JP6062733B2 - Cell culture composition and cell culture vessel - Google Patents

Description

  The present invention relates to a cell culture composition and a cell culture vessel.

  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 carried out in a general polystyrene cell culture dish, the cells only form a monolayer structure and have a structure similar to that found in highly differentiated cells. The body cannot be formed.

  For example, when a vascular endothelial cell is cultured in a monolayer, it proliferates but dedifferentiates and cannot form a luminal (tubular) structure seen in this cell. Functions such as secretion of antithrombotic substances that cells have are often lost. Similarly, in the case of chondrocytes, a pellet-like cell structure found in these cells cannot be formed, and the function as chondrocytes is not expressed.

  Regarding the above problem, for example, a cell culture method is known in which cells are differentiated by giving appropriate stimulation to the cells (for example, adding VEGF or the like to vascular endothelial cells). 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.

  On the other hand, as a technique for recovering a cultured cell structure from a cell incubator, a cell incubator in which poly (N-isopropylacrylamide) (PNIPAAm), which is a temperature-responsive polymer, is introduced onto the surface by radiation graft polymerization is known. (For example, refer to Patent Document 1). Here, the temperature-responsive polymer is soluble in water at a temperature lower than a predetermined temperature (lower critical solution temperature, hereinafter also referred to as “cloud point”), but insolubilized at a predetermined temperature or higher. Refers to the polymer that precipitates.

  In the radiation graft polymerization, PNIPAAm is covalently bonded and fixed to the surface of the petri dish by polymerizing N-isopropylacrylamide as a monomer using radical carbon generated on the surface of the polystyrene petri dish by irradiation. Therefore, it is possible to prevent the polymer layer from being redissolved and lost when cell culture is performed in a clean bench at room temperature (about 25 ° C.) with respect to the cell culture vessel having PNIPAAm.

  According to this cell culture vessel, cells are cultured in a general 37 ° C. incubator to form a tissue, and then subjected to cell detachment operation such as trypsin treatment under conditions of 32 ° C. or lower. And cell structures can be recovered. Therefore, for example, cells having a cell sheet-like structure can be recovered while retaining the extracellular matrix.

Japanese Patent No. 4475847

  However, in a cell culture vessel in which the conventional poly (N-isopropylacrylamide) is introduced onto the surface by radiation graft polymerization, after the radical polymerization reaction by radiation irradiation, unreacted monomers must be washed and further sterilized. Could not be used. Therefore, those who can produce the cell culture device are only researchers in a limited research facility, and the cell culture device is generally expensive (RepCell (registered trademark), Cellseed, reference).

  In addition, when cells are cultured using a general cell culture vessel, in order to form these cell structures, addition of a substance that stimulates the cells (eg, VEGF) and formation of the cell structures Scaffolding preparation, additional sterilization to cell incubators, application of physical, chemical or mechanical stimuli (eg pulsation to the pulse frequency or blood flow shear force on vascular endothelial cells) Such as load application).

Therefore, the present invention provides a cell culture composition containing a temperature-responsive polymer with a cloud point reduced to room temperature or lower, and a cell culture vessel, which enables operation of cell culture under room temperature conditions. The purpose is to.
The present invention also provides a composition for cell culture and a cell culture vessel that can easily form a cell structure while maintaining the differentiated state of the cultured cells equivalent to that in the living body without requiring the above operations. It is also intended to provide.
In the present invention, the “cloud point” of the composition does not necessarily mean a strict meaning, “the temperature at which it dissolves below a predetermined temperature but becomes insoluble and precipitates above a predetermined temperature”. In addition, it 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 a condition lower than a predetermined temperature”.

The gist of the present invention is as follows.
The composition for cell culture of the present invention comprises a polymer of 2-N, N-dimethylaminoethyl methacrylate (hereinafter also referred to as “DMAEMA” ) and 2-amino-2-hydroxymethyl-1,3-propanediol. (hereinafter, also referred to as "tris".) and, seen containing nucleic acid, heparin, polyacrylic acid, polymethacrylic acid, and one or more anionic material selected from the group consisting of alkali metal salts,
The ratio of the 2-amino-2-hydroxymethyl-1,3-propanediol to the polymer is 1.0 or less,
The C / A ratio is 0.5 to 16 .

  The cell culture device of the present invention is characterized in that the culture surface is coated with any of the cell culture compositions of the present invention.

Also, in the cell culture device of the present invention, the culture surface of the cell culture vessel is, 2-N having per unit area, the amount of N- dimethylaminoethyl methacrylate click relay bets polymer, in 5~200ng / mm ² Preferably there is.

The present invention is a temperature-responsive polymer in which the cloud point is reduced to room temperature (about 25 ° C.) or less (a polymer insolubilized at a temperature higher than the cloud point is hardly re-dissolved at room temperature (about 25 ° C.) or less). A cell culture composition and a cell culture vessel are provided to enable operation of cell culture under conditions of about room temperature.
Moreover, this invention can provide the composition for cell cultures and a cell culture device which can form a cell structure simply, without requiring the said various operation.

FIG. 4 is a photograph of a vascular endothelial cell derived from rat subcutaneous fat having a tubular (tubular) structure cultured using the cell culture composition and cell culture vessel of the present invention when 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 the photograph when the vascular endothelial cell derived from the rat subcutaneous fat which has a pellet-like structure cultured using the composition for cell culture and cell culture device of the present invention is observed with a stereomicroscope. FIG. 4 is a photograph of a state where a myriad of fibroblast-like cells diffuse from the pellet-like structure shown in FIG. 3 on the surface of a commercially available cell culture dish when observed with a phase contrast microscope. (A) shows a state in which a cell structure of stromal cells derived from rat subcutaneous fat having a pellet-like structure, which has been cultured using the cell culture composition and cell culture vessel of the present invention, is adhered to a biotube. It is a photograph when it observes with a stereomicroscope, (b) is when the cross section when the biotube which the cell structure adhered is cut | disconnected along XX 'in (a) is observed with a fluorescence microscope. It is a photograph. Phase contrast of rat vascular endothelial cells derived from rat subcutaneous fat, prepared by changing the ratio of Tris to polymer in the cell culture composition and cultured using the cell culture composition and cell culture device of the present invention It is a photograph when observed with a microscope. Rat vascular endothelial cells derived from rat subcutaneous fat and cultured using the cell culture composition of the present invention and the cell culture vessel prepared by changing the C / A ratio in the cell culture composition were analyzed with a phase contrast microscope. It is a photograph when observed. It is a photograph when the vascular endothelial cell derived from rat subcutaneous fat cultured with various cell densities using the composition for cell culture and cell culture device of the present invention is observed with a phase contrast microscope and the naked eye. Photographs obtained by observing vascular endothelial cells derived from rat subcutaneous fat with a phase-contrast microscope, which were prepared by changing the content of the polymer in the cell culture composition and cultured using the cell culture device of the present invention. It is. 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 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 using the composition for cell cultures and cell culture device of this invention, and culture conditions. 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 inventors have added 2-amino-2-hydroxymethyl-1,3- known as a buffering agent to 2-N, N-dimethylaminoethyl methacrylate (DMAEMA), a temperature-responsive polymer having a cloud point of 32 ° C. When a composition was prepared by adding propanediol (Tris), the time until the insolubilized product formed at a temperature higher than the cloud point was redissolved under room temperature (about 25 ° C.) was delayed. I found out. If cells are cultured using this composition, the cell culture operation under room temperature conditions becomes possible.
Next, the inventors added an anionic substance to the above composition to prepare a cell culture composition, and prepared a cell culture device in which the culture surface was coated with the cell culture composition. Then, when cell culture was performed in a general cell incubator (at 37 ° C.) using this cell culture vessel, the cells gathered together to form a tubular (tubular) or pellet-shaped cell structure. Found to form.

(Composition for cell culture)
The composition for cell culture of the present invention 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), (3) nucleic acids, heparin, hyaluronic acid, dextran sulfate, polystyrene sulfonic acid, polyacrylic acid, polymethacrylic acid, polyphosphoric acid, sulfated polysaccharides, curdlan and polyalginic acid, and these And one or more anionic substances selected from the group consisting of alkali metal salts.

  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 its derivative and Tris interact with each other (for example, a cross-linking action), and the polymer is likely to aggregate.

  Regarding temperature responsiveness, in the case of the mixture of (1) and (2), that is, in the case of (3) the mixture not containing an anionic substance, the same behavior as described above occurs.

  In addition, according to the composition for cell culture, since the composition remains solidified from the start of operation in a clean bench at room temperature until the operation is completed and returned to the 37 ° C. incubator again, Cells in culture can continue to adhere to a cell culture vessel in which the culture surface is coated with this composition.

Furthermore, according to this composition for cell culture, a cell structure can be formed by culturing cells under appropriate culture conditions.
This is because the hydrophobic side chain of the polymer of DMAEMA and / or a derivative thereof contained in the composition for cell culture and the amino group of Tris are stimulating the cells while performing some interaction. Presumed. In addition, the anionic substance contained in the composition preferably balances the positive and negative charges in the composition and suppresses cytotoxicity (the surface of the mammalian cell membrane is negatively charged). Therefore, it is presumed that cationic substances often have cytotoxicity) and that the balance between the hydrophilicity and hydrophobicity of the above composition is suitable to enable cell migration and orientation. The
In addition, it is estimated that the composition for cell culture of the present invention is suitable for cell culture because the cytotoxicity and cytotoxicity are suppressed as compared with the composition consisting only of the above (1) and (2). The

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

According to the composition having the above ratio, the cell structure can be easily formed.
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.

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

In the cell culture composition of the present invention, the C / A ratio (positive charge / negative charge) is preferably 0.5 to 32.
Note that 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.

When the C / A ratio is 0.5 to 32, the above effect of reducing the cloud point to room temperature (25 ° C.) or less is easily obtained.
According to the composition having the C / A ratio, since there is an excess of the polymer in the composition, the cationic DMAEMA and / or the derivative thereof arranged so as to be adsorbed by the anionic substance. This is presumed to be a result of the cohesive force exhibited by the polymer in response to temperature changes.

Moreover, if the C / A ratio is 0.5 to 32, the above effect of facilitating the formation of a cell structure can be easily obtained.
According to the composition having the C / A ratio, it is estimated that the balance between the positive charge and the negative charge in the composition is particularly suitable, and the cytotoxicity due to the positive charge can be suppressed. In addition, it is presumed that the balance between hydrophilicity and hydrophobicity of the composition can be made particularly suitable to facilitate cell migration and orientation.

  For the same reason as above, the C / A ratio is more preferably 0.5 to 16, and the C / A ratio is most preferably around 8.

(Cell culture device)
The cell culture device of the present invention has a culture surface coated with the composition for cell culture.

  According to this cell incubator, when the cells are cultured, for example, when operated in a clean bench at room temperature, the composition remains solidified for a certain period of time, specifically 5 to 40 minutes. Therefore, it is possible to keep the composition, and thus the cells in culture, adhered to the cell culture vessel.

In the cell culture device of the present invention, the culture surface of the cell culture device has (1) 2-N, N-dimethylaminoethyl methacrylate (DMAEMA) and / or contained in the composition for cell culture, which is contained per unit area. Or it is preferable that the quantity of the polymer of its derivative (s) is 5-200 ng / mm < ² >.

When the amount per unit area of (1) is 5 to 200 ng / mm ² , the effect of the composition for cell culture of the present invention that facilitates the formation of 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 5 to 50 ng / mm ² .

In the cell culture device of the present invention, for example, an aqueous solution of the composition for cell culture of the present invention cooled to a temperature below the cloud point is cast on the bottom surface of the well of the cell culture device and statically kept in a 37 ° C. incubator. It can be prepared by removing unnecessary water (supernatant) used to coat the composition and then solidifying the composition.
Therefore, it is not necessary to perform a special treatment for coating the surface of the cell culture vessel with the temperature-responsive polymer, for example, radiation graft polymerization. That is, the cell culture device of the present invention can be prepared by a researcher using the culture device by a simple method (without requiring a special apparatus) at a low cost.

(Cell culture method)
The cell culture composition and the cell culture method using the cell culture vessel of the present invention are described below.

  This cell culture method comprises a seeding step for seeding cells in the cell culture device of the present invention and a culture step for culturing the seeded cells.

According to this cell culture method, as described above, even when operated in a clean bench at room temperature, the cells being cultured can be kept adhered to the cell culture vessel. Moreover, according to this cell culture method, a cell structure can be formed similarly to the above.
The above cell culture method makes it possible to reproduce a cell structure having a structure similar to that of a differentiated cell in a cell culture vessel, and information on DNA, mRNA, protein, etc. expressed in the differentiated cell. Can provide useful tools for

This cell culture method is not limited to cell types, but includes various types of cells such as vascular cells, adipose stem cells, hepatocytes, chondrocytes, fibroblasts, cardiomyocytes, kidney cells, neurons, smooth muscle cells, chondrocytes, etc. Can be applied to cells.
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.

(Culture method of tubular (tubular) cells)
Here, in one example of the cell culture method, the density of cells to be seeded is less than 1.0 × 10 ³ cells / mm ² (when seeded by adding 1.0 mL of cell suspension to a 24-well cell culture plate, It is preferable that it is less than 20 × 10 ⁴ pieces / mL). The cells to be seeded are living cells.
With the cell density, a cell structure having a tubular (tubular) structure can be formed.

  The cell culture method of the above example 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.

(Pellet cell culture method)
Here, in another example of the cell culture method, the density of cells to be seeded is 1.0 × 10 ³ cells / mm ² or more (adding 1.0 mL of cell suspension to a 24-well cell culture plate) In the case of seeding by (2), seed at 20 × 10 ⁴ cells / mL or more) on the culture surface of the cell culture device (seeding step).
With the cell density, a cell structure having a pellet-like structure can be formed. This cell structure has living cells in the pellet-like structure.

  The cell culture method of the other example can be particularly suitably applied to mesenchymal cells such as chondrocytes, fibroblasts, vascular endothelial cells, cardiomyocytes and the like. 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.

  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) Manufacture of cell structure (Test A1a) Preparation of composition for cell culture (A1a-1) Synthesis of temperature-responsive polymer In a transparent vial made of soft glass having a capacity of 50 mL, 2- (N, N -Dimethylaminoethyl) methacrylate 7.0g was added, and it stirred using the 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 irradiated with ultraviolet rays for 21 hours using a round black fluorescent lamp (manufactured by NEC, model number: FCL20BL, 18W) without using a solvent (that is, Polymerization (under neat conditions). 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) Measurement of cloud point of temperature-responsive polymer Hereinafter, DNase and RNase-free sterile water were used.
A 3% aqueous solution of the temperature-responsive polymer synthesized in (A1a-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.

(A1a-3) Preparation of Polymer / Tris Mixture The temperature-responsive polymer 1% aqueous solution prepared in (A1a-2)) was cooled to 4 ° C. to make this solution liquid. Under cooling, granules of 2-amino-2-hydroxymethyl-1,3-propanediol (Tris) (manufactured by Wako Pure Chemicals, Tris999) were added to this solution and rapidly dissolved. And it filtered with a 0.2 micrometer mixed cellulose membrane filter, water was added to this mixture, and the aqueous solution (final concentration is a polymer: 100 microgram / mL, a tris: 100 microgram / mL) of the polymer / Tris mixture was obtained.

(A1a-4) Measurement of cloud point of polymer / Tris mixture The absorbance at 660 nm of the aqueous solution of the polymer / Tris mixture prepared in (A1a-3) was measured between 20 ° C and 40 ° C.
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-3) 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.

(A1a-5) Preparation of polymer / Tris / anionic substance mixture Firefly luciferase reporter vector (DNA) (Promega, 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 8.0.

(A1a-6) 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-4) 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, regarding the temperature responsiveness, the behavior was almost the same as in the case of not mixing an anionic substance, that is, in the case of (A1a-4).

(A1a-7) Coating of cell culture composition on cell culture vessel By cooling the aqueous solution of the cell culture composition prepared in (A1a-5) to 4 ° C., this solution is made into a liquid state and made of polystyrene. 200 μL of this solution was added to each well of a 24-well cell culture plate (Iwaki, microplate, model number: 3815-024, bottom area per well: 200 mm ² ) at room temperature, and the bottom of the hole was quickly Cast all over. 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 A1b) Cultivation of tubular (tubular) cells (Test A1b-1) Cultivation of tubular (tubular) cells on plate Under the conditions of room temperature, (A1a-7) of the above test (1) In each well of the obtained 24-well cell culture plate, vascular endothelial cells derived from rat subcutaneous fat (primary culture cells) were added to a complete medium (Dulbecco's modified Eagle medium (DMEM) + 10% fetal calf serum (FCS) solution, DMEM). 1 ml each of a culture solution suspended in Gibco, model number 11965, FCS: Invitrogen, lot number 852546) and adjusted to a cell density of 10 × 10 ⁴ cells / mL (cell density: 5.0). × 10 ² pieces / mm ² ). 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, a part of the cells moved on the plate, and the cells gathered together, and the exposure of the plate surface was confirmed.
On the fifth day, the exposed areas became visible with the naked eye, while the cells gathered together formed a network-like 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).

(Test A1b-2) Culture of blood vessel-like cell structure using tubular (tubular) cells A plurality of grooves almost parallel to each other were provided on the plate surface to limit the migration and migration of cultured cells. . 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 A1b-1), vascular endothelial cells (primary cultured cells) derived from rat subcutaneous fat were seeded on these plates, and the cells were cultured (Test Example A35, Test Example A36, Test Example A37). 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.

(Test A1c) Cultivation of pellet-like cells In each well of the 24-well cell culture plate obtained in (A1a-7) of the above test (1), vascular endothelial cells (primary culture cells) derived from rat subcutaneous fat were completely added. Suspended in a 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), and the cell density was 15 × The culture solution adjusted to 10 ² cells / mm ² was added by 1 mL each (cell density: 30 × 10 ⁴ cells / mL). This operation took 5 to 10 minutes including taking a photomicrograph in the initial sowing stage. The cells were cultured in a cell culture incubator at 37 ° C. (Test Example A2).
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. Cells that gathered together formed a three-dimensional pellet-like structure (FIG. 3). When this structure was observed in more detail with a phase-contrast microscope, it was shown that these cells assembled spontaneously as if they formed some tissue (Test Example A2).

  When the obtained pellet-like structure was transferred to a normal 24-well cell culture plate that had not been subjected to coating treatment and further cultured, countless fibroblast-like cells were found from the pellet-like structure. Then, it diffused radially so as to bleed, and spread and adhered to the entire surface of the culture dish. As a result, in a cell mass that is large enough to be visually recognized by the naked eye, the cells in the pellet-like structure usually survive, as opposed to necrosis of the internal cells and death. (Fig. 4).

(Test A2) Examination of the ratio of Tris 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, preparation of cell incubator Polymer / Tris with a Tris ratio of 0, 1, 2, 4, 8 by changing only the Tris content An aqueous solution of the mixture was prepared in the same manner as (A1a-3) in Test A1a (Test Example A3, Test Example A4, Test Example A5, Test Example A6, and Test Example A7, respectively).

(Test A2b) Tubular cell culture Cell culture was performed in the same manner as in Test A1b except that adherent rat subcutaneous fat-derived vascular endothelial cells (primary cultured cells) 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 A3 and A4, it was confirmed that the aggregate of cells formed a tubular (tubular) structure (FIG. 6). On the other hand, in Test Examples A5 to A7, the proportion of cells with poor morphology was large, and some of the cells had necrosis (FIG. 6).

(Test A3) Examination of ratio of anionic substance (examination of C / A ratio)
The conditions were the same as in Test 1 except for the conditions shown below. Detailed conditions are shown in Table 1.
(Test A3a) Preparation of cell culture composition, preparation of cell culture device Preparation of cell culture composition (adjustment step)
As an anionic substance, GFP expression vector pQBI25 (DNA) was used. Further, the ratio of Tris to the polymer was set to 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) of Test A1a (Test Example A8, Test Example A9, Test Example A10, Test Example A11, Test Example A12, respectively).

(Test A3b) Tubular cell culture Cell culture was performed in the same manner as in Test 1b, except that vascular endothelial cells (primary cultured cells) derived from adherent rat subcutaneous fat were used. Here, the cell density was 7.5 × 10 ² cells / mm ² (15 × 10 ⁴ cells / mL). FIG. 7 shows a view observed with a phase contrast microscope.
On the fifth day, in Test Example A11, it was confirmed that the cell aggregate formed a tubular (tubular) structure (FIG. 7). On the other hand, in Test Example A8 and Test Example A9, the adherent cells showed good adhesion extensibility as observed in normal culture (FIG. 7), and in Test Example A10, some cells The phenomenon that the cells moved on the plate and the cells gathered to each other occurred to form many small aggregates (FIG. 7), and most of the cells were killed in Test Example A12 (FIG. 7).

(Test A4) Examination of cell density Except for the conditions shown below, it was the same as Test A1. Detailed conditions are shown in Table 1.
(Test A4a) Preparation of cell culture composition, preparation of cell incubator An aqueous solution of a polymer / Tris mixture having a Tris ratio to the polymer of 0.1 by changing only the content of Tris was changed to Test A1a. It was prepared in the same manner as (A1a-3).
The cell culture composition had a C / A ratio of 8.4.

(Test A4b, A4c) Culture of tube-like cells / pellet-like cells 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 pieces / mm ² (30 × 10 ⁴ pieces / mL) (Test Example A13, Test Example A14, Test Example A15, respectively).
Except for the above, cell culture was performed in the same manner as in Tests A1b and A1c. FIG. 8 shows a diagram observed with a phase contrast microscope and the naked eye.
After 70 hours, the cells became confluent. When the culture was further continued, on the fifth day, in Test Example A13 and Test Example A14, a phenomenon in which some cells moved on the plate and the cells gathered to each other occurred. A (tubular) structure was formed (FIG. 8). On the other hand, in Test Example A15, on the fifth day of culture, all the cells were detached from the end of the culture dish at once, and the cells were contracted while being rounded, so that the cells were in one place with each other. Aggregation occurred. The above phenomenon was sufficiently visible with the naked eye. The cells that gathered together formed a three-dimensional pellet-like structure (FIG. 8). Further observation of this structure with a phase-contrast microscope showed that these cells were spontaneously assembled as if they formed some tissue.

(Test A5) Examination of content of polymer contained in aqueous solution of cell culture composition The same as Test 1 except for the conditions shown below. Detailed conditions are shown in Table 1.
(Test A5a) Preparation of cell culture composition and cell incubator An aqueous solution of a polymer / Tris mixture in which the ratio of Tris to polymer was 0.1 by changing only the Tris content was changed to that of Test A1a ( Prepared in the same manner as A1a-3).
The cell culture composition had a C / A ratio of 8.4.

The content of the temperature-responsive polymer contained in the aqueous solution of the cell culture composition added to each well of the polystyrene 24-well cell culture plate is 1.0, 2.0, 4.0, and 8.0 μg. As in (A1a-7) of Test Example A1a, 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 5.0, 10 and 10 respectively). 20, 40 ng / mm ² ). 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 A16, Test Example A17, Test Example A18, Test Example A19, respectively).

(Test A5b, A5c) Culture of tube-like cells / pellet-like cells 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).
Except for the above, cell culture was performed in the same manner as in Test A1b. FIG. 9 shows a view observed with a phase contrast microscope.
On the fifth day, in Test Examples A18 and A19, the aggregate of cells formed a tubular (tubular) structure (FIG. 9). In Test Example 17, the cell aggregate formed a pellet-like structure (FIG. 9). On the other hand, in Test Example A16, the adherent cells showed good adhesion extensibility as observed in normal culture (FIG. 9).

(Test A6) Examination of anionic substances (when heparin is used)
The conditions were the same as those in Test A1 except for the conditions shown below.
(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 A1) Uncoated cell culture vessel It was the same as test 1 except for the conditions shown below. Detailed conditions are shown in Table 1.
In each well of an untreated or fibronectin-coated polystyrene 24-well cell culture plate, cell seeding (cell density: 7.5 × 10 ² cells / mm ² (15 ×), as in tests A1b and A1c. 10 ⁴ cells / mL)), 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. 11 below was obtained). As in Test Example A1b, no exposure of the plate surface, cell lumen (tube shape), or pellet structure was confirmed.

(Comparative Test A2) Cell culture vessel coated with a composition containing no anionic substance The same conditions as in Test 1 except for the conditions shown below. Detailed conditions are shown in Table 1.
Using an aqueous solution of the polymer / Tris mixture prepared in (A1a-3) that does not contain an anionic substance, the cell culture vessel is coated with the composition for cell culture in the same manner as in (A1a-7) of test A1a. did. Then, a 24-well cell culture plate (cell culture device) having a culture surface coated with the composition for cell culture 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. At the initial stage of culture (after 6 hours of culture), most cells did not adhere, and on the second day of culture, it was confirmed that most cells were necrotic (FIG. 11).

(Test A10) Production of Cell Structure (Test A10a) Preparation of Composition for Cell Culture (A10a-1) 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.

(A10a-2) Coating of cell culture composition on cell culture vessel Using the aqueous solution of the composition for cell culture prepared in (A10a-1), similarly to (A1a-7), 24-well cells made of polystyrene Each well of a culture plate (bottom area per well: 200 mm ² ) was coated.
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 A10b) Culture of luminal (tubular) cells Using stromal cells (primary cultured cells) derived from rat subcutaneous fat, the cell density was 10 × 10 ⁴ cells / mL (cell density: 5.0 × 10 6). Cell culture was carried out in the same manner as in (Test A1b) except that ² pieces / mm ² ) (Test Examples A20 to A34).
Cells were observed after 6 days. In Test Example A20 where the C / A ratio was 0.5, no change was observed in the cells, and in Test Examples A21 and A22 where the C / A ratio was 1, 2, the cell aggregates contained many small aggregates. In Test Example A23, which was formed and the C / A ratio was 4, most of the cells were formed in Test Example A24 in which the aggregate of cells formed a tubular (tube-like) structure, and the C / A ratio was 6. Died (Fig. 10, top). In Test Example A25 in which the C / A ratio was 0.5, no change was observed in the cells. In Test Example A26 in which the C / A ratio was 1, the cell aggregates formed many small aggregates. In Test Example A27 with a C / A ratio of 2, the aggregate of cells formed a tubular (tubular) structure, and in Test Examples A28 and A29 with a C / A ratio of 4 and 6, Cells were killed (FIG. 10, middle panel). Furthermore, in Test Example A30 in which the C / A ratio was 0.5, no change was observed in the cells, and in Test Example A31 in which the C / A ratio was 1, the cell aggregate formed many small aggregates. In Test Examples A32, A33, and A34 with C / A ratios of 2, 4, and 6, most cells were killed (FIG. 10, lower panel).

(Test A10c) Pellet-like cell culture Using a rat subcutaneous fat-derived stromal cell (primary culture cell) and the GFP expression vector pQBI 25 (DNA), the cell density was 10.0 × 10 ⁴ cells / mL (cell Cell culture was performed in the same manner as in (Test A1c) except that the density was set to 5.0 × 10 ² pieces / mm ² (Test Example A38). Detailed conditions are shown in Table 1. One day later, a pellet-like cell structure (diameter of about 1 mm) was formed in the same manner as in (Test A1c).
10 mL of medium was placed in a 100 mm diameter cell culture dish, and a biotube was submerged therein. The pellet-like cell structure obtained in the above (Test A1c) was grasped with tweezers and placed on the biotube. After 5 minutes, the cell structure adhered to the biotube and was not peeled off (FIG. 5 (a )reference). When incubated at 37 ° C. for 7 days, the volume of the pellet-like cell structure was reduced, