an excellent method capable of producing a cell structure in a lump shape (pellet shape) or a luminal shape (tube shape), which is easily sized with the naked eye, There was room for improvement in order to realize the formation of a complex cell structure including a cell structure having a plurality of quasi-organized structures.

  Therefore, an object of the present invention is to produce a cell structure including a tubular cell structure extending like a mesh inside a massive cell structure.

The gist of the present invention is as follows.
The method for producing the cell structure of the present invention comprises:
Preparing a temperature-responsive polymer or temperature-responsive polymer composition; and
A first coated region and a second coated region, each of which covers the first culture surface and the second culture surface of the cell culture vessel with the temperature-responsive polymer composition, and has a surface zeta potential of 0 to 50 mV, respectively. Preparing the preparation process,
A seeding step comprising: a first seeding step of seeding the first cells on the first coating region; and a second seeding step of seeding a second cell on the second coating region;
A first culturing step of culturing the seeded first cell to prepare a luminal cell structure, and culturing the seeded second cell to prepare a sheet-like cell structure. A pre-culture process comprising two culture processes;
Affixing the luminal cell structure on the sheet-like cell structure to prepare a composite cell structure;
And a post-culturing step for culturing the composite cell structure.

  Moreover, in the manufacturing method of the cell structure of this invention, it is preferable that the contact angle of water with respect to a said 1st coating area | region and a 2nd coating area | region is 50-90 degrees.

Furthermore, in the method for producing a cell structure of the present invention, the cell density of the first cells in the first seeding step is 100 to 300 cells / mm ^2, and the cells of the second cells in the second seeding step The density is preferably 400 to 1,200 pieces / mm ² .

  Furthermore, in the method for producing a cell structure of the present invention, it is preferable that the first cell is a vascular endothelial cell and the second cell is a smooth muscle cell.

  Furthermore, in the method for producing a cell structure of the present invention, the temperature-responsive polymer or the temperature-responsive polymer composition includes a 2-N, N-dimethylaminoethyl methacrylate (DMAEMA) unit and an anionic monomer unit. Temperature-responsive polymer; temperature-responsive polymer comprising N-isopropylacrylamide (NIPAM) units, cationic monomer units, and anionic monomer units; 2-N, N-dimethylaminoethyl methacrylate (DMAEMA) and / or its Derivative polymer, 2-amino-2-hydroxymethyl-1,3-propanediol (Tris), nucleic acid, heparin, hyaluronic acid, dextran sulfate, polystyrene sulfonate, polyacrylic acid, polymethacrylic acid, polyphosphoric acid , Sulfated polysaccharides, curdlan and polyarginine And at least one temperature-responsive polymer or temperature-responsive polymer selected from the group consisting of: a temperature-responsive polymer composition comprising one or more anionic substances selected from the group consisting of these alkali metal salts; A composition is preferred.

  Furthermore, in the method for producing a cell structure of the present invention, the anionic monomer is at least selected from the group consisting of acrylic acid, methacrylic acid, and a vinyl derivative having a carboxyl group, a sulfonic acid group, and a phosphoric acid group in the side chain. One type is preferable.

  Furthermore, in the method for producing a cell structure of the present invention, the cationic monomer is 3- (N, N-dimethylaminopropyl) -meth (a) acrylamide, 3- (N, N-dimethylaminopropyl) -meta. It is selected from the group consisting of (a) acrylate, aminostyrene, 2- (N, N-dimethylaminoethyl) -meth (a) acrylamide, 2- (N, N-dimethylaminoethyl) -meth (a) acrylate. It is preferable that there is at least one.

  The cell structure of the present invention is produced by the method for producing a cell structure of the present invention described above.

  The cell structure of the present invention is characterized by including a luminal cell structure extending in a mesh shape inside a massive cell structure.

  In the cell structure of the present invention, it is preferable that the tubular cell structure includes vascular endothelial cells, and the massive cell structure includes smooth muscle cells.

  ADVANTAGE OF THE INVENTION According to this invention, the cell structure containing the tubular cell structure extended in a mesh shape inside a lump-like cell structure can be manufactured.

(I)-(ix) is a figure shown about the outline | summary of the manufacturing method of the cell structure of embodiment of this invention. It is the photograph when the composite cell structure which contains the vascular endothelial cell extended in mesh shape inside the smooth muscle cell prepared in the Example of this invention was observed. (A) is a photograph when the complex cell structure is observed with a stereomicroscope, and (b) is a photograph when the complex cell structure is observed with a fluorescence phase contrast microscope. In (b), the vascular endothelial cells emit green fluorescence, and the smooth muscle cells emit red fluorescence.

  Hereinafter, with reference to drawings, the manufacturing method of the cell structure of the present invention and the embodiment of the cell structure of the present invention will be described in detail.

(Method for producing cell structure)
A method for producing a cell structure according to an embodiment of the present invention includes:
Preparing a temperature responsive polymer composition or temperature responsive polymer composition; and
A first coated region and a second coated region, each of which covers the first culture surface and the second culture surface of the cell culture vessel with the temperature-responsive polymer composition, and has a surface zeta potential of 0 to 50 mV, respectively. Preparing the preparation process,
A seeding step comprising: a first seeding step of seeding the first cells on the first coating region; and a second seeding step of seeding a second cell on the second coating region;
A first culturing step of culturing the seeded first cell to prepare a luminal cell structure, and culturing the seeded second cell to prepare a sheet-like cell structure. A pre-culture process comprising two culture processes;
Affixing the luminal cell structure on the sheet-like cell structure to prepare a composite cell structure;
A post-culture step of culturing the composite cell structure.

  1 (i) to (ix) show an outline of a method for producing a cell structure according to an embodiment of the present invention.

  In the method for producing a cell structure according to an embodiment of the present invention, the cell structure has a plurality of quasi-organized structures with extremely high viability (activity), which are formed after progressing while growing on the culture surface. Cell structures can be used. Therefore, the composite cell structure produced also has extremely high viability (activity), and it is possible to perform tests according to various assays on tissues.

  Details of each step will be described below.

  In the method for producing a cell structure according to an embodiment of the present invention, first, a temperature-responsive polymer or a temperature-responsive polymer composition is prepared (preparation step).

Examples of the temperature-responsive polymer and the temperature-responsive polymer composition used in the cell culture vessel of the embodiment of the present invention include (A) 2-N, N-dimethylaminoethyl methacrylate (DMAEMA) unit, anionic monomer unit, Temperature-responsive polymer containing (B) N-isopropylacrylamide (NIPAM) unit, cationic monomer unit, and anionic monomer unit, (C) 2-N, N-dimethylaminoethyl Polymer of methacrylate (DMAEMA) and / or its derivative, 2-amino-2-hydroxymethyl-1,3-propanediol (Tris), nucleic acid, heparin, hyaluronic acid, dextran sulfate, polystyrene sulfonate, polyacryl Acid, polymethacrylic acid, polyphosphoric acid, sulfated polysaccharide, card Emissions and polyalginic acid and the temperature-responsive polymer composition comprising one or more anionic material selected from the group consisting of alkali metal salts and the like.
Here, as the above (A), for example, (A-1) a temperature-responsive polymer obtained by a method of polymerizing DMAEMA in the presence of water, (A-2) a polymer block mainly containing DMAEMA (polymer chain α-terminal) And a temperature-responsive polymer mainly containing DMAEMA and an anionic monomer (polymer chain ω terminal).
In the embodiment of the present invention, these may be used alone or in combination of two or more.
Details of the temperature-responsive polymers (A-1), (A-2), and (B) will be described later.

  Next, in the method for producing a cell structure according to the embodiment of the present invention, the first cultivating surface and the second culturing surface of the cell culture vessel are coated with the temperature-responsive polymer composition, respectively, and the first coated region, respectively. And preparing a second coating region (preparation step). Here, the surface zeta potential of the first culture surface and the second culture surface is 0 to 50 mV.

In the example shown in FIG. 1, for convenience in a pasting step described later, a cell culture vessel used for preparing a massive cell structure is provided with a substrate having a smaller bottom surface compared to the bottom surface of the cell culture device, and the substrate. Cell culture used for preparing a sheet-like cell structure using a jig provided on the top and having a projection having a top surface and a side surface (see FIGS. 1 (i) and (ii)) This jig is not used in the vessel.
In this example, the top surface of the jig shown in FIG. 1 (ii) is the first culture surface, and the culture surface of the cell incubator shown in FIG. 1 (v) is the second culture surface. The temperature responsive polymer composition is coated to form a first coated region and a second coated region, respectively.

  Examples of the material of the cell incubator include polystyrene, polyethylene terephthalate (PET), polypropylene, polybutene, polyethylene, polycarbonate, etc., precise molding is easy, and various sterilization methods can be applied. Polystyrene and polyethylene terephthalate (PET) are preferable from the viewpoint that they are transparent and suitable for microscopic observation.

Of the jig, the portions other than the top surfaces of the protrusions coated with the temperature-responsive polymer composition, that is, the side surfaces of the substrate and the protrusions are preferably non-cell-adhesive.
“Cell non-adhesiveness” refers to the property that adherent cells (eg, fibroblasts, hepatocytes, vascular endothelial cells, etc.) do not adhere or are difficult to adhere under normal culture conditions.
According to such a configuration, it is possible to suppress the seeded cells from adhering to an uncoated region other than the coated region on the culture surface. Non-adherent cells can be removed from the cell incubator by a simple operation such as medium exchange, which suppresses adverse effects on the growth of adherent cells such as apoptosis and heat shock protein production by these cells. It becomes possible to do.

The surface zeta potential of the first coating region and the second coating region is set to 0 to 50 mV. If the surface zeta potential is 0 mV or more, negatively charged cells can be adhered, and if it is 50 mV or less, cytotoxicity can be reduced.
For the same reason as described above, the surface zeta potential is preferably 0 to 35 mV, and more preferably 10 to 25 mV.

According to the cell culture device provided with the first coating region and the cell culture device provided with the second coating region, after culturing the cells in a general 37 ° C. incubator to form a tissue, the cloud point Under the following conditions, for example, cells having a cell sheet-like structure can be collected without performing a cell detachment operation such as trypsin treatment.
Therefore, if cells are cultured using this cell culture device, the cell culture operation can be performed under conditions of about room temperature.

Furthermore, according to the first coating region and the second coating region having the surface zeta potential in the above specific range, a lumpy structure (pellet shape) or a luminal shape (pellate shape) can be obtained simply by culturing cells under appropriate culture conditions. A cell structure having a (tubular) structure can be easily formed.
This is presumed that the surface zeta potential in the specific range gives a special stimulus to the cells. For example, when a temperature-responsive polymer having a cationic group and an anionic group in the side chain described later is used, it is presumed that these side chain functional groups are stimulating the cell while interacting in some way. The In addition, the temperature-responsive polymer having a surface zeta potential in the specific range described above suppresses cytotoxicity by optimizing the balance between positive charge and negative charge (the surface of the cell membrane of mammalian cells is negatively charged). Therefore, it is presumed that the cationic substance often has cytotoxicity), and that the charge balance of the temperature-responsive polymer is suitable to allow cell migration and orientation.

  Since the cell structure produced in the first coated region and the second coated region is formed after passing through the growth surface and growing on the culture surface, the extracellular matrix produced between the cells. Is abundant. Therefore, the viability (activity) of the cell structure itself is extremely high.

In the first coating region and second coating region has a per unit area, the amount of temperature-responsive polymer is preferably 5.0~50ng / mm ^2, further to be 15-40 / mm ² preferable. If it is the said range, the effect of making it easy to form a cell structure will be easy to be acquired.

  In the first coating region and the second coating region, the temperature-responsive polymer is precipitated from an aqueous solution state, solution coating and solvent drying, radiation irradiation, low-temperature plasma irradiation, corona discharge, glow discharge, ultraviolet light, radical generator. The culture surface of the cell culture vessel may be coated by a technique such as graft polymerization using

Moreover, in the manufacturing method of the cell structure of embodiment of this invention, it is preferable that the contact angle of the water with respect to a 1st coating area | region and a 2nd coating area | region is 50-90 degrees from a viewpoint of improving the effect of this invention. 60 to 80 ° is more preferable, and 62 to 78 ° is particularly preferable.
In addition, the contact angle of water with respect to the coating region refers to an average value of contact angles measured at an arbitrary number of points in the coating region in accordance with JIS R3257.

  Subsequently, in the method for producing a cell structure according to the embodiment of the present invention, cells are seeded on the aforementioned culture surface (seeding step). Specifically, first cells are seeded on the first coated region (first seeding step), and second cells are seeded on the second coated region (second seeding step).

  In this step, for example, the cell culture device provided with the first culture surface and the cell culture device provided with the second culture surface, which have been allowed to stand in a 37 ° C. incubator, are taken out to a clean bench at room temperature, It can be performed by adding cells and a cell culture medium to each of them (see FIG. 1).

Cell density of the first cell in the first seeding step is preferably 100-300 / mm ^2, further preferably 120 to 300 pieces / mm ^2, is 150 to 300 pieces / mm ² It is particularly preferred.
Cell density of the first cell in the second seeding step, more preferably from preferably from 400～1,200 pieces / mm ^2, a 800 to 1,200 pieces / mm ^2, 1,000 pieces It is especially preferable that it is ˜1,200 pieces / mm ² .

Here, as the first cells that can be introduced inside the cell structure in the method for producing the cell structure of the embodiment of the present invention, vascular endothelial cells, vascular stromal cells, Flk-1 positive stem cells, CD31 positive cells In particular, vascular endothelial cells and vascular stromal cells are preferred from the viewpoint of forming a luminal network in a self-organizing manner without adding additives such as growth hormones and cytokines. A 1st cell may be used individually by 1 type, and may use 2 or more types together.
In addition, examples of the second cell that can be located mainly outside the cell structure include smooth muscle cells, fibroblasts, mesenchymal stem cells, and the like. From the viewpoint of facilitating imitation of the blood vessel structure, mesenchymal stem cells and smooth muscle cells are preferred. A 2nd cell may be used individually by 1 type, and may use 2 or more types together.

  In addition, a culture medium, the additive included in this, etc. can be appropriately determined by those skilled in the art based on the cell type and the purpose of the experiment.

  And in the manufacturing method of the cell structure of embodiment of this invention, the seeded cell is cultured (preculture process). The seeded first cells are cultured to prepare a tubular cell structure (first culture step), and the seeded second cells are cultured to prepare a sheet-like cell structure. (Second culture step).

  This step can be performed using, for example, a general 37 ° C. cell incubator (see FIG. 1).

In the pre-culture process (the first culture process and the second culture process), it is preferable that the seeded cells are subjected to stationary culture.
Although it does not specifically limit as culture | cultivation temperature in a 1st culture process and a 2nd culture process, For example, it is preferable that it is 30 degreeC or more, it is further more preferable that it is 35 degreeC or more, and it is 38 degrees C or less. Preferably, it is 37 degreeC.
The culture time in the first culture step and the second culture step is not particularly limited because the growth rate differs depending on the cell type, and the growth rate varies depending on the state and the number of passages even for the same type of cells. It is preferable that the time does not exceed the state of the conjugation determined by the trader. For example, it is usually preferable that the time is about 3 to 168 hours.

  In the first culturing step, the first cells adhered to the first coated region contract in some places, are detached from the first coated region along with the contraction, and have a network having a tubular structure on the first coated region. A network composed of a cellular structure is formed (see FIG. 1 (iii)).

  In the second culturing step, the second cells adhered to the second coating region extend and proliferate on one surface of the second coating region, sometimes forming a single-layer sheet-like cell structure (FIG. 1 (vi )reference).

  In particular, the culturing in the second culturing step is preferably completed before the phenomenon of aggregation of sheet-like cell structures occurs in the below-described pasting step. Usually, it is preferably completed within about 3 to 21 hours.

  Next, in the method for producing a cell structure according to an embodiment of the present invention, a luminal cell structure is pasted on a sheet-like cell structure to prepare a composite cell structure (pasting step).

In this step, for example, a jig provided with a luminal cell structure cultured in the first covering region is turned upside down so that a top surface thereof becomes a bottom surface, and a luminal cell structure is obtained by It sticks on the sheet-like cell structure culture | cultivated in the 2 covering area | region (refer FIG. 1 (vii), (viii)).
Here, when a luminal cell structure is affixed to a sheet-like cell structure, the luminal cell structure may be brought into contact with or pressed against the sheet-like cell structure. It may be transferred using a spatula or the like accordingly.

  In the example shown in FIG. 1, the area of the top surface of the jig is smaller than the area of the culture surface of the cell culture vessel for culturing the second cell, but in the embodiment of the present invention, In order to spread the vascular network to be embedded in the obtained composite cell structure throughout the cell structure, the area of the top surface of the jig is the area of the culture surface of the cell incubator for culturing the second cell It is preferable that they are substantially the same. In this case, the luminal cell structure is preferably attached to the entire sheet-like cell structure.

Finally, in the method for producing a cell structure according to the embodiment of the present invention, the composite cell structure is cultured (post-culture step).
This step can be performed, for example, using a general 37 ° C. cell incubator.

  In the post-culture process, the second cells that have adhered to the second coating region shrink while curling from the edge of the top surface of the jig toward the center. It peels from the 2nd covering field. And a 2nd cell aggregates in the center part of a 2nd coating area | region, entraining the tubular cell structure stuck in the sticking process. Then, the cells gathered together spontaneously gather as if to form some kind of tissue, forming a three-dimensional massive cell structure in which luminal cell structures are stretched (FIG. 1). (See (ix)).

  Hereinafter, the temperature-responsive polymer composition used in the method for producing a cell structure according to an embodiment of the present invention will be described in detail.

  Hereinafter, the temperature-responsive polymer (A-1) and the production method thereof will be described.

(Method for producing temperature-responsive polymer)
In the method for producing a temperature-responsive polymer (A-1), first, a mixture containing 2-N, N-dimethylaminoethyl methacrylate (DMAEMA) is prepared (preparation step). Here, the mixture further includes a polymerization inhibitor and water.

  A commercially available product can be used as 2-N, N-dimethylaminoethyl methacrylate (DMAEMA). Examples of the polymerization inhibitor include methylhydroquinone (MEHQ), hydroquinone, p-benzoquinoline, N, N-diethylhydroxylamine, N-nitroso-N-phenylhydroxylamine (Cupperron), t-butylhydroquinone, and the like. Further, MEHQ or the like contained in commercially available DMAEMA may be used as it is. Examples of water include ultrapure water.

The weight ratio of the polymerization inhibitor to the mixture is preferably 0.01% to 1.5%, and more preferably 0.1% to 0.5%. If it is the said range, the runaway of radical polymerization reaction can be suppressed, the uncontrollable bridge | crosslinking can be reduced, and the solubility with respect to the solvent of the temperature-responsive polymer manufactured can be ensured.
The weight ratio with respect to the water mixture is preferably 1.0% to 50%, and more preferably 9.0% to 33%. If it is the said range, the reaction rate of the hydrolysis reaction of a side chain and the reaction rate of the growth reaction of the polymer chain to superpose | polymerize can be harmonized in good balance. Thereby, the temperature-responsive polymer whose ratio (copolymerization ratio) of DMAEMA in which the side chain is not hydrolyzed with respect to DMAEMA in which the side chain is hydrolyzed can be obtained.

Next, in the method for producing a temperature-responsive polymer (A-1), the mixture is irradiated with ultraviolet rays (irradiation step). Here, ultraviolet rays are irradiated in an inert atmosphere. DMAEMA undergoes radical polymerization upon irradiation with ultraviolet rays to form a polymer.
In this step, for example, after the mixture is added to a transparent sealed vial and the inside of the vial is made an inert atmosphere by bubbling an inert gas, ultraviolet rays are irradiated from the outside of the vial using an ultraviolet irradiation device.

The wavelength of the ultraviolet light is preferably 210 nm to 600 nm, and more preferably 360 nm to 380 nm. If it is the said range, a polymerization reaction can be advanced efficiently and the polymeric material which has a desired copolymerization ratio can be obtained stably. In addition, the manufactured polymer material can be prevented from being colored.
Examples of the inert gas include nitrogen, argon, helium, neon and the like.

Regarding the reaction conditions, the temperature conditions are preferably 15 ° C. to 50 ° C., more preferably 20 ° C. to 30 ° C. If it is set as the said range, the start reaction by a heat | fever can be suppressed and the start reaction by light irradiation can be advanced preferentially. Further, the reaction rate of the hydrolysis reaction can be balanced with respect to the reaction rate of the growth reaction of the polymer chain.
The reaction time is preferably 7 hours to 24 hours, and more preferably 17 hours to 21 hours. If it is the said range, a temperature-responsive polymer can be obtained with a high yield, and radical polymerization can be performed, suppressing a photodecomposition reaction and an unnecessary crosslinking reaction.

  In addition, it is preferable that it is 10 minutes-1 hour after completing preparation of a mixture in a preparation process until it starts irradiation of an ultraviolet-ray in an irradiation process.

  It takes about 10 minutes to replace the gas inside the vial to which the mixture has been added to create an inert atmosphere inside the vial. Therefore, if the time is less than 10 minutes, there is a possibility that an inert atmosphere necessary for radical polymerization cannot be obtained. Further, in the mixture, the hydrolysis reaction of DMAEMA is started before the irradiation with ultraviolet rays is started. Therefore, if the above time is longer than 1 hour, a large number of methacrylic acid that is inert to the radical polymerization reaction is generated in the mixture.

In the method for producing a temperature-responsive polymer of (A-1), since water is contained in the mixture, the radical polymerization reaction of DMAEMA and the ester bond of the side chain of poly-2-N, N-dimethylaminoethyl methacrylate (PDMAEMA) The hydrolysis reaction can be antagonized.
The product obtained by this antagonism is the repeating unit (A) represented by the formula (I)
And the repeating unit (B) represented by the formula (II)
It becomes a polymer containing.
Therefore, both the cationic functional group that the polymer has, that is, the dimethylamino group, and the anionic functional group that the polymer has, that is, the carboxyl group that is formed by hydrolyzing the ester bond of the side chain must be provided in a balanced manner. it can. And according to the manufacturing method of the temperature-responsive polymer of (A-1), there are few polymers derived from poly (2-N, N-dimethylaminoethyl methacrylate) having a cationic functional group and an anionic functional group. It can be easily manufactured in the process.

In addition, even if it is not the same manufacturing method as the manufacturing method of the temperature-responsive polymer of (A-1), if DMAEMA, a polymerization inhibitor, and water coexist in the reaction system at the time of ultraviolet irradiation, The same effect as the above-described effect of the method for producing a temperature-responsive polymer can be obtained.
For example, a mixture containing DMAEMA and a polymerization inhibitor and water are prepared separately, and then an inert gas is bubbled into the mixture and water, and then the mixture and water are mixed under an inert atmosphere and at the same time UV The method for producing a temperature-responsive polymer in which A is irradiated can also be included in the temperature-responsive polymer of (A-1).

(Temperature responsive polymer)
The temperature-responsive polymer (A-1) is produced by the production method (A-1).

Here, the temperature-responsive polymer (A-1) is preferably a molecule having a number average molecular weight (Mn) of 10 kDa to 500 kDa. Further, a molecule having a ratio (Mw / Mn) of the weight average molecular weight (Mw) to the number average molecular weight (Mn) is preferably 1.1 to 10.0.
The molecular weight of the temperature-responsive polymer can be adjusted as appropriate depending on the conditions of irradiation time and irradiation intensity of ultraviolet rays.

  According to the temperature-responsive polymer (A-1), the cloud point can be lowered to, for example, room temperature (25 ° C.) or lower.

  In the temperature-responsive polymer (A-1), the time until the insolubilized product of the temperature-responsive polymer formed at a temperature equal to or higher than the cloud point is redissolved under room temperature (about 25 ° C.) conditions is remarkably delayed. To do. It is presumed that this is because the obtained temperature-responsive polymer has high self-aggregation properties because of the presence of a cationic functional group and an anionic functional group in the molecule.

  Further, using the temperature-responsive polymer of (A-1), as described later, it is possible to prepare a cell culture vessel in which the culture surface is coated with this temperature-responsive polymer.

  Further, according to the temperature-responsive polymer of (A-1), as described later, a cell structure having a tubular (tubular) structure is formed by culturing cells under appropriate culture conditions. be able to.

  The ratio of the number of functional groups of the cationic functional group (2-N, N-dimethylamino group) and the number of functional groups of the anionic functional group (carboxyl group) of the temperature-responsive polymer of (A-1) (C / A ratio) is preferably 0.5 to 32, and more preferably 4 to 16.

  When the C / A ratio is in the above range, the above effect of reducing the cloud point can be easily obtained. In the temperature-responsive polymer having the C / A ratio, the cationic functional group and the anionic functional group act on the intermolecular and / or intramolecular aggregation in an ionic bond in the temperature-responsive polymer, This is presumed to be a result of increasing the cohesive force of the temperature-responsive polymer.

  In addition, when the C / A ratio is in the above range, the balance between the positive charge and the negative charge in the temperature-responsive polymer can be particularly suitable, and the cytotoxicity due to the positive charge can be suppressed. It is presumed that the balance between hydrophilicity and hydrophobicity of the temperature-responsive polymer can be made particularly suitable to facilitate cell migration and orientation.

(Method for producing temperature-responsive polymer)
In the method for producing a temperature-responsive polymer (A-2), first, ultraviolet light is irradiated to a first mixture containing 2-N, N-dimethylaminoethyl methacrylate (DMAEMA) (first polymerization step).
Here, in addition to DMAEMA, the first mixture may optionally include, for example, other monomers, solvents, and the like.
Moreover, ultraviolet rays may be irradiated in an inert atmosphere.

DMAEMA may be a commercially available product.
Examples of other monomers that can be included in the first mixture include N, N-dimethylacrylamide, acrylic acid and methacrylic acid esters having a polyethylene glycol side chain, N-isopropylacrylamide, and 3-N, N-dimethylaminopropyl. Examples include acrylamide, 2-N, N-dimethylaminoethyl methacrylamide and the like, and in particular, from the viewpoint of enabling stable adjustment of ion balance, N, N-dimethylacrylamide and polyethylene glycol side chains. Preference is given to esters of acrylic acid and methacrylic acid and N-isopropylacrylamide. These may be used alone or in combination of two or more. Here, the ratio (number of moles) of the usage amount of the other monomer to the usage amount of DMAEMA is preferably 0.001-1, and more preferably 0.01-0.5.

  Examples of the solvent include toluene, benzene, chloroform, methanol, ethanol, and the like. In particular, toluene and benzene are preferable because they are inert to the ester bond of DMAEMA. These may be used alone or in combination of two or more.

  In this step, for example, the first mixture is added to a transparent sealed vial and the inside of the vial is made an inert atmosphere by bubbling an inert gas, and then irradiated with ultraviolet rays from the outside of the vial using an ultraviolet irradiation device. To do.

The ultraviolet wavelength is preferably 210 to 600 nm, and more preferably 360 to 380 nm. If it is the said range, a polymerization reaction can be advanced efficiently and the polymeric material which has a desired copolymerization ratio can be obtained stably. In addition, the manufactured polymer material can be prevented from being colored.
The irradiation intensity of ultraviolet light is preferably 0.01m~50mW / cm ^2, further preferably 0.1m~5mW / cm ^2. If it is the said range, a polymerization reaction can be advanced stably at a suitable speed (time), suppressing decomposition | disassembly by the cutting | disconnection of a useless chemical bond, etc.
Examples of the inert gas include nitrogen, argon, helium, neon and the like.

As temperature conditions, it is preferable that it is 10-40 degreeC, and it is still more preferable that it is 20-30 degreeC. If it is the said range, it can react at room temperature of a normal laboratory, and it becomes possible to suppress reaction by means (heating etc.) different from light.
The reaction time is preferably 10 minutes to 48 hours, and more preferably 60 minutes to 24 hours.

  In this step, DMAEMA undergoes radical polymerization by irradiation with ultraviolet rays to form a polymer (poly (2-N, N-dimethylaminoethyl methacrylate) (PDMAEMA)), which contains 2-N, N-dimethylaminoethyl methacrylate. A homopolymer block is formed. When other monomers are also used, a polymer block containing DMAEMA and other monomers is formed.

Next, in the method for producing a temperature-responsive polymer of (A-2), the number average molecular weight of the polymer (specifically, polymerized 2-N, N-dimethylaminoethyl methacrylate) in the first polymerization step is predetermined. When it becomes more than the value, an anionic monomer is added to the first mixture to prepare a second mixture (addition step).
Here, in addition to the first mixture after the first polymerization step and the anionic monomer, the second mixture is, for example, another monomer, a solvent (toluene, benzene, methanol, etc.) that can be included in the first mixture, and the like. May be included.
The anionic monomer may be added under an inert atmosphere.

Examples of the anionic monomer include acrylic acid, methacrylic acid, vinyl derivatives having a carboxyl group, a sulfonic acid group, and a phosphoric acid group in the side chain. Particularly, from the viewpoint of chemical stability, acrylic acid, methacrylic acid, and the like. Acid is preferred.
These may be used alone or in combination of two or more.

  Examples of other monomers that can be included in the second mixture include N, N-dimethylacrylamide, acrylic acid and methacrylic acid esters having a polyethylene glycol side chain, N-isopropylacrylamide, and 3-N, N-dimethylaminopropyl. Examples thereof include acrylamide and 2-N, N-dimethylaminoethyl methacrylamide, and particularly, N, N-dimethylacrylamide which is electrically neutral and hydrophilic is preferable. These may be used alone or in combination of two or more. Here, the ratio (mole) of the use amount of the other monomer to the use amount of DMAEMA is preferably 0.01 to 10, and more preferably 0.1 to 5.

  In this step, for example, the second mixture is added while keeping the inside of the vial in an inert atmosphere by flowing an inert gas through the vial.

The predetermined value of the number average molecular weight is preferably 5,000, more preferably 20,000, and particularly preferably 100,000 from the viewpoint of sufficiently obtaining the effect of reducing the cloud point.
The number average molecular weight of the polymerized PDMAEMA in the first mixture after the first polymerization step is obtained by collecting a small amount of the reaction mixture from the polymerization system at a predetermined time, and performing gel permeation chromatography (GPC) or light scattering method. It can be measured by a method well known to those skilled in the art such as (SLS).

  In this step, in addition to the homopolymer containing DMAEMA during polymerization, an anionic monomer is also included in the polymerization system, and the polymerization system in the vial is changed from the DMAEMA homopolymerization system to the co-polymerization of DMAEMA and the anionic monomer. It will change to a polymerization system.

And in the manufacturing method of the temperature-responsive polymer of (A-2), a 2nd mixture is irradiated with an ultraviolet-ray (2nd polymerization process).
Here, the ultraviolet rays may be irradiated in an inert atmosphere.

  In this step, for example, ultraviolet rays are irradiated from the outside of the vial after adding the second mixture using an ultraviolet irradiation device.

  Various conditions such as the wavelength of ultraviolet rays, the irradiation intensity of ultraviolet rays, the inert gas used, the reaction temperature, and the reaction time in the second polymerization step may be the same as those in the first polymerization step.

  In this step, DMAEMA and anionic monomer are radically polymerized by irradiation with ultraviolet rays, and DMAEMA and anionic are continuously formed in the polymer chain α-terminal of the homopolymer block containing DMAEMA formed in the first polymerization step. A copolymer block containing monomers is formed. When other monomers are used, a copolymer block containing DMAEMA, an anionic monomer, and other monomers is formed.

  As described above, a temperature-responsive polymer including a homopolymer block containing DMAEMA and a copolymer block of DMAEMA and an anionic monomer is obtained.

  In addition, in the production method of (A-2), as understood by those skilled in the art, a mixture of polymers having various molecular weights and molecular structures is formed. A homopolymer block containing DMAEMA, DMAEMA and an anionic monomer, From the viewpoint of obtaining a temperature-responsive polymer containing the copolymer block as a main component, it is preferable to perform polymerization under the same conditions throughout the first polymerization step, the addition step, and the second polymerization step.

(Temperature responsive polymer)
The temperature-responsive polymer (A-2) is produced by the production method (A-2).

The temperature-responsive polymer of (A-2) mainly contains 2-N, N-dimethylaminoethyl methacrylate, and optionally a hydrophilic monomer such as acrylic acid or methacrylic acid having dimethylacrylamide and polyethylene glycol side chains. Polymer block containing other monomer units (polymer chain α-terminal), mainly comprising 2-N, N-dimethylaminoethyl methacrylate and anionic monomer (polymer chain ω-terminal), optionally other monomer units And a copolymer block containing.
Preferably, the temperature-responsive polymer of (A-2) comprises a DMAEMA homopolymer block and a copolymer block of DMAEMA and an anionic monomer, and more preferably consists of these blocks.

  Here, as the temperature-responsive polymer of (A-2), the number average molecular weight of the polymer block at the polymer chain α-terminal (for example, DMAEMA homopolymer block) is preferably 5000 Da or more, and 20000 Da or more. Is more preferable.

The temperature-responsive polymer (A-2) is preferably a molecule having a number average molecular weight (Mn) of 10 to 500 kDa. Further, a molecule having a ratio (Mw / Mn) of the weight average molecular weight (Mw) to the number average molecular weight (Mn) is preferably 1.1 to 10.0.
The molecular weight of the temperature-responsive polymer can be adjusted as appropriate depending on the conditions of irradiation time and irradiation intensity of ultraviolet rays.

  According to the temperature-responsive polymer (A-2), the cloud point can be lowered to, for example, room temperature (25 ° C.) or lower.

  In the temperature-responsive polymer of (A-2), the time until the insolubilized product of the temperature-responsive polymer formed at a temperature higher than the cloud point is redissolved at room temperature (about 25 ° C.) is remarkably delayed. To do. It is presumed that this is because the obtained temperature-responsive polymer has high self-aggregation properties because of the presence of a cationic functional group and an anionic functional group in the molecule.

  In particular, the temperature-responsive polymer of (A-2) has a DMAEMA homopolymer block having a high molecular weight (for example, 5000 Da or more) at the α-terminal of the polymer chain, so that the temperature-dependent globule transition of the side chain of DMAEMA Therefore, it is considered that the cloud point can be effectively reduced.

  In addition, using this temperature-responsive polymer, as described later, it is possible to prepare a cell culture vessel in which the culture surface is coated with this temperature-responsive polymer.

  Furthermore, according to the temperature-responsive polymer of (A-2), as described later, by culturing cells under appropriate culture conditions, it has a tubular (tubular) or massive (pellet-like) structure. Cell structures can be formed.

  The ratio of the number of functional groups of the cationic functional group (2-N, N-dimethylamino group) and the number of functional groups of the anionic functional group (carboxyl group) of the temperature-responsive polymer of (A-2) (C / A ratio) is preferably 0.5 to 32, and more preferably 4 to 16.

  When the C / A ratio is in the above range, the above effect of reducing the cloud point can be easily obtained. In the temperature-responsive polymer having the C / A ratio, the cationic functional group and the anionic functional group act on the intermolecular and / or intramolecular aggregation in an ionic bond in the temperature-responsive polymer, This is presumed to be a result of increasing the cohesive force of the temperature-responsive polymer.

  In addition, when the C / A ratio is in the above range, the balance between the positive charge and the negative charge in the temperature-responsive polymer can be particularly suitable, and the cytotoxicity due to the positive charge can be suppressed. It is presumed that the balance between hydrophilicity and hydrophobicity of the temperature-responsive polymer can be made particularly suitable to facilitate cell migration and orientation.

(Method for producing temperature-responsive polymer)
The method for producing the temperature-responsive polymer (B) is also referred to as N-isopropylacrylamide (NIPAM) (hereinafter also referred to as “monomer (A)”) and a cationic monomer (hereinafter referred to as “monomer (B)”). ) And an anionic monomer (hereinafter also referred to as “monomer (C)”). Optionally, other monomers may be added to the above three types of monomers for polymerization.

  N-isopropylacrylamide (NIPAM) may be a commercially available product.

Examples of the cationic monomer include monomers having a cationic functional group. Examples of the cationic functional group include amino groups such as primary to quaternary amino groups, guanidine groups, and the like. Tertiary amino groups are preferred from the viewpoints of the properties, low cytotoxicity, sterilization stability, and strong positive chargeability.
More specifically, the cationic monomer preferably has a high stability even when it supports a physiologically active substance or is subjected to alkaline conditions. For example, 3- (N, N-dimethylaminopropyl) -(Meth) acrylamide, 3- (N, N-dimethylaminopropyl)-(meth) acrylate, aminostyrene, 2- (N, N-dimethylaminoethyl)-(meth) acrylamide, 2- (N, N- Dimethylaminoethyl)-(meth) acrylate and the like.
Among these, 3- (N, N-dimethylaminopropyl) acrylamide is particularly preferable because it has a high positive charge strength and facilitates loading of an anionic substance.
In addition, aminostyrene has a high positive charge strength, so that it is easy to support an anionic substance and the aromatic ring in the molecule interacts with the hydrophobic structure of other substances in an aqueous solution. This is preferable because it expands the variation of possible anionic substances.
Furthermore, 2- (N, N-dimethylaminoethyl) -methacrylamide has a weak positive charge at a neutral pH, and its solubility in water is not affected by temperature, so it was supported once. This is preferable because it facilitates release of the anionic substance.
These may be used alone or in combination of two or more.

Examples of the anionic monomer include monomers having an anionic functional group, and examples of the anionic functional group include a carboxylic acid group, a sulfonic acid group, a sulfuric acid group, a phosphoric acid group, and a boronic acid group. From the viewpoints of physical stability, cell affinity, and high purity, carboxylic acid groups, sulfonic acid groups, and phosphoric acid groups are preferred.
More specifically, acrylic acid, methacrylic acid, vinyl benzoic acid and the like can be mentioned, and methacrylic acid and vinyl benzoic acid are particularly preferable from the viewpoint of chemical stability and cell affinity.
These may be used alone or in combination of two or more.

Examples of the other monomer include neutral hydrophilic monomers such as dimethylacrylamide and acrylic acid or methacrylic acid having a polyethylene glycol side chain.
These may be used alone or in combination of two or more.
Other monomers can be used to adjust the balance of hydrophilicity / hydrophobicity other than electric charge, and variations can be expanded.

  Here, the usage amount of NIPAM, the usage amount of the cationic monomer, and the usage amount of other monomers in the method for producing the temperature-responsive polymer of (B) with respect to the total usage amount of the monomers (A) to (C). The ratio (mole) can be appropriately adjusted by those skilled in the art so as to obtain a desired ratio of the monomer component in consideration of the reactivity in the polymerization reaction of the monomer.

Here, radical polymerization, ionic polymerization, etc. are mentioned as a polymerization method.
As the radical polymerization, living radical polymerization is preferable, and as living radical polymerization, reversible addition-fragmentation chain transfer (RAFT) polymerization, atom transfer radical polymerization (ATRP), iniferter polymerization and the like can be mentioned, and iniferter polymerization is preferable.
As ionic polymerization, living anionic polymerization is preferred.

An example of a method for producing the temperature-responsive polymer (B) is a method using radical polymerization.
In an example of this production method, first, a first mixture containing N-isopropylacrylamide (NIPAM) is irradiated with ultraviolet rays (first polymerization step).
Here, in addition to DMAEMA, the first mixture may optionally contain, for example, other monomers, solvents, chain transfer agents, stabilizers, surfactants, and the like.
Moreover, ultraviolet rays may be irradiated in an inert atmosphere.

  In this step, for example, the first mixture is added to a transparent sealed vial and the inside of the vial is made an inert atmosphere by bubbling an inert gas, and then irradiated with ultraviolet rays from the outside of the vial using an ultraviolet irradiation device. To do.

  Examples of the solvent include benzene, toluene, chloroform, methanol, water, and the like. In particular, benzene and toluene are preferable from the viewpoint of solubility and inertness to polymerization. These may be used alone or in combination of two or more.

  In this step, for example, the first mixture is added to a transparent sealed vial and the inside of the vial is made an inert atmosphere by bubbling an inert gas, and then irradiated with ultraviolet rays from the outside of the vial using an ultraviolet irradiation device. To do.

The ultraviolet wavelength is preferably 210 to 600 nm, and more preferably 360 to 380 nm. If it is the said range, a polymerization reaction can be advanced efficiently and the polymeric material which has a desired copolymerization ratio can be obtained stably. In addition, the manufactured polymer material can be prevented from being colored.
The irradiation intensity of ultraviolet light is preferably 0.01~50mW / cm ^2, further preferably 0.1~5mW / cm ^2.
Examples of the inert gas include nitrogen, argon, helium, neon and the like.

As temperature conditions, it is preferable that it is 10-40 degreeC, and it is still more preferable that it is 20-30 degreeC. With the above range, it is possible to perform the polymerization reaction at room temperature in a normal laboratory, and it is also possible to control the reaction by means of heating different from the means of light irradiation. .
The reaction time is preferably 10 minutes to 48 hours, and more preferably 60 minutes to 24 hours.

  In this step, NIPAM undergoes radical polymerization by irradiation with ultraviolet rays to become a polymer (poly (N-isopropylacrylamide) (PNIPAM)), and a homopolymer block containing N-isopropylacrylamide is formed. When other monomers are also used, a polymer block containing NIPAM and other monomers is formed.

Next, in the method for producing a temperature-responsive polymer (B), a cationic monomer and an anionic monomer are added to the first mixture after the first polymerization step to prepare a second mixture (addition step).
Here, the second mixture contains, in addition to the first mixture after the first polymerization step, the cationic monomer, and the anionic monomer, for example, other monomers, a solvent, a chain transfer agent, a stabilizer, a surfactant, and the like. It's okay.
Further, the cationic monomer and the anionic monomer may be added under an inert atmosphere.

  In this step, for example, the cationic monomer and the anionic monomer are added while keeping the inside of the vial in an inert atmosphere by flowing an inert gas through the vial.

  In this step, in addition to the homopolymer containing NIPAM during polymerization, a cationic monomer and an anionic monomer are also included in the polymerization system, and the polymerization system in the vial is changed from NIPAM homopolymerization system to NIPAM and cationic polymer. It will change to the copolymerization system of a monomer and an anionic monomer.

And in the manufacturing method of the temperature-responsive polymer of (B), an ultraviolet-ray is irradiated to a 2nd mixture (2nd polymerization process).
Here, the ultraviolet rays may be irradiated in an inert atmosphere.

  In this step, for example, ultraviolet rays are irradiated from the outside of the vial after adding the cationic monomer and the anionic monomer using an ultraviolet irradiation device.

The ultraviolet wavelength is preferably 210 to 600 nm, and more preferably 360 to 380 nm. If it is the said range, a polymerization reaction can be advanced efficiently and the polymeric material which has a desired copolymerization ratio can be obtained stably. In addition, the manufactured polymer material can be prevented from being colored.
The irradiation intensity of ultraviolet light is preferably 0.01~50mW / cm ^2, further preferably 0.1~5mW / cm ^2.
Examples of the inert gas include nitrogen, argon, helium, neon and the like.

As temperature conditions, it is preferable that it is 10-40 degreeC, and it is still more preferable that it is 20-30 degreeC. With the above range, it is possible to perform the polymerization reaction at room temperature in a normal laboratory, and it is also possible to control the reaction by means of heating different from the means of light irradiation. .
The reaction time is preferably 10 minutes to 48 hours, and more preferably 60 minutes to 24 hours.

  In this step, NIPAM, the cationic monomer, and the anionic monomer are radically polymerized by irradiation with ultraviolet rays, and are continuous with the polymer chain α-terminal of the homopolymer block containing NIPAM formed in the first polymerization step. A copolymer block comprising NIPAM, a cationic monomer and an anionic monomer is formed. When other monomers are used, a polymer block containing NIPAM and other monomers and / or a copolymer block containing NIPAM, cationic monomers, anionic monomers and other monomers are formed.

  As described above, a temperature-responsive polymer including a homopolymer block containing NIPAM and a copolymer block of NIPAM, a cationic monomer, and an anionic monomer is obtained.

  In the manufacturing method of this example, it is preferable to irradiate ultraviolet rays over the first polymerization step, the addition step, and the second polymerization step from the viewpoint of realizing an efficient reaction.

Another example of the method for producing the temperature-responsive polymer of (B) is a method using radical polymerization, and N-isopropylacrylamide (NIPAM), a cationic monomer, an anionic monomer, and optionally other The mixture containing the monomer is irradiated with ultraviolet rays.
Here, the said mixture may contain a solvent, a chain transfer agent, a stabilizer, surfactant, etc., for example.
Moreover, ultraviolet rays may be irradiated in an inert atmosphere.
Other conditions may be the same as in the above-described example manufacturing method.

(Temperature responsive polymer)
The temperature-responsive polymer (B) is produced by the production method (B).

The temperature-responsive polymer of (B) includes N-isopropylacrylamide (NIPAM) units, cationic monomer units, and anionic monomer units, and optionally other monomer units. This polymer can be manufactured by the manufacturing method of the above-mentioned example and another example.
Preferably, the temperature responsive polymer of (B) comprises mainly a polymer block (polymer chain α-terminal) comprising N-isopropylacrylamide (NIPAM) units and optionally other monomer units, and a cationic monomer. And a copolymer block containing anionic monomer units and optionally other monomer units. More preferably, the temperature-responsive polymer of (B) includes a homopolymer block of NIPAM and a copolymer block of NIPAM, a cationic monomer and an anionic monomer, and particularly preferably comprises these blocks. This polymer can be manufactured by the manufacturing method of the above-mentioned example.

For example, in the temperature-responsive polymer described in Patent Document 1, DMAEMA that imparts temperature responsiveness to the polymer is a cationic monomer that is necessary for forming a cell structure (along with an anionic monomer) at the same time. DMAEMA related to responsiveness is contained at the α-terminal of the polymer chain as a polymer block.
In such a temperature-responsive polymer, a cationic monomer always exists at the α-terminal of the polymer chain, so that the degree of freedom in adjusting the position of the cationic site in the polymer chain is not high, and the cationic monomer is mainly contained in DMAEMA. Therefore, it is not always easy to adjust the positive charge intensity of the cationic site and the pH of the temperature-responsive polymer aqueous solution.
For example, when a temperature-responsive polymer is used for drug delivery (DDS), there is a possibility that the types and amounts of drugs that can be carried are limited. As a technique of DDS, for example, by applying a temperature-responsive polymer carrying a drug in a cell culture vessel and culturing cells or tissues in the cell culture vessel after application, For example, a method of gradual release of the drug can be mentioned. Here, since the temperature-responsive polymer of Patent Document 1 includes DMAEMA having a small positive charge intensity, it is not always easy to load a drug of an anionic substance, and the types and amounts of drugs that can be loaded are limited. There was a possibility.

On the other hand, in the temperature-responsive polymer of the embodiment of the present invention, NIPAM that imparts temperature responsiveness to the polymer is a neutral monomer, and the cationic monomer that is necessary for the formation of the cell structure (along with the anionic monomer) is NIPAM. Is a different monomer.
In the temperature-responsive polymer of the embodiment of the present invention, it is not always necessary to have a cationic monomer at the α-terminal of the polymer chain, and the position of the cationic site in the polymer chain can be freely adjusted, Since a wide range of cationic monomers can be used, it is possible to easily adjust the positive charge intensity at the cationic site and the pH of the aqueous solution of the temperature-responsive polymer.
According to the temperature-responsive polymer of the embodiment of the present invention, for example, when the temperature-responsive polymer is used for drug delivery (DDS), it is possible to increase the amount of the drug that can be carried while expanding the type of drug that can be carried. Thus, the application range of the temperature-responsive polymer can be expanded.

In the temperature-responsive polymer (B), the ratio (mole) of the NIPAM unit to the total of the NIPAM unit, the cationic monomer unit, and the anionic monomer unit is preferably 0.6 to 0.9. It is more preferable that it is 7-0.9, and it is especially preferable that it is 0.9.
When other monomers are also used, the ratio (mole) of the other monomer units to the total of NIPAM units, cationic monomer units, anionic monomer units is preferably 0.001 to 0.2, More preferably, it is 0.01-0.1.

  As the temperature-responsive polymer (B), the polymer block α-terminal polymer block (for example, NIPAM homopolymer block) preferably has a number average molecular weight of 5000 Da or more, and more preferably 20000 Da or more.

The temperature-responsive polymer (B) is preferably a molecule having a number average molecular weight (Mn) of 10 to 500 kDa. Further, a molecule having a ratio (Mw / Mn) of the weight average molecular weight (Mw) to the number average molecular weight (Mn) is preferably 1.1 to 10.0.
The molecular weight of the temperature-responsive polymer can be appropriately adjusted depending on the polymerization conditions.

  According to the temperature-responsive polymer (B), the cloud point can be lowered to, for example, room temperature (25 ° C.) or lower.

  In the above-described temperature-responsive polymer, the time until the insolubilized product of the temperature-responsive polymer formed at a temperature equal to or higher than the cloud point is redissolved under room temperature (about 25 ° C.) conditions is significantly delayed. It is presumed that this is because the obtained temperature-responsive polymer has high self-aggregation properties because of the presence of a cationic functional group and an anionic functional group in the molecule.

  In particular, the preferred temperature-responsive polymer of (B) described above has a high molecular weight NIPAM homopolymer block at the polymer chain α-terminal, so that temperature-dependent globule transition of the side chain of NIPAM is likely to occur. It is considered that the cloud point can be effectively reduced.

  In addition, using this temperature-responsive polymer, as described later, it is possible to prepare a cell culture vessel in which the culture surface is coated with this temperature-responsive polymer.

  Furthermore, according to the temperature-responsive polymer of (B), as described later, a cell structure having a tubular (tubular) or massive (pellet-like) structure by culturing cells under appropriate culture conditions. The body can be formed.

  The ratio (C / A ratio) between the number of functional groups of the cationic functional group and the number of functional groups of the anionic functional group of the temperature-responsive polymer (B) is preferably 0.5 to 32. More preferably, it is ~ 16.

  When the C / A ratio is in the above range, the above effect of reducing the cloud point can be easily obtained. In the temperature-responsive polymer having the C / A ratio, the cationic functional group and the anionic functional group act on the intermolecular and / or intramolecular aggregation in an ionic bond in the temperature-responsive polymer, This is presumed to be a result of increasing the cohesive force of the temperature-responsive polymer.

  In addition, when the C / A ratio is in the above range, the balance between the positive charge and the negative charge in the temperature-responsive polymer can be particularly suitable, and the cytotoxicity due to the positive charge can be suppressed. It is presumed that the balance between hydrophilicity and hydrophobicity of the temperature-responsive polymer can be made particularly suitable to facilitate cell migration and orientation.

  Hereinafter, the temperature-responsive polymer (C) and the production method thereof will be described.

(Method for producing temperature-responsive polymer composition)
In the method for producing a temperature-responsive polymer composition (C), first, a mixed-type temperature-responsive polymer composition is prepared (mixture preparation step). Specifically, (1) 2-N, N-dimethylaminoethyl methacrylate (DMAEMA) and / or a polymer thereof and (2) 2-amino-2-hydroxymethyl-1,3-propanediol ( Tris), and (3) nucleic acid, heparin, hyaluronic acid, dextran sulfate, polystyrene sulfonic acid, polyacrylic acid, polymethacrylic acid, polyphosphoric acid, sulfated polysaccharide, curdlan and polyalginic acid, and alkali metal salts thereof. One or more anionic substances selected from the group are mixed ((2) Tris is optionally included).

  The polymer of (1) DMAEMA and / or its derivative is a temperature-responsive polymer, and its cloud point is 32 ° C. The tris in (2) serves to reduce the cloud point slightly, and / or reduce the rate at which a polymer formed at a higher temperature than the cloud point re-dissolves when cooled below the cloud point, and In addition, it is presumed to play a role of stimulating cells by positive charges derived from amino groups while maintaining hydrophilicity even in the hydrophobic polymer layer. The anionic substance (3) is presumed to play a role of enabling migration and orientation of cells to be cultured and a role of suppressing cytotoxicity.

According to this mixed temperature-responsive polymer composition, the cloud point can be reduced to room temperature (25 ° C.) or lower.
In the composition, it is presumed that the side chain of the polymer of DMAEMA and / or its derivative and Tris interact with each other (for example, a cross-linking action), and the polymer is likely to aggregate.

  Here, with respect to the above (1), as the polymer of DMAEMA and / or a derivative thereof, a molecule having a number average molecular weight (Mn) of 10 kDa to 500 kDa is preferable. Moreover, the ratio (Mw / Mn) of the weight average molecular weight (Mw) and the number average molecular weight (Mn) is preferably 1.1 to 6.0.

  In addition, as the DMAEMA derivative (1), for example, a derivative in which the hydrogen atom of the methyl group of the methacrylate is substituted with a halogen, a derivative in which the methyl group of the methacrylate is substituted with a lower alkyl group, and a hydrogen atom of the methyl group in the dimethylamino group Examples include halogen-substituted derivatives and derivatives in which the methyl group of the dimethylamino group is substituted with a lower alkyl group.

  Regarding the above (2), it is preferable that Tris is a pure substance having a purity of 99.9% or more, or that the Tris aqueous solution is made neutral or basic at the time of use by adding an alkaline substance or the like. Tris is commercially available in the form of a hydrochloride. When this is used, the pH of the aqueous Tris solution is lowered, so that the cloud point of the composition rises to about 70 ° C. Therefore, tris hydrochloride is not preferred.

  Among the anionic substances listed in (3) above, examples of the nucleic acid include DNA, RNA, and other artificial nucleic acids such as single-stranded, double-stranded, oligo, and hairpin.

The anionic substances listed in (3) above preferably have a certain size, for example, a molecular weight (M) of 1 kDa to 5,000 kDa.
When the molecular weight is within the above range, the anionic substance can ionically bond with the cationic substance and can capture the cationic substance for a long time, thereby forming stable ionic complex fine particles. . Moreover, the cytotoxicity which a cationic substance generally has by the electrostatic interaction with respect to the cell membrane surface of a cell can also be relieved.

  In addition to the anionic substances listed in (3), for example, by dehydrating and condensing a dicarboxylic acid such as oxalic acid to the 4-position amino group of poly (4-aminostyrene) which is a cationic polymer. In addition, a polymer derivative having an anionic functional group introduced and substantially functioning as an anionic substance can also be used.

  Two or more anionic substances listed in (3) above may be included.

Here, (1) 2-N, N-dimethylaminoethyl methacrylate (DMAEMA) and / or its derivative polymer (2) 2-amino-2-hydroxymethyl-1,3-propanediol (Tris) It is preferable to use a mixed temperature-responsive polymer composition having a ratio ((2) / (1)) of 1.0 or less.
The ratio ((2) / (1)) is assumed to be a weight ratio.

When the mixed-type temperature-responsive polymer composition in the above ratio is used, a cell structure can be easily formed during the culture.
According to this composition, the balance between the hydrophilicity and the hydrophobicity of the composition can be further improved. And it is estimated that this suitable balance has adjusted the adhesiveness of the cell to a culture surface suitably, and has activated the migration and orientation of a cell.

The ratio ((2) / (1)) is preferably 0.1 or more.
By setting the ratio to 0.1 or more, the above effect of reducing the cloud point can be easily obtained. In addition, the above effect of facilitating formation of a cell structure is easily obtained.

  For the same reason as described above, the ratio ((2) / (1)) is more preferably 0.1 to 0.5.

Here, the C / A ratio (positive charge / negative charge) in the temperature-responsive polymer composition is preferably 0.5 to 16.
Note that in this specification, the C / A ratio refers to the ratio of the positive charge of a substance contained in the composition to the negative charge of the substance contained in the composition. Specifically, the C / A ratio is as follows: (1) The number of moles of the polymer of DMAEMA and / or its derivative is N1, and (3) The number of moles of the anionic substance is N3. The positive charge per molecule) × N1} / {(negative charge per molecule of anionic substance) × N3}.
In this specification, when the anionic substance is DNA, the number of negative charges per molecule of the anionic substance is calculated by the number of DNA base pairs (bp number) × 2, and the molecular weight (Da) is , Bp number × 660 (average molecular weight of AT pair and CG pair).

By setting the C / A ratio to 0.5 to 16, the above effect of facilitating the formation of a tubular cell structure can be easily obtained.
It is presumed that the cytotoxicity due to the positive charge can be suppressed by suitably balancing the positive charge and the negative charge in the composition. In addition, it is presumed that the balance between hydrophilicity and hydrophobicity of the composition can be further improved to facilitate cell migration and orientation.

  For the same reason as described above, the C / A ratio is more preferably 2 to 10, and the C / A ratio is most preferably around 8.

(Cell structure)
The cell structure of the embodiment of the present invention can be produced by the method for producing a cell structure of the embodiment of the present invention.
The cell structure of the embodiment of the present invention includes a luminal cell structure extending in a mesh shape inside a massive cell structure.

  Here, the luminal cell structure preferably contains vascular endothelial cells, more preferably consists of vascular endothelial cells, and the massive cell structure preferably contains smooth muscle cells, and smooth muscle cells. More preferably, it consists of. In this case, the obtained cell structure is a spheroid-like structure in which capillaries are embedded.

EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to the following Example at all.
In the following tests, commercially available reagents were used without further purification unless otherwise specified.

(Test 1) Production of Polymer Production of Intramolecular Ion Complex Type Temperature Responsive Polymer (Example Production Method 1)
To a transparent vial made of soft glass having a capacity of 50 mL, 10.0 g of 2-N, N-dimethylaminoethyl methacrylate (DMAEMA) was added and stirred using a magnetic stirrer.
The mixture (liquid) was deoxygenated by purging nitrogen gas of G1 grade high purity (purity: 99.99995%) for 10 minutes (flow rate: 2.0 L / min).
Thereafter, the reaction product was polymerized by irradiating the mixture with ultraviolet rays using a round black fluorescent lamp (manufactured by NEC, model number: FCL20BL, 18W) for 5 hours.

Here, when a part of the reaction product was sampled and the number average molecular weight of the polymerized DMAEMA was measured, it was 97,0000 (dispersion = 4.1).
Immediately after confirming that the number average molecular weight is 5,000 or more, 1.4 g of methacrylic acid was added and magnetic stirring was continued again while keeping the inside of the vial in an inert atmosphere by flowing an inert gas through the vial. The mixture was stirred using a vessel. The methacrylic acid added here was previously bubbled with an inert gas.

  The reaction product was polymerized by irradiating the mixture with ultraviolet rays for 16 hours using a round black fluorescent lamp (manufactured by NEC, model number: FCL20BL, 18W). The reaction product became viscous immediately after mixing and solidified after 1 hour to obtain a polymer as a reaction product. The reaction product was dissolved in 2-propanol and the solution was transferred to a dialysis tube. Then, dialysis was performed for 72 hours to purify the reaction product.

The solution containing the reaction product was filtered through a 0.2 μm filter (manufactured by Toyo Roshi Kaisha, model number: 25AS020) made of cellulose mixed ester, and the obtained filtrate was freeze-dried to obtain a DMAEMA homopolymer block and a DMAEMA. A temperature-responsive polymer composed of a copolymer block of methacrylic acid was obtained (yield: 6.8 g, conversion 60%).
The number average molecular weight (Mn) of this polymer was measured using GPC (manufactured by Shimadzu Corporation, model number: LC-10vp series) with polyethylene glycol (manufactured by Shodex, TSK series) as a standard substance, and Mn = 450,0000 (Mw / Mn = 2.2) was determined (Example polymer 1).

The nuclear magnetic resonance spectrum (NMR) of Example polymer 1 was measured using a nuclear magnetic resonance apparatus (manufactured by Varian, model number: Gemini 300) using heavy water (D ₂ O) as a standard substance. The typical peaks common to Example polymer 1 are shown below.
¹ H-NMR (in D ₂ O) δ 0.8-1.2 (br, 3H, -CH ₂ -C (CH ₃ )-), 1.6-2.0 (br, 2H, -CH ₂ -C (CH ₃ )-) , 2.2-2.4 (br, 6H, -N (CH ₃ ) ₂ ), 2.5-2.7 (br, 1.9H, -CH ₂ -N (CH ₃ ) ₂ ), 4.0-4.2 (br, 1.9H, -O -CH _2- ).
Here, the number of protons of the methyl group (δ 0.8-1.2) bonded to the α-position (three in the case of the DMAEMA unit and the methacrylic acid unit) A and the ethyl bonded to the oxygen of the ester bond of the side chain From the number of methyl protons of the group (δ 4.0-4.2) (two in the case of the DMAEMA unit and zero in the case of the methacrylic acid unit) B, the number of functional groups of the amino group of the side chain of DMAEMA, and methacrylic acid The ratio with the number of functional groups of the carboxyl group in the side chain was calculated.
As a result, in the case of Example polymer 1, it was 94: 6. This is C / A ratio = 15.6 in terms of the C / A ratio referred to as an ionic complex in a two-component mixed system containing a cationic polymer and an anionic polymer.

(Test 2) Measurement of cloud point of polymer A 3% aqueous solution of Example polymer 1 was prepared, and the absorbance at 660 nm of this aqueous solution was measured between 20 ° C and 40 ° C.
As a result, the aqueous solution was transparent at 20 ° C. to 30 ° C. and the absorbance was almost 0. However, white turbidity was observed in the aqueous solution from around 31 ° C., and the absorbance rapidly increased at 32 ° C. This confirmed that the polymer had a cloud point of about 32 ° C.
When the example polymer was heated to 37 ° C., the polymer aqueous solution was suspended with good responsiveness, and then the entire aqueous solution was solidified. When this solidified product was maintained at room temperature (25 ° C.), it remained solidified for several tens of hours. Thereafter, the solidified product was gradually dissolved and changed into a homogeneous aqueous solution. When the solidified polymer was cooled to 4 ° C., it quickly dissolved. And even if it repeated the said temperature raising and temperature-decreasing operation, the change in responsiveness did not occur, and it was confirmed that the polymer reversibly causes a phase transition.

(Test 3) Measurement of particle diameter of polymer aggregate Example polymer 1 was made into a liquid at 20 ° C., and polymer molecules were obtained by light scattering using a static light scattering apparatus (manufactured by Sysmex Corporation, model number: Zetasizer Nano). The particle diameter of the aggregate was measured and found to be 250 nm. It was suggested that even at 20 ° C., which is a temperature lower than the cloud point, an aggregate having a relatively large particle size, that is, an aggregate that easily precipitates and hardly diffuses after precipitation. It was suggested that Example Polymer 1 could be easily coated on a cell culture vessel.

(Test 4) Preparation of cell culture device and jig As a cell culture device, a 35 mm cell culture plate (made by Iwaki, model number: 3000-035-MYP, bottom area per well: 9 cm ² ) made of potystyrene was used. .
The jig shown in FIG. 1 was used as a jig used in the first seeding step and the first culture step. The diameter of the substrate was 59 mm, the diameter of the protrusion was 34 mm, and the height of the protrusion was 10 mm.
The jig was placed in a cell culture vessel used for the first seeding process and the second culture process.

An aqueous solution (concentration: 15 ng / mL) of a temperature-responsive polymer cooled to a cloud point or less was prepared, and 750 μL was applied to the bottom surface of the cell culture vessel as the second culture surface. Moreover, 750 μL of the aqueous solution was applied to the top surface of the jig serving as the first culture surface. Then, the applied temperature-responsive polymer aqueous solution was dried by leaving the jig and the cell culture vessel in a clean bench.
Further, the side surfaces of the substrate and the protrusions of the jig were coated with a nonionic surfactant (Asahi Denka Co., Ltd., Pluronic F-68) to make the cells non-adhesive.

(Test 5) Measurement of zeta potential Zeta potential meter (manufactured by Otsuka Electronics Co., Ltd., model number) was used to measure the surface zeta potential of the coated region provided on the jig piece and the cell culture plate piece according to the same procedure as in (Test 4). : ELSZ) and a cell unit for flat plate samples.
Specifically, a small sample was brought into close contact with the lower surface of the quartz cell, and a monitor particle suspension was injected into the cell. Here, particles (zeta potential: −5 mV to +5 mV) obtained by coating polystyrene latex (particle diameter: about 500 nm) with hydroxypropylcellulose (Mw = 30,000) were used as standard monitor particles. As a solvent, a 10 mM sodium chloride aqueous solution was used under the conditions of pH = 7 and 37 ° C. The zeta potential was calculated using the Smoluchowski equation.
The zeta potential of the surface of the uncoated jig piece and the surface of the uncoated cell culture plate piece is both -68 mV, which is a value well known to those skilled in the art as the zeta potential of a general thermoplastic resin solid surface. there were.
On the other hand, the zeta potential of the surface of the small piece of the jig covered with the temperature-responsive polymer was +20 mV. Moreover, the zeta potential of the surface of the small piece of the cell culture plate coated with the temperature-responsive polymer was +20 mV.
As is well known to those skilled in the art, in the current technology, the measured value of the zeta potential on the solid surface has a variation of about ± 10%, and also in the sample preparation process, the coating operation itself varies. Therefore, the measured value of the zeta potential may have a certain amount of error.

(Test 6) Measurement of contact angle The contact angle of water with respect to the second coating region, which is the coating region of the jig, and the contact angle of water with respect to the first coating region, which is the coating region of the cell culture plate, conform to JIS R3257. When measured using a contact angle meter (trade name: DMs-400, manufactured by Kyowa Interface Science Co., Ltd.), they were 70 ° ± 10 ° and 72 ° ± 10 °, respectively.

Here, on the top surface of the jig, vascular stromal cells derived from rat subcutaneous fat modified so as to express GFP permanently are treated with a complete medium (Dulbecco's modified Eagle medium (DMEM) + 10% fetal bovine serum (FBS) solution). DMEM: manufactured by Gibco, model number: 1195-065, FCS: manufactured by Inbirotogen, lot number: 926696), and the cell density was 3.0 × 10 ⁵ cells / plate (310 cells / mm ² ). So as to be.
The seeded vascular stromal cells were cultured for 120 hours in a cell culture incubator at 37 ° C. and 5% CO ₂ atmosphere.
After 72 hours of culture, a luminal cell structure was formed.

In addition, mesenchymal stem cells derived from rat subcutaneous fat, which were fluorescently labeled (red) with a cell linker kit on the culture surface of the cell incubator, were cultured in a complete medium (Dulbecco's modified Eagle medium (DMEM) + 10% fetal bovine serum (FBS). ) Solution, DMEM: manufactured by Gibco, model number: 119905-065, FCS: manufactured by Inbirotogen, lot number: 926696), and the cell density is 5.0 × 10 ⁵ cells / plate (520 cells / mm) ² ) Sowing was carried out.
The seeded mesenchymal stem cells were cultured for 8 hours in a cell culture incubator at 37 ° C. and 5% CO ₂ atmosphere.
After 3 hours of culture, a monolayer sheet-like cell structure was formed.

  Here, the jig in which the tubular cell structure was formed on the coated top surface was taken out using tweezers and turned upside down. When the luminal cell structure was pressed against the sheet-like cell structure, the luminal cell structure was adhered to the sheet-like cell structure.

The sheet-like cell structure to which the tubular cell structure was adhered was cultured for 16 hours in a cell culture incubator at 37 ° C. and 5% CO ₂ atmosphere.
The cells that make up the sheet-like cell structure are detached from the outer edge of the culture surface all at once, and the sheet-like cell structure is engulfed in such a way that the sheet shrinks while curling. However, the phenomenon of aggregation in the center of the culture surface occurred. The above phenomenon was sufficiently visible with the naked eye.
The cells collected from each other formed one three-dimensional lump (pellet-like) structure including a luminal cell structure extending in a mesh shape.
The cell structure formed as described above was then spontaneously detached from the second coating region.
When the cellular structure was observed using a fluorescence phase contrast microscope, green fluorescence derived from vascular endothelial cells was confirmed in almost the entire cell mass, and a mesh-like structure was derived from the smooth muscle cell inside the cell mass. Red fluorescence could be confirmed (FIG. 2).

  FIG. 2 shows a photograph of a composite cell structure prepared in the example of the present invention, which includes vascular endothelial cells extending in a mesh shape inside smooth muscle cells, and (a) shows a composite cell structure. Is a photograph when observed with a stereomicroscope, and (b) shows a photograph when the composite cell structure is observed with a fluorescence phase contrast microscope. Here, the vascular endothelial cells emit green fluorescence, and the smooth muscle cells emit red fluorescence.

  According to the present invention, it is possible to produce a cell structure including a tubular cell structure extending like a mesh inside a massive cell structure, for example, a spheroid in which capillaries are embedded.

Claims (10)

Preparing a temperature-responsive polymer or temperature-responsive polymer composition; and
A first coated region and a second coated region, each of which covers the first culture surface and the second culture surface of the cell culture vessel with the temperature-responsive polymer composition, and has a surface zeta potential of 0 to 50 mV, respectively. Preparing the preparation process,
A seeding step comprising: a first seeding step of seeding the first cells on the first coating region; and a second seeding step of seeding a second cell on the second coating region;
A first culturing step of culturing the seeded first cell to prepare a luminal cell structure, and culturing the seeded second cell to prepare a sheet-like cell structure. A pre-culture process comprising two culture processes;
Affixing the luminal cell structure on the sheet-like cell structure to prepare a composite cell structure;
A method for producing a cell structure, comprising a post-culture step of culturing the composite cell structure.   The method for producing a cell structure according to claim 1, wherein a contact angle of water with respect to the first coating region and the second coating region is 50 to 90 °. The cell density of the first cells in the first seeding step is 100 to 300 cells / mm ² ,
The cell density of the second cells in the second seeding step is 400 to 1,200 cells / mm ² .
The manufacturing method of the cell structure of Claim 1 or 2. The first cell is a vascular endothelial cell,
The second cell is a smooth muscle cell,
The manufacturing method of the cell structure as described in any one of Claims 1-3.   The temperature-responsive polymer or temperature-responsive polymer composition comprises 2-N, N-dimethylaminoethyl methacrylate (DMAEMA) units and anionic monomer units; N-isopropylacrylamide (NIPAM) units Responsive polymer comprising: a cationic monomer unit; and an anionic monomer unit; a polymer of 2-N, N-dimethylaminoethyl methacrylate (DMAEMA) and / or a derivative thereof, and 2-amino-2-hydroxy Methyl-1,3-propanediol (Tris), nucleic acids, heparin, hyaluronic acid, dextran sulfate, polystyrene sulfonic acid, polyacrylic acid, polymethacrylic acid, polyphosphoric acid, sulfated polysaccharides, curdlan and polyalginic acid and these Group of alkali metal salts of At least one temperature-responsive polymer or temperature-responsive polymer composition selected from the group consisting of one or more anionic substances selected from the group consisting of: 5. The method for producing a cell structure according to any one of 4 above.   The cell structure according to claim 5, wherein the anionic monomer is at least one selected from the group consisting of acrylic acid, methacrylic acid, and a vinyl derivative having a carboxyl group, a sulfonic acid group, and a phosphoric acid group in the side chain. Body manufacturing method.   The cationic monomer includes 3- (N, N-dimethylaminopropyl) -meth (a) acrylamide, 3- (N, N-dimethylaminopropyl) -meth (a) acrylate, aminostyrene, 2- (N, The N-dimethylaminoethyl) -meth (a) acrylamide and the 2- (N, N-dimethylaminoethyl) -meth (a) acrylate are at least one selected from the group consisting of: A method for producing a cell structure.   A cell structure produced by the method for producing a cell structure according to any one of claims 1 to 7.   A cellular structure comprising a luminal cellular structure extending in a mesh form inside a massive cellular structure. The luminal cell structure includes vascular endothelial cells,
The massive cell structure includes smooth muscle cells,
The cell structure according to claim 9.