JP2014027917A - Production method of cell structure, and cell structure produced by the same - Google Patents

Abstract

The present invention provides a cell structure suitable for spontaneously forming a tubular (tubular) structure in a cell.
A polymer of 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof, 2-amino-2-hydroxymethyl-1,3-propanediol, nucleic acid, heparin, hyaluronic acid, dextran sulfate. Mixing one or more anionic substances selected from the group consisting of polystyrene sulfonic acid, polyacrylic acid, polymethacrylic acid, polyphosphoric acid, sulfated polysaccharides, curdlan and polyalginic acid and their alkali metal salts A preparation step for preparing a composition for cell culture, a seeding step for coating the culture surface of the cell culture device with the composition for cell culture, and seeding on the culture surface of the coated cell culture device, and culturing the seeded cells A manufacturing method of a cell structure provided with a culture process.
[Selection] Figure 1

Description

  The present invention relates to a method for producing a cell structure, and a cell structure produced by the method, and in particular, production of a cell structure suitable for producing a cell structure having a tubular (tubular) structure. The present invention relates to a method and a cell structure produced by the 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.

  In general, cells taken out of the body are often dedifferentiated under various stresses that cause disturbance in their gene regulation, and may be dedifferentiated to proliferate the cells. It is often necessary. As a result, even if cells collected from a patient are cultured under simple culture conditions, the cells often cannot maintain the original gene expression state, and thus cannot form a cell structure and thus a tissue. There is a problem that the advanced function of the cell cannot be exhibited. For example, when cell culture is performed in a general polystyrene cell culture dish, the cells only form a monolayer structure, and a structure found in highly differentiated cells, such as vascular endothelial cells, is generated. It is difficult to form a cell structure having a structure similar to a tubular (tubular) structure, which is a shape when existing in the body, and an antithrombotic substance of vascular endothelial cells Specific functions such as secretion are also lost.

  In addition, cell culture methods that construct a three-dimensional structure that mimics the structure of a tissue, such as spheroid culture, cluster culture, pellet culture, and three-dimensional carrier culture, have been developed. A cell culture method for producing cells having a three-dimensional structure by using an extracellular matrix having a three-dimensional structure as a scaffold for cell culture (for example, see Patent Document 1) is also known. .

JP 2010-524458 A

However, even if a cell structure having a tube-like structure is physically created by culturing vascular endothelial cells using a polystyrene cell culture vessel having a tube-like flat surface, the cell structure is The gene expression state and advanced functions inherent in vascular endothelial cells cannot be provided.
In order to form a cell structure using various conventional cell culture methods, it is necessary to give physical, chemical or mechanical stimulation to the cells in culture, for example, vascular endothelial cells On the other hand, it is necessary to devise such as culturing while applying pulsation according to the pulse frequency and load according to the shear force of blood flow.

Therefore, the present invention enables a cell structure to be easily produced without depending on the skill of the operator by spontaneously forming a tubular (tubular) structure in the cell. An object of the present invention is to provide a method for producing a cell structure.
Another object of the present invention is to provide a cell structure produced by the above production method.

The gist of the present invention is as follows.
The method for producing a cell structure of the present invention comprises a polymer of 2-N, N-dimethylaminoethyl methacrylate (hereinafter also referred to as “DMAEMA”) and / or a derivative thereof, and 2-amino-2-hydroxymethyl- 1,3-propanediol (hereinafter also referred to as “Tris”), nucleic acid, heparin, hyaluronic acid, dextran sulfate, polystyrene sulfonic acid, polyacrylic acid, polymethacrylic acid, polyphosphoric acid, sulfated polysaccharide, curdlan And a preparation step of preparing a cell culture composition by mixing one or more anionic substances selected from the group consisting of polyalginic acid and alkali metal salts thereof, and the cell culture composition with the cell culture composition. A preparatory step of coating a culture surface and preparing a coated cell incubator; and the coated cell culture at less than 1.0 × 10 ³ cells / mm ² A seeding step for seeding on the culture surface of the vessel, and a culture step for culturing the seeded cells.
The cell density is the cell density of living cells.
In addition, the correspondence between the unit of cell density “pieces / mm ² ” used in the present specification and the unit of “cells / mL” of general cell density is as follows. It will be clearly understood by those skilled in the art when considering the area of the culture surface of a 150 mm diameter dish, 6-well to 96-well plate, T25-T160 flask, etc.) and its appropriate amount of medium. For example, the above-mentioned “cell density of less than 1.0 × 10 ³ cells / mm ² ” means “20 × 10 ⁴ cells / mm2” when a 24-well cell culture plate well known to those skilled in the art is used as a cell incubator. Corresponds to “cell density below mL”.

  In the method for producing a cell structure of the present invention, in the cell culture composition, the ratio of the 2-amino-2-hydroxymethyl-1,3-propanediol to the polymer is 1.0 or less. It is preferable that

  Furthermore, in the manufacturing method of the cell structure of this invention, it is preferable that C / A ratio is 4.5-15 in the said composition for cell culture. In the present invention, “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.

Furthermore, in the method for producing a cell structure of the present invention, in the coated cell culture vessel, the culture surface of the coated cell culture vessel has 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof per unit area. The amount of the polymer is preferably 10 to 200 ng / mm ² .

  The cell structure of the present invention is produced by any one of the production methods described above.

According to the method for producing a cell structure of the present invention, the cell structure can be easily formed without depending on the skill of the operator by spontaneously forming a tubular structure in the cell. Can be manufactured.
Moreover, according to the cell structure of the present invention, a tubular (tubular) cell structure produced by the above production method can be provided.

It is a photograph when the vascular endothelial cell derived from the rat subcutaneous fat which has a luminal structure (tube shape) cultured according to the manufacturing method of the cell structure of the present invention is observed with a phase contrast microscope. (A), (b), (c) Rat subcutaneously having a tubular (tubular) structure cultured using the cell culture composition of the present invention and the cell culture device of the present invention provided with a groove It is a photograph when vascular endothelial cells derived from fat are observed with a fluorescence microscope. It is a photograph when the vascular endothelial cell derived from the rat subcutaneous fat origin blood vessel which has a pellet-like structure cultured according to the manufacturing method of the cell structure of the present invention is observed with a stereomicroscope. It is the photograph when the vascular endothelial cell derived from rat subcutaneous fat cultured with various cell densities according to the manufacturing method of the cell structure of the present invention is observed with a phase contrast microscope and the naked eye. Rat vascular endothelium derived from rat subcutaneous fat, cultured according to the method for producing a cell structure of the present invention, using the composition for cell culture prepared by changing the ratio of Tris to the polymer in the composition for cell culture It is a photograph when a cell is observed with a phase contrast microscope. A rat subcutaneous fat-derived vascular endothelial cell cultured according to the method for producing a cell structure of the present invention using a cell culture composition prepared by changing the C / A ratio in the cell culture composition. It is a photograph when observed with a phase contrast microscope. Rat vascular endothelial cells derived from rat subcutaneous fat were cultured in accordance with the method for producing a cell structure of the present invention using a cell culture vessel prepared by changing the polymer content in the cell culture composition. It is a photograph when observed with a phase contrast microscope. It is a photograph when vascular endothelial cells derived from rat subcutaneous fat cultured using a cell culture vessel coated with a composition containing no anionic substance are observed with a phase contrast microscope. (A), (b) It is a figure which shows the correlation of the structure of the cell cultured according to the manufacturing method of the cell structure of this invention, and culture conditions. Stromal cells derived from rat subcutaneous fat and cultured using the cell culture composition and cell culture vessel of the present invention prepared by changing the C / A ratio in the cell culture composition were obtained using a phase contrast microscope. It is a photograph when observed. It is a figure which shows a time-dependent change of the light transmittance (turbidity) when the polymer / Tris mixture aggregated at the temperature more than a cloud point is made into 25 degreeC conditions. Were prepared by changing the ratio of Tris for polymer in the composition for cell culture, the cell culture compositions of the present invention, is a diagram showing the results of ^{1 H} NMR measurements at different temperatures. It is a figure shown about the result of the differential scanning calorimetry (DSC) of the composition for cell cultures of this invention prepared by changing the ratio of Tris with respect to the polymer in the composition for cell cultures. It is a figure shown about the result of having measured the zeta potential of the surface (solid surface) of the cell culture device of the present invention prepared by changing the C / A ratio in the composition for cell culture. It is a figure shown about the result of having measured the zeta potential of fine particles before making it settle on the surface of the cell culture device of the present invention prepared by changing C / A ratio in the composition for cell culture. (A) is a figure which shows the image which measured the surface of the cell culture plate in dynamic tapping mode, (b) is a figure which shows the image which measured the surface of the cell culture plate in phase mode. It is a photograph when the surface of the cell culture device of the present invention prepared by changing the C / A ratio in the cell culture composition is observed with a fluorescence microscope (the fluorescence is fluorescently labeled as an anionic substance). Derived from DNA). It is a photograph when the surface of the cell culture device of the present invention prepared by changing the C / A ratio in the cell culture composition is observed with an AFM in water.

The polymer of DMAEMA is known as a temperature-responsive polymer, and its lower critical solution temperature (hereinafter also referred to as “cloud point”) is about 32 ° C. Therefore, when a cell culture vessel formed by coating a composition for cell culture containing a DMAEMA polymer is used, for example, when performing cell culture operations in a clean bench at room temperature (about 25 ° C.) There was a problem that the coated polymer layer was dissolved again and washed away.
In the present specification, the “cloud point” of the composition does not necessarily mean strictly, “the temperature at which it dissolves below a predetermined temperature but becomes insoluble and precipitates above a predetermined temperature”. Instead, it also refers to “the temperature at which the time required for dissolution is 10 minutes or more when the insolubilized and precipitated composition is dissolved under conditions below a predetermined temperature”.

In solving the problems of the present invention, as a result of intensive studies, the inventors have succeeded in preparing a cell culture composition having a cloud point of about room temperature (25 ° C.). This cell culture composition comprises (1) a polymer of 2-N, N-dimethylaminoethyl methacrylate (DMAEMA) and / or a derivative thereof, and (2) 2-amino-2-hydroxymethyl-1,3- Propanediol (Tris), and (3) nucleic acids, heparin, hyaluronic acid, dextran sulfate, polystyrene sulfonic acid, polyacrylic acid, polymethacrylic acid, polyphosphoric acid, sulfated polysaccharides, curdlan and polyalginic acid and their alkali metals One or more anionic substances selected from the group consisting of salts.
In addition, the inventors can continue to adhere cells in culture to the cell culture vessel even when operated in a clean bench at room temperature, and the cultured cell structures can be recovered as they are. A cell culture vessel formed by coating the composition for cell culture was prepared.

  Then, when cells such as vascular endothelial cells, adipose stem cells, chondrocytes, hepatocytes, fibroblasts, etc. were cultured in a general cell incubator (at 37 ° C.) using this cell incubator, Collected to form a tubular (tubular) or pellet-like cell structure. Here, it was found that the shape of the cell structure tends to depend on the density of cells seeded in the cell culture vessel.

(Method for producing cell structure)
The cell culture method of the present invention comprises a polymer of 2-N, N-dimethylaminoethyl methacrylate (DMAEMA) and / or a derivative thereof, 2-amino-2-hydroxymethyl-1,3-propanediol (Tris), , A nucleic acid, heparin, hyaluronic acid, dextran sulfate, polystyrene sulfonic acid, polyacrylic acid, polymethacrylic acid, polyphosphoric acid, sulfated polysaccharide, curdlan and polyalginic acid, and a kind selected from the group consisting of these alkali metal salts A preparation step of preparing a cell culture composition by mixing the above anionic substances, a preparation step of preparing a coated cell culture vessel by coating the culture surface of the cell culture vessel with the cell culture composition, A seeding process in which cells are seeded on a culture surface of a coated cell culture device at a cell density of less than 1.0 × 10 ³ cells / mm ^2; And a culturing step for culturing the vesicle.
Details of each step will be described below.

(Preparation process)
In the cell culture method of an example of the present invention, first, a cell culture composition is prepared (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.

  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 composition for cell culture, 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 a derivative thereof and Tris interact with each other (for example, a crosslinking 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.

  Examples of the DMAEMA derivative (1) include, for example, a derivative in which a hydrogen atom of a methyl group of a methacrylate is substituted with a halogen, a derivative in which a methyl group of a methacrylate is substituted with a lower alkyl 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. A polymer derivative having an anionic functional group introduced and substantially functioning as an anionic substance can also be used in the present invention.

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

In the method for producing a cell structure of the present invention, (1) 2-amino-2-hydroxymethyl-1 for a polymer of (1) 2-N, N-dimethylaminoethyl methacrylate (DMAEMA) and / or a derivative thereof. , 3-propanediol (Tris) ratio ((2) / (1)) is preferably 1.0 or less.
The ratio ((2) / (1)) is assumed to be a weight ratio.

According to the method for producing a cell structure using the composition for cell culture in the above ratio, the cell structure can be easily formed in the culturing step.
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.

Moreover, it is preferable that the said ratio is 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.

On the other hand, since the tris compound itself of (2) has a tertiary amine, it has cytotoxicity to cells, and if the re-dissolution of the polymer layer can be delayed, it cannot be added in a large amount. The ratio ((2) / (1)) is 1.0 or less, that is, the ratio ((2) / (1)) is more preferably 0.1 to 1.0.
For the same reason as described above, the ratio ((2) / (1)) is more preferably 0.1 to 0.5.

In the method for producing a cell structure of the present invention, the C / A ratio (positive charge / negative charge) in the cell culture composition is preferably 4.5-15.
In the present invention, the C / A ratio refers to the ratio of the positive charge possessed by the substance contained in the composition to the negative charge possessed by 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 the present invention, 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. The number x 660 (average molecular weight of AT pair and CG pair) shall be calculated.

By setting the C / A ratio to 4.5 to 15, the above effect of easily forming a 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 above, the C / A ratio is more preferably 6 to 12, and the C / A ratio is most preferably around 8.

(Preparation process)
Next, a coated cell culture device is prepared by coating the prepared cell culture composition on the culture surface of the cell culture device (preparation step).

  According to this coated cell culture vessel, the composition is solidified within a certain period of time, specifically 5 to 40 minutes, even when operating in a clean bench at room temperature during the operation of seeding cells. Therefore, the coating layer can be kept fixed to the cell culture vessel after the unnecessary water (supernatant) used for coating is removed and the cell suspension is added.

According to this coated cell culture device, the cell structure can be spontaneously formed in the cell by culturing the cell under an appropriate culture condition in the culturing step.
In the coated cell culture vessel, in the culturing process, the hydrophobic side chain of the polymer of DMAEMA and / or a derivative thereof contained in the composition and the amino group of Tris are stimulated to cells while interacting in some way. It is estimated that
In addition, the anionic substance contained in the composition suppresses cytotoxicity by optimizing the balance between positive and negative charges in the composition during the culturing step (the surface of the cell membrane of mammalian cells is negative). Cationic substances are often cytotoxic because they are charged), and the balance between hydrophilicity and hydrophobicity of the above composition is preferred to enable cell migration and orientation It is estimated to be.
In addition, the coated cell culture device has suppressed cytotoxicity and cytotoxicity compared to a coated cell culture device in which the culture surface is coated with the composition consisting only of the above (1) and (2). It is presumed to have a surface state suitable for cell culture.

Here, in the above-mentioned coated cell culture device, (1) 2-N, N-dimethylaminoethyl methacrylate (DMAEMA) contained in the composition for cell culture that the culture surface of the cell culture device has per unit area and It is preferable that the amount of the derivative polymer is 10 to 200 ng / mm ² .

When the amount per unit area of the above (1) is 10 to 200 ng / mm ² , the effect of the composition for cell culture of the present invention that makes it easy to form a cell structure is easily obtained.

For the same reason as above and cost, the amount of the polymer per unit area is more preferably 10 to 100 ng / mm ² .

Here, the coated cell culture device is, for example, casting an aqueous solution of the cell culture composition cooled to a temperature below the cloud point to the bottom surface of the well of the coated cell culture device, and then in a 37 ° C. incubator. To remove unnecessary water (supernatant) used for coating the composition, and then solidify the composition.
It is preferable to remove the supernatant using an aspirator. Thereby, dilution of a culture medium can be avoided and the influence of the trace amount component in a supernatant liquid can be excluded.

(Seeding process)
Then, the cells are seeded on the culture surface of the coated cell culture device in the prepared coated cell culture device at a cell density of less than 1.0 × 10 ³ cells / mm ² (seeding step).
This step can be performed, for example, by removing the coated cell culture vessel that has been allowed to stand in an incubator at 37 ° C. to a clean bench at room temperature.

With respect to the cell density, for example, when seeding in a 24-well plate having a culture surface area of 200 mm ² and a medium volume of 1 mL, seeding is performed at a cell density of less than 1.0 × 10 ³ cells / mm ^2. Cell suspension must have a cell density of less than 20 × 10 ⁴ cells / mL.

  The cell culture method of the present invention can be particularly suitably applied to mesenchymal cells such as vascular endothelial cells, adipocytes, adipose stem cells, and fibroblasts. In the case of primary cells, adherent cells that form colonies may be selected and can be appropriately selected by those skilled in the art.

If cells are seeded at the above cell density, the detailed mechanism is unknown, but a cell structure having a tubular (tubular) structure can be formed in the culture process.
In addition, the luminal (tubular) cell structure in the present invention refers to a structure in which cultured cells spontaneously gather to form a lumen. In this cell structure, it is considered that a state similar to the gene expression state in vivo is reproduced. In this respect, the cell structure of the present invention is completely different from the tube-shaped cell structure obtained by a conventional method of culturing cells using a tube-shaped scaffold.

Moreover, although the said cell density changes with cell types, it is preferable to set it as 50 pieces / mm < ² > or more which is a common-sense lower limit of a cell culture technique. In particular, since it is easy to form a tubular (tubular) cell structure, the cell density is preferably 250 cells / mm ² or more.

The cell density is more preferably 250 to 1,000 cells / mm ² . In particular, it is preferably 450 to 800 pieces / mm ² , more preferably 500 to 600 pieces / mm ² . When the cell density is within the above range, the above effect of facilitating formation of a cell structure having a tubular (tubular) structure in the culturing step can be easily obtained.

(Culture process)
Finally, the seeded cells are cultured (culture process).
This step can be performed using, for example, a general 37 ° C. cell incubator.

  Moreover, those skilled in the art can appropriately determine the culture conditions such as the culture time and the culture medium to be used based on the cell type and the purpose of the experiment.

  According to the method for producing a cell structure of the present invention, a tubular (tubular) cell structure having the same structure as the tubular (tubular) structure found when vascular endothelial cells and the like are differentiated. The body can be reproduced in a cell culture vessel, and information on DNA, mRNA, protein, etc. expressed in differentiated cells can be obtained. Specifically, it becomes possible to analyze the interaction between proteins in cells and the response of cells to physiologically active substances. Therefore, the method for producing a cell structure of the present invention can provide a useful tool for pharmaceutical research and molecular biology basic research.

(Cell structure)
The cell structure of the present invention can be produced by the above-described method for producing a cell structure of the present invention.

  Hereinafter, the present invention will be described in more detail with reference to test examples, but the present invention is not limited to the following test examples.

(Test A)
(Test A1) Production of cell structure (Test A1a) Preparation of cell culture composition (adjustment step)
(A1a-1) Synthesis of temperature-responsive polymer To a transparent vial made of soft glass having a capacity of 50 mL, 7.0 g of 2- (N, N-dimethylaminoethyl) methacrylate was added and stirred using a magnetic stirrer. did. 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 is irradiated with ultraviolet rays for 21 hours using a round black fluorescent lamp (manufactured by NEC, model number FCL20BL, 18W), so that the reaction product is used without using a solvent (ie, neat). Polymerization). The reaction product became viscous after 5 hours and solidified after 15 hours to obtain a polymer as a reaction product. This reaction product was dissolved in chloroform and then reprecipitated using n-hexane. The reprecipitation using chloroform / n-hexane was repeated a total of 6 times to purify the reaction product.
The purified polymer was evaporated to remove the solvent remaining in it. Thereafter, the polymer is dissolved in 150 mL of benzene, filtered through a PTFE 0.45 μm filter (manufactured by Pall Corp., model number: Ecodisk 25CR), and the resulting filtrate is freeze-dried to obtain a cationic temperature responsiveness. A (homo) polymer was obtained (yield 4.3 g, conversion: 61.4%). 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 = 97,000. It was determined that (Mw / Mn = 4.1).
Further, the nuclear magnetic resonance spectrum (NMR) of this polymer was measured in heavy water (D ₂ O) using a nuclear magnetic resonance apparatus (manufactured by Varian, model number: Gemini 300). The results are as follows.
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, 2H, -CH ₂ -N (CH ₃ ) ₂ ), 4.0-4.2 (br, 2H, -O-CH _2- )

(A1a-2) Preparation of polymer / Tris / anionic substance mixture DNase and RNase-free sterile water were used below.
An aqueous solution of the temperature-responsive polymer synthesized in (A1a-1) was prepared, and this aqueous solution was cooled to 4 ° C. to make this solution a liquid state. Under cooling, 2-amino-2-hydroxymethyl-1, Add granules of 3-propanediol (Tris) (manufactured by Wako Pure Chemicals Co., Ltd., Tris999), dissolve quickly, filter through a 0.2 μm mixed cellulose membrane filter, and add an aqueous solution of polymer / Tris mixture (final concentration is polymer: 100 μg / mL, Tris: 100 μg / mL).
On the other hand, firefly luciferase reporter vector (DNA) (manufactured by Promega, pGL3-Control, model number: E1741) was used as an anionic substance, and this DNA was dissolved in sterile water (final concentration: 33 μg / mL). To 300 μL of this DNA solution, 400 μL of the polymer / Tris mixture aqueous solution was added dropwise little by little and mixed.
Then, the mixture is diluted with 1,300 μL of water and adjusted so that the total amount of the solution becomes about 2,000 μL, and the polymer / tris / anionic substance mixture, that is, the method for producing the cell structure of the present invention is used. An aqueous solution of the composition for cell culture to be used (final concentration, polymer: 20 μg / mL, Tris: 20 μg / mL, DNA: 5 μg / mL) was obtained.
The cell culture composition had a C / A ratio (positive charge / negative charge) of 8.4.

(A1a-3) Measurement of cloud point of polymer / tris / anionic substance mixture The absorbance at 660 nm of the aqueous solution of the polymer / tris / anionic substance mixture prepared in (A1a-2) is between 20 ° C. and 40 ° C. Thus, the measurement was performed from the low temperature side to the high temperature side.
As a result, the aqueous solution was transparent at 20 ° C. to 28 ° C. and the absorbance was almost 0, but white turbidity was observed in the aqueous solution from around 29 ° C., and the absorbance increased rapidly from around 30 ° C. . This confirmed that the polymer had a cloud point of about 30 ° C.
Moreover, when the mixture aqueous solution adjusted in (A1a-2) which was cooled in a refrigerator and returned to a transparent aqueous solution was heated to 37 ° C., the mixture aqueous solution was suspended with good responsiveness, and then the aqueous solution. The whole solidified. When this solidified product was maintained at room temperature (25 ° C.), it remained in a solidified state for 24 to 48 hours. Thereafter, the solidified product was gradually dissolved and changed into a homogeneous aqueous solution. Thereby, it was confirmed that the speed of the phase transition from the solid phase to the liquid phase near the room temperature was reduced.
In the measurement of the cloud point of the temperature-responsive polymer alone in (A1a-2) above, the phase transition occurred with good responsiveness to temperature changes (both temperature rise and temperature drop rates were 1 ° C./min). In consideration of this, the delay in responsiveness was remarkable.

(Test A1b) Coating of cell culture composition on cell culture vessel (coating process)
By cooling the aqueous solution of the composition for cell culture prepared in (A1a-2) to 4 ° C., this solution is brought into a liquid state, and a polystyrene 24-well cell culture plate (manufactured by Iwaki Co., Ltd., microplate, model number: 3815). -024, 200 μL of this solution was added to each well at a bottom area of 200 mm ² ) at room temperature, and was quickly cast on the entire bottom surface of the hole. The cell culture dish was then incubated for 6 hours in a cell culture incubator (37 ° C., 5% CO ₂ ).
In this way, a 24-well cell culture plate (cell culture device) was obtained in which the well on the culture surface was coated with the composition for cell culture.

(Test A1c, A1d) Cell culture (seeding process, culture process)
(Test A1c-1, A1d-1) Culture of cells in plate Under the conditions of room temperature, vascular endothelial cells (primary cultured cells) derived from rat subcutaneous fat were placed in each well of the 24-well cell culture plate prepared in test 1b. The cell density was suspended in a complete medium (Dulbecco's modified Eagle medium (DMEM) + 10% fetal calf serum (FCS) solution, DMEM: manufactured by Gibco, model number: 11965, FCS: manufactured by Invitrogen, lot number: 852546). The culture solution adjusted to 5 × 10 ² cells / mm ² (10 × 10 ⁴ cells / mL) was added in an amount of 1 mL. This operation took 5 to 10 minutes including taking a photomicrograph in the initial sowing stage. Then, the cells were cultured in a cell culture incubator at 37 ° C. (Test Example A1).
After 70 hours, the cells became confluent. When the culture was further continued, on the 5th day of culture, all the cells were detached from the end of the culture dish at once, and the cells were agglomerated in one place as if the sheets were curling and shrinking. The phenomenon that occurs. The above phenomenon was sufficiently visible with the naked eye. The cells that gathered together formed a network structure when viewed in a two-dimensional plane. When this network structure was observed in more detail, it was confirmed that the aggregate of cells formed a tubular (tubular) structure (FIG. 1), and these cells were as if they were blood vessels. It was shown that they were spontaneously assembled as if forming (Test Example A1).

(Tests A1c-2, A1d-2) Cultivation of blood vessel-like cell structure using tubular (tubular) cells A plurality of grooves substantially parallel to each other are provided on the plate surface for migration and migration of cultured cells. Added restrictions. Specifically, the groove width was about 0.03 mm and the groove depth was about 0.05 mm. A plate having a gap between grooves in the range of 0.1 mm to 10 mm (hereinafter also referred to as “inter-groove distance”) was prepared (see FIGS. 2A, 2B, and 2C).
Similar to (Test A1c-1) and (Test A1d-1), vascular endothelial cells derived from rat subcutaneous fat (primary culture cells) were seeded on these plates, and the cells were cultured (Test Example A19, Test Example). A20, Test Example A21). Detailed conditions are shown in Table 1.

The figure which observed the cell on the 6th day is shown to Fig.2 (a), (b) and (c). 2A shows a case where the distance between the grooves is about 200 μm, FIG. 2B shows a case where the distance between the grooves is about 400 μm, and FIG. 2C shows a case where the distance between the grooves is about 1,400 μm. The photograph which observed the cell adhere | attached on each area | region between each groove | channel in the case of doing using the fluorescence microscope is shown.
It was found that the cells did not adhere to the groove but only adhered to the inter-groove region and proliferated. As described later in (Test C2), a commercially available polystyrene cell culture plate is prepared by performing plasma discharge in a mixed gas containing ammonia and oxygen, and by this treatment, an amino group derived from ammonia ( -NH ₂₎ and oxygen derived from the carboxyl group of (-COOH) in which has been introduced. The phenomenon that the cell does not adhere to the groove and the phenomenon that the cell itself recognizes the groove (surface irregularities) and moves away from the groove are surface plasma in the groove formed by carving the surface of the cell culture dish in this embodiment. This may be because the polystyrene portion in the deep part of the base material that has not been treated is exposed. When the inter-groove distance was 0.5 mm or more, the cells formed a lumen in the inter-groove region, and the lumen extended in all directions. When the inter-groove distance was 0.2 mm or less, the cells formed one lumen in the inter-groove region, and the lumen extended in one direction.
From these results, it can be said that one blood vessel-like cell structure could be formed by using a plate provided with grooves. These indicate the possibility of producing longer and thicker linear blood vessels, and the possibility of producing branched blood vessels (for example, blood vessels having a Y-shaped structure). I can say that.

(Comparative test A1)
(Comparative tests A1a and A1b) Preparation of cell culture composition and preparation of cell culture device A cell culture composition was prepared in the same manner as in test A1a, and a cell culture device was prepared in the same manner as in test A1b.

(Comparative Test A1c, A1d) Cell Culture In each well of this 24-well cell culture plate prepared in Comparative Test A1b, vascular endothelial cells derived from rat subcutaneous fat (primary cultured cells) were suspended in the complete medium. The cell density was adjusted to 15 × 10 ² cells / mm ² (30 × 10 ⁴ cells / mL), and 1 mL of the culture solution was added. The cells were cultured in a cell culture incubator (Comparative Test Example A1).
After 70 hours, the cells became confluent. When the culture was further continued, on the fifth day of culture, all the cells suddenly detached from the end of the culture dish at one time, and the cells were contracted as if the sheets were curled up. The phenomenon of aggregation occurs. The above phenomenon was sufficiently visible with the naked eye. The cells collected from each other formed a three-dimensional pellet-like structure rather than a tubular (tubular) structure (FIG. 3). When this structure was observed in more detail with a phase-contrast microscope, it was shown that these cells were spontaneously assembled as if they formed some tissue (Comparative Test Example A1).

(Test A2) Examination of cell density It was the same as Test 1 except for the conditions shown below. Detailed conditions are shown in Table 1.
(Test A2a) Preparation of cell culture composition (adjustment step)
As an anionic substance, GFP expression vector pQBI25 (DNA) was used. An aqueous solution of the polymer / Tris mixture was prepared in the same manner as (Aa2) of Test A1a, with the Tris ratio to the polymer being 0.1 by changing only the Tris content.
This cell culture composition had a C / A ratio (positive charge / negative charge) of 8.4.

(Test A2c, A2d) Cell culture (seeding process, culture process)
Adhesive rat subcutaneous fat-derived vascular endothelial cells (primary cultured cells) were used. Then, the cell density, 3.8 × 10 ² pieces ^/ mm 2 (7.5 × 10 ⁴ pieces /ML),7.5×10 ² pieces / ^mm 2 (15 × 10 ⁴ pieces / mL), 15 × 10 ² / mm ² (30 × 10 ⁴ / mL) (Test Example A2, Test Example A3, Test Example A4, respectively).
Except for the above, cell culture was performed in the same manner as in tests A1c and A1d. FIG. 4 shows a diagram observed with a phase contrast microscope.
After 70 hours, the cells became confluent. When the culture was further continued, in Test Example A2 and Test Example A3, on the fifth day, a part of the cells moved on the plate and the cells gathered together (FIG. 4). A luminal (tubular) structure was formed (FIG. 4). On the other hand, in Test Example A4, cell aggregates aggregated to form a pellet-like structure (FIG. 4). From FIG. 3, the luminal structure of Test Example A2 has higher homogeneity than the luminal structure of Test Example A3, and some of the cell layers were completely detached in Test Example A3. It can be seen that a pellet structure is being formed.

(Test A3) Examination of the ratio of Tris It was the same as Test A1 except for the conditions shown below. Detailed conditions are shown in Table 1.
(Test A3a) Preparation of cell culture composition (adjustment step)
By changing only the Tris content, the polymer / Tris mixture aqueous solution in which the ratio of Tris to the polymer was set to 0, 1, 2, 4, 8 was the same as (A1a-2) in Test A1a. Prepared (Test Example A5, Test Example A6, Test Example A7, Test Example A8, Test Example A9, respectively).
This cell culture composition had a C / A ratio (positive charge / negative charge) of 8.4.

(Test A3c, A3d) Cell culture (seeding process, culture process)
Cell culture was performed in the same manner as in Tests A1c and A1d, except that vascular endothelial cells (primary cultured cells) derived from adhesive rat subcutaneous fat were used. Here, the cell density was 7.5 × 10 ² cells / mm ² (15 × 10 ⁴ cells / mL). FIG. 6 shows a view observed with a phase contrast microscope.
On the fifth day, in Test Examples A5 and A6, it was confirmed that the aggregate of cells formed a tubular (tubular) structure (FIG. 5). On the other hand, in Test Examples A7 to A9, the proportion of cells having no normal shape was large, and necrosis occurred in some cells (FIG. 5).

(Test A4) Examination of ratio of anionic substance (examination of C / A ratio)
The conditions were the same as in Test Example A1 except for the conditions shown below. Detailed conditions are shown in Table 1.
(Test A4a) Preparation of cell culture composition (adjustment step)
The ratio of Tris to the polymer of this cell culture composition was 0.1.
By changing only the content of the anionic substance, the C / A ratio (positive charge / negative charge) in the cell culture composition was set to 0.5, 2, 4, 8, 16 and the polymer / tris / anion The active substance mixture was prepared in the same manner as (A1a-2) in Test A1a (Test Example A10, Test Example A11, Test Example A12, Test Example A13, Test Example A14, respectively).

(Test A4c, A4d) Cell culture (seeding process, culture process)
Cell culture was carried out in the same manner as in test A1b except that vascular endothelial cells (primary cultured cells) derived from adhesive rat subcutaneous fat were used. Here, the cell density was 7.5 × 10 ² cells / mm ² (15 × 10 ⁴ cells / mL). FIG. 6 shows a view observed with a phase contrast microscope.
On the fifth day, in Test Example A13, it was confirmed that the cell aggregate formed a tubular (tubular) structure (FIG. 6). On the other hand, in Test Example A10 and Test Example A11, the adherent cells showed good adhesion extensibility as observed in normal culture (FIG. 6). In Test Example A12, some cells moved on the plate. However, a phenomenon in which the cells gather together occurred, and many small aggregates were formed (FIG. 6). In Test Example A14, most cells were killed (FIG. 6).

(Test A5) Examination of content of polymer contained in aqueous solution of cell culture composition The same as test A1 except for the conditions shown below. Detailed conditions are shown in Table 1.
(Test A5a) Preparation of cell culture composition (adjustment step)
An aqueous solution of a polymer / Tris mixture was prepared in the same manner as (A1a-2) of Test A1a by changing only the Tris content, so that the ratio of Tris to the polymer was 0.1.
This cell culture composition had a C / A ratio (positive charge / negative charge) of 8.4.

(Test 5b) Preparation of cell culture device (preparation process)
Tested by setting the content of the temperature-responsive polymer contained in the aqueous solution of the cell culture composition to be added to each well of the polystyrene 24-well cell culture plate to 1.0, 2.0, 4.0, and 8.0 μg. Similar to A1b, the cell culture device was coated with the cell culture composition (the amount of the temperature-responsive polymer per unit area of the well was 5, 10, 20, 40 ng / mm ² , respectively). And the 24-well cell culture plate (cell culture device) which coat | covers the composition for cell culture on the culture surface was obtained (Test Example A15, Test Example A16, Test Example A17, Test Example A18, respectively).

(Test A5c, A5d) Cell culture (seeding process, culture process)
Adhesive rat subcutaneous fat-derived vascular endothelial cells (primary cultured cells) were used. The cell density was 3.8 × 10 ² cells / mm ² (7.5 × 10 ⁴ cells / mL).
Other than the above, cell culture was performed in the same manner as in Test A1d. FIG. 7 shows a view observed with a phase contrast microscope.
On the fifth day, in Test Examples A17 and A18, the cell aggregate formed a tubular (tubular) structure (FIG. 7). On the other hand, in Test Example A15, the adherent cells showed good adhesion extensibility as observed in normal culture (FIG. 7), and in Test Example A16, the cell aggregate formed a pellet-like structure. (FIG. 7).

(Test A6) Examination of anionic substances: When heparin was used The same procedure as in Test A1 was conducted except for the conditions shown below. Detailed conditions are shown in Table 1.
(Test A6a) Preparation of cell culture composition and cell culture vessel Heparin was used as an anionic substance. The polymer / Tris / anionic substance mixture can be confirmed to have a cloud point of 30 to 32 ° C., and the above mixture, which has been solidified by raising the temperature to 37 ° C., is maintained at room temperature (25 ° C.). Although it remained in a solidified state for a period of 24 minutes, it was confirmed that the solidified product was gradually dissolved and changed into a homogeneous aqueous solution.
And the cell culture device which coat | covers the said mixture on a culture surface was obtained. This cell incubator had high cell affinity and antithrombotic properties.
The inventors have described the composition for cell culture according to the above test A6 as non-patent literature “Nakayama, Y., Yamaoka, S., Nemoto, Y., Alexey, B., Uchida, K., Thermoresponsive Heparin Bioconjugate as Novel Aqueous Anti throrombogenic Coating Material, Bioconjugate Chem., 2011, 22, 193-199. ”

(Comparative test A2) Uncoated cell culture vessel It was the same as test A1 except for the conditions shown below. Detailed conditions are shown in Table 1.
Each well of a 24-well cell culture plate made of polystyrene untreated or coated with fibronectin was seeded in the same manner as in test A1c, and the cells were cultured in a cell culture incubator.
After 3 days, the cells became confluent. When the culture was further continued, most of the cells stopped growing, but a small part of the cells grew so as to overlap the cells adhered to the culture surface. Then, the number of cells that cause necrosis, fusion, exfoliation, etc. increased, and many cells died (this cell death is presumed to be the effect of degrading enzymes and HSP (heat shock protein) released from dead cells. A result similar to the result shown in FIG. 8 below was obtained). As in Test A1, exposure of the plate surface, cell lumen (tube shape), and pellet structure were not confirmed.

(Comparative test A3) Cell culture vessel coated with a composition containing no anionic substance The same conditions as in test A1 except for the conditions shown below. Detailed conditions are shown in Table 1.
Using the aqueous solution of the polymer / Tris mixture prepared in (A1a-2) that does not contain an anionic substance, the cell culture composition was coated on the culture surface of the cell culture vessel in the same manner as in Test A1b. Then, a 24-well cell culture plate (cell culture device) obtained by coating the cell culture composition was obtained.

Vascular endothelial cells (primary cultured cells) derived from rat subcutaneous fat were used. The cell density was 7.5 × 10 ² cells / mm ² (15 × 10 ⁴ cells / mL).
Except for the above, cell culture was performed in the same manner as in Tests A1b and A1c. Most cells did not adhere at the initial stage of culture (after 6 hours of culture), and it was confirmed that most cells were necrotic on the second day of culture (FIG. 8).

(Test A10) Production of cell structure (Test A10a) Preparation of cell culture composition (adjustment step)
(A10a-2) Preparation of polymer / anionic substance mixture Sodium polyacrylate (PAANA) was used as the anionic substance. Polyacrylic acid (number average molecular weight Mn = 4.0 × 10 ⁶ g / mol) was weighed, and this was mixed with a carboxyl group in the molecule and a stoichiometrically equimolar amount of sodium hydroxide, and water was further added. In addition, an aqueous solution was obtained. This aqueous solution was stirred for 72 hours and then freeze-dried to obtain a powder, which was used in the following experiment. This powder preparation method is well known to those skilled in the art. 20 μL of an aqueous polymer solution (85 μg / mL), 30 μL of a sodium polyacrylate aqueous solution (33 μg / mL), and physiological saline were prepared. In the same manner as in test A1, these were mixed to prepare a mixture having C / A ratios of 0.5, 1, 2, 4, and 6 for different polymers and sodium polyacrylate amounts. Then, this mixture was diluted with an appropriate amount of physiological saline under each condition to adjust the total amount of the solution to 200 μL, and a polymer / anionic substance mixture, that is, an aqueous solution of a cell culture composition was obtained. Detailed conditions are shown in Table 1.

(A10b) Coating of cell culture composition with cell culture composition (coating process)
Using the aqueous solution of the cell culture composition prepared in (A10a-2), each well of a polystyrene 24-well cell culture plate (bottom area per well: 200 mm ² ) is coated in the same manner as (A1b). did.
In this way, a 24-well cell culture plate (cell culture device) was obtained in which the well on the culture surface was coated with the composition for cell culture.

(Test A10c, 10d) Cell culture (seeding process, culture process)
(Test A1c) except that stromal cells (primary cultured cells) derived from rat subcutaneous fat were used and the cell density was 10 × 10 ⁴ cells / mL (cell density: 5.0 × 10 ² cells / mm ² ). ), Cell culture was performed (Test Examples A22 to A36).
Cells were observed after 6 days. In Test Example A22 where the C / A ratio was 0.5, no change was observed in the cells, and in Test Examples A23 and A24 where the C / A ratio was 1, 2, the cell aggregates contained many small aggregates. In Test Example A25, where the C / A ratio was 4 and the aggregate of cells formed a tubular (tubular) structure, and in Test Example A26 with a C / A ratio of 6, most cells Died (Fig. 10, top). In Test Example A27 in which the C / A ratio was 0.5, no change was observed in the cells. In Test Example A28 in which the C / A ratio was 1, the cell aggregates formed many small aggregates. In Test Example A29 with a C / A ratio of 2, the aggregate of cells formed a tubular (tube-like) structure, and in Test Examples A30 and A31 with a C / A ratio of 4 and 6, Cells were killed (FIG. 10, middle panel). Furthermore, in Test Example A32 in which the C / A ratio was 0.5, no change was observed in the cells, and in Test Example A33 in which the C / A ratio was 1, the cell aggregate formed many small aggregates. In Test Examples A34, A35, and A36 with C / A ratios of 2, 4, and 6, most cells were killed (FIG. 10, lower panel).

  The results of Test Example A and Comparative Test Example A are shown in Table 1. FIG. 9 shows the correlation between the structure of cells cultured using the cell culture composition and the cell culture device of the present invention and the culture conditions.

(Test B) Analysis of the polymer / Tris mixture (Test B1) Measurement of cloud point of the polymer / Tris mixture 0.1% of the final concentration of the polymer, and the ratio of Tris to the polymer is 0, 0.5, 1, 2, 4 and 8, as in (Test A) (Test A1a) and (A1a-3), an aqueous solution of a polymer / Tris mixture was prepared (Test Example B1, Test Example B2, Test Example B3, Test Example B4, respectively). Test Example B5, Test Example B6).

The absorbance at 600 nm of the aqueous solution of the polymer / Tris mixture prepared in (Test A1a) (A1a-3) of (Test A) was measured between 20 ° C. and 37 ° C.
Specifically, 2 mL of each aqueous solution was added to a 10 mm × 10 mm square quartz cell (thickness 1 mm). And the light absorbency in each temperature was measured, heating up 1 degreeC at a time from initial temperature 20 degreeC. Here, the absorbance at each temperature was determined by measuring the absorbance after maintaining the temperature for 30 minutes so as not to be affected by the temperature responsiveness of the polymer.
As a result, when Tris is not included (Test Example B1), the cloud point of the polymer is 32 ° C., and the cloud point of the polymer / Tris mixture (Test Example B2 to Test Example B6) is 31.0 ° C. to The temperature dropped to 30.5 ° C.

Moreover, it analyzed about the temperature responsiveness of the aqueous solution of the said polymer / Tris mixture. Specifically, the aqueous solution of each polymer / Tris mixture is allowed to stand at 37 ° C. for 30 minutes to become cloudy, and immediately maintained at 25 ° C. until the aqueous solution has a transmittance of 100% (control is water). The time required for was measured.
The results are shown in FIG. It was confirmed that the rate of phase transition from the solid phase to the liquid phase decreased as the ratio of Tris to the polymer increased.

(Test B2) Analysis of polymer / Tris mixture by ¹ H NMR The final concentration of the polymer was 4.1 mg / mL, and the ratio of Tris to the polymer was 1, 2, 4, 8 (Test A1a) (Test A1a) A heavy water (D ₂ O) solution of a polymer / Tris mixture was prepared according to the method described in (A1a-2) (Test Example B7, Test Example B8, Test Example B9, Test Example B10, respectively).
The nuclear magnetic resonance spectrum (NMR) of this polymer / Tris mixture was measured under the conditions of 4 ° C., 25 ° C., and 37 ° C. using a nuclear magnetic resonance apparatus (manufactured by Varian, model number: Gemini 300). Detailed conditions are shown in Table 2.

The results are shown in FIG. The integral value of the ¹ H NMR signal of the polymer refers to the integral value of -N (CH ₃ ) ₂ protons in the polymer side chain, and the integral value of Tris refers to the integral of protons of -C (CH ₂ OH) ₃ groups. Points to the value. At 4 ° C., the weight ratio (relative ratio) of Tris to the polymer and the ratio of the integral value of Tris ¹ H NMR signal to the polymer showed values that were almost theoretical values. At 25 ° C., as the weight ratio (relative ratio) of Tris to polymer increased, the ratio of the integrated value of Tris ¹ H NMR signal to polymer increased rapidly. At 37 ° C, no polymer side chain proton peak was detected. From these results, when the polymer / Tris mixture exceeds about 25 ° C., it forms agglomerates due to hydrophobic aggregation, thereby shielding the polymer that aggregates in a temperature-responsive manner from the magnetic field environment. It was found that in any polymer / Tris mixture, Tris is present in the solvent in a higher proportion at any temperature compared to the polymer (of course, some of the Tris molecules are attached to the polymer by hydrogen bonding with the polymer). It is thought that it is taken in the agglomerate).

(Test B3) Analysis of Polymer / Tris Mixture by Differential Scanning Calorimeter Assuming that the final concentration of polymer is 4.1 mg / mL and the ratio of Tris to polymer is 0, 0.5, 1, 2, 4, 8, (Test In the same manner as (Test A1a) and (A1a-2) in A), an aqueous solution of a polymer / Tris mixture was prepared (Test Example B11, Test Example B12, Test Example B13, Test Example B14, Test Example B15, and Test Example, respectively) B16).
The temperature control described below was performed using a program of the thermal analyzer. The polymer / Tris mixture aqueous solution was heated to 40 ° C. and maintained for 3 minutes to cause precipitation in a closed measuring cell, and then the mixture was instantaneously cooled to 25 ° C. and maintained at 25 ° C. as it was. Then, this mixture was subjected to differential differential scanning calorimetry (dDSC) using a differential scanning calorimeter (manufactured by Shimadzu Corporation, model number: DSC60). Detailed conditions are shown in Table 2.

  The results are shown in FIG. The polymer / Tris mixture melts around 25 ° C., where an endotherm occurs, and as the weight ratio of Tris to the polymer increases, the time from cooling to 25 ° C. until the endotherm occurs increases. It was.

  From the results of (Test B1) to (Test B3), it was revealed that the temperature responsiveness of the composition containing the polymer is changed by adding Tris to the polymer having temperature responsiveness. More specifically, by adding Tris, once at 37 ° C, the composition containing the polymer on the bottom of the culture dish is agglomerated, precipitated and applied to a cell incubator prepared at room temperature (about 25 ° C). ) (For example, in a clean bench), it is possible to prevent the polymer layer coated on the cell culture vessel from being redissolved and washed away due to its temperature responsiveness.

  The results of Test Example B and Comparative Test Example B are shown in Table 2.

(Test C) Measurement of the zeta potential on the surface of the cell culture vessel (Test C1) Measurement of the zeta potential on the entire surface of the cell culture vessel (Test C1-1) Coating of the cell culture composition on the cell culture vessel Cell culture plate made of polystyrene (Nunc, Nunclon (registered trademark) Δ Surface, 100 m diameter) was cut into small pieces having a size of 35 mm × 14 mm × 1 mm. The cell culture plate is prepared by performing plasma discharge in a mixed gas containing ammonia and oxygen. By this treatment, an ammonia-derived amino group (—NH ₂ ) and an oxygen-derived carboxyl group (—COOH) are prepared. Is introduced.
Here, similar to (Test A4) of (Test A), a polymer / Tris / anionic material mixture was prepared with a C / A ratio of 0.5, 2, 4, 8, 16 and (Test As in (Test A1b) of A), each polymer / Tris mixture was coated on the cut cell culture plate (Test Example C1, Test Example C2, Test Example C3, Test Example C4, Test, respectively). Example C5).
Specifically, an aqueous solution of a polymer / Tris / anionic substance mixture was cast on the entire surface of the cell adhesion surface of the small piece, and the small piece of the cell culture plate was placed in another cell culture plate and covered.
Incubate for 6 hours in a cell culture incubator (37 ° C., 5% CO ₂ ) while preventing drying in the presence of humidification water, then stop using the humidification water and remove the lid of the cell culture plate at the same time. The inside was dried and incubated for 24 hours. As a result, after about 21 hours, the water on the surface on which the mixture was cast was almost evaporated. Observation with a scanning electron microscope revealed that a coating layer of the polymer / tris / anionic substance mixture was uniformly formed on almost the entire surface of the small piece casted with the aqueous solution of the polymer / tris mixture.

(Test C1-2) Zeta potential measurement 1
(Test C1-2a) Relationship with C / A Ratio The zeta potential of the coated surface of the coated piece was measured using a zeta potentiometer (manufactured by Otsuka Electronics Co., Ltd., model number: ELSZ) and a cell unit for flat plate samples. did.
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 cell culture plate was −68 mV, which was a value well known to those skilled in the art as the zeta potential of a general thermoplastic resin solid surface (Comparative Test Example C1).
On the other hand, it was found that the zeta potential on the surface of the cell culture plate coated with the polymer / Tris / anionic substance mixture showed a positive value and increased depending on the C / A ratio. Specifically, in test example C1 with C / A ratio = 0.5, in test example C3 with -28 mV and C / A ratio = 2, in test example C3 with C / A ratio = 4, In Test Example C4 of 19 mV and C / A ratio = 8, it was 20 mV and in Test Example C5 of C / A ratio = 16, it was 22 mV.
By comparing this result with the result of (Test A3) in (Test A), it was suggested that the zeta potential on the surface of the cell culture vessel may affect cell differentiation.
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. The results are shown in FIG. The zeta potential across the cell culture vessel surface tended to saturate as the C / A ratio increased.
(Test C1-2b) Relationship with pH In the same manner as (Test C1-2a), the zeta potential of the coated surface of the coated piece was measured.
The solvent of the monitor particle suspension was used under the condition of pH = 5. First, the zeta potential of the surface of the uncoated cell culture plate was measured and found to be −40 mV (Comparative Test Example C2). This is because, under acidic conditions, dissociation of protons of carboxyl groups (—COOH) introduced to the surface of the culture plate is suppressed, and protonation of amino groups (—NH ₂ ) is promoted, resulting in positive charge. Is estimated to be the result of the increase. On the other hand, it was found that the zeta potential on the surface of the cell culture plate coated with the polymer / Tris / anionic substance mixture showed a positive value and increased depending on the C / A ratio. Specifically, in Test Example C6 where the C / A ratio = 2, it was 36 mV. Compared with the result of Test Example C2 (pH = 7, 0.5 mV), the potential was greatly increased.
This result is presumed to be a result of an increase in the positive charge by promoting the protonation of the tertiary amino group of the polymer. The zeta potential of 36 mV at pH = 5 and C / A ratio = 2 is higher than the zeta potential of 22 mV at pH = 7 and C / A ratio = 16 (Test Example C5).
When the solvent of the monitor particle suspension was used under the condition of pH = 9, the zeta potential on the surface of the uncoated cell culture plate was −71 mV (Comparative Test Example C3), and the polymer / Tris / anionic property The zeta potential on the surface of the cell culture plate coated with the substance mixture was −12 mV in Test Example C10 with a C / A ratio = 2, and turned negative.
From these results, it was shown that the zeta potential on the surface of the cell culture vessel, which may affect cell differentiation, can also be prepared by the pH of the cell culture composition.

(Test C1-3) Zeta potential measurement 2
The zeta potential was measured in the same manner as in (Test C2) except that protein particles in FCS (fetal bovine serum, manufactured by Invitrogen, Australia, lot number 852546) solution were used as standard monitor particles.
Since FCS is a mixture as is well known to those skilled in the art, the strongest light scattering peak was observed in the neutral range when the FCS solution was irradiated with laser light (in most cases, it was observed as a single peak). The particles were used as monitor particles.
The zeta potential of the standard monitor particle measured in 10% FCS aqueous solution was about −20 mV.
The zeta potential on the surface of the uncoated cell culture plate is -19 mV (Comparative Test Example C4), while the zeta potential on the surface of the cell culture plate coated with the polymer / Tris / anionic mixture is any C The / A ratio was also about -14 mV (Test Examples C11 to C14).
In the case of an uncoated cell culture plate, the zeta potential on the surface thereof is almost the same as the zeta potential of the FCS monitor particles, so that the protein component contained in the FCS may be adsorbed on the plate surface. It was suggested. On the other hand, in the case of a cell culture plate coated with a composition for cell culture, the zeta potential of the surface is smaller than the zeta potential of the FCS monitor particles, and thus is included in the FCS. It was suggested that inhibition of the adsorption of protein components to the plate surface or selective adsorption of some protein components to the plate surface occurred.

(Test C2) Measurement of zeta potential of fine particles on cell culture vessel surface Polymer / anionic substance mixture, that is, cell of the present invention in accordance with the above description of (A1a-2) Preparation of polymer / tris / anionic substance mixture An aqueous solution of the cell culture composition used in the method for producing the structure was prepared.
As an anionic substance, firefly luciferase reporter vector (DNA) (manufactured by Promega, model number: E1741) was desalted by a method well known to those skilled in the art, and DNA was dissolved in DNase-free water (final concentration: 33 μg / mL). .
The aqueous polymer solution was adjusted to a final concentration of 100 μg / mL, and 50 μL, 100 μL, 200 μL, 400 μL, and 800 μL were added dropwise to 300 μL of the DNA solution and mixed. Then, this mixture is diluted with water to adjust the total amount of each solution to about 2,000 μL, and the cell culture used in the method for producing a polymer / anionic substance mixture, that is, the cell structure of the present invention. An aqueous solution of the composition was obtained (Test Example C15, Test Example C16, Test Example C17, Test Example C18, Test Example C19, respectively). The CA ratios are 1, 2, 4, 8, and 16, respectively. The prepared solution was measured for the zeta potential of the fine particles that were in Brownian motion in the solution using a zeta electrometer (Sysmex Corporation, Zeta Sizer Nano).

  FIG. 15 shows the result. As for the fine particles before aggregation and precipitation on the surface of the cell culture vessel, the zeta potential tended to saturate as the C / A ratio increased. This result had a correlation with the measurement result of the zeta potential of the entire cell culture device surface.

  The results of Test Example C and Comparative Test Example C are shown in Table 3.

(Test D) Surface structure analysis by atomic force microscope (AFM) (Test D-1) Surface structure analysis by atomic force microscope (AFM) FIG. 16 (a) was uncoated or coated with a composition for cell culture. The image which measured the surface of the cell culture plate in the contact tapping mode is shown. From this result, it was confirmed that the unevenness on the nanoscale of the surface of the plate changes depending on the C / A ratio (Test Example D1, Test Example D2, Test Example D3, Test Example D4).
FIG. 16 (b) shows an image of the surface of a cell culture plate uncoated or coated with a composition for cell culture in phase mode. From this result, it was confirmed that the viscoelastic or adsorptive property of the surface of the plate also changes depending on the C / A ratio, as with the nanoscale irregularities on the plate surface (Test Example D1, Test Example D2, Test Example D3, Test Example D4).

(Test D-2) Surface observation with fluorescence microscope As an anionic substance, 2.7 kb DNA (Mirus Bio, model number: MIR7904) fluorescently labeled with Cy3 was used. Similarly to (A0a), a polymer / anionic substance mixture having C / A ratios of 2, 4, 8, and 16 (Test Example D5, Test Example D6, Test Example D7, and Test Example D8, respectively) was prepared. Then, each well of a polystyrene 24-well cell culture plate (bottom area per well: 200 mm ² ) was coated in the same manner as (A1b). After incubating the plate at 37 ° C. for 6 hours, each well was observed using a fluorescence microscope (Nikon Corporation, trade name: TE2000-U) (excitation wavelength: 488 nm, fluorescence wavelength: 570 nm). Detailed conditions are shown in Table 4.
FIG. 17 shows the observation results. There was a tendency for the fluorescence intensity to decrease from Test Example D5 with a C / A ratio of 2 to Test Example D8 with a C / A ratio of 16. This result suggests that when the C / A ratio is large, Cy3-labeled DNA is coated on the polymer, so that the laser light is blocked by the polymer.

(Test D-3) Surface observation by underwater AFM As an anionic substance, firefly luciferase reporter vector (DNA) (manufactured by Promega, model number: E1741) is desalted by a method well known to those skilled in the art and dissolved in DNase-free water. Used. Similarly to (A1a-5), a polymer / anionic substance mixture having a C / A ratio of 2, 4, 8, 16 (Test Example D9, Test Example D10, Test Example D11, Test Example D12, respectively) was prepared. did. Then, each well of a 24-well cell culture plate made of polystyrene (bottom area per well: 200 mm ² ) was coated in the same manner as (A1a-7). After incubating the plate at 37 ° C. for 3 hours, each well was observed using an atomic force microscope (SPM-9700, manufactured by Shimadzu Corporation) (resonance frequency 75 kHz to 145 kHz, spring constant 0.1 N / m, silicon nitride) ). Detailed conditions are shown in Table 4.
FIG. 18 shows the observation results. The upper part of FIG. 18 shows an image in the dynamic tapping mode, and the lower part of FIG. 18 shows an image in the phase mode. There was a tendency for the number (density) of fine particles to increase from Test Example D9 with a C / A ratio of 2 to Test Example D12 with a C / A ratio of 16. The fine particles had a diameter of 50 nm to 500 nm. From this result, it was found that the polymer / anionic substance mixture was deposited and deposited as fine particles on the bottom surface of the culture dish without casting into a film at the time of coating, so that there was an area where polystyrene was exposed to the well. It was confirmed to exist.

  The results of Test Example D and Comparative Test Example D are shown in Table 4.

The method for producing a cell structure according to the present invention is obtained by using a tubular (tubular) cell structure having the same structure as that of a differentiated cell. It can be easily reproduced in a cell culture vessel without depending on the above. In addition, it is possible to provide a useful tool for obtaining information on DNA, mRNA, protein and the like expressed in differentiated cells.
For example, when the present invention is applied to vascular endothelial cells, vascular cells having a luminal structure can be cultured. By attaching the vascular cells to the heart, it may be possible to generate capillaries from the cells and provide collateral blood flow in the vicinity of the blocked blood vessel. Further, for example, by co-culturing the vascular cells and cardiomyocytes, it may be possible to construct cardiomyocytes having a vascular system.

Claims (5)

2-N, N-dimethylaminoethyl methacrylate and / or derivatives thereof, 2-amino-2-hydroxymethyl-1,3-propanediol, nucleic acid, heparin, hyaluronic acid, dextran sulfate, polystyrene sulfonic acid A composition for cell culture by mixing one or more anionic substances selected from the group consisting of polyacrylic acid, polymethacrylic acid, polyphosphoric acid, sulfated polysaccharides, curdlan and polyalginic acid, and alkali metal salts thereof A preparation process for preparing the product,
A step of preparing a coated cell culture device by coating the culture surface of the cell culture device with the cell culture composition;
A seeding step of seeding cells on the culture surface of the coated cell culture vessel at a cell density of less than 1.0 × 10 ³ cells / mm ² ;
And a culture step of culturing the seeded cells.   2. The cell culture composition according to claim 1, wherein a ratio of the 2-amino-2-hydroxymethyl-1,3-propanediol to the polymer is 1.0 or less. A method for producing a cell structure.   The method for producing a cell structure according to claim 1 or 2, wherein the composition for cell culture has a C / A ratio of 4.5 to 15. In the coated cell culture vessel, the amount of 2-N, N-dimethylaminoethyl methacrylate and / or its derivative polymer per unit area on the culture surface of the coated cell culture vessel is 10 to 200 ng / mm ^2. The method for producing a cell structure according to any one of claims 1 to 3, wherein   A cell structure produced by the method for producing a cell structure according to any one of claims 1 to 4.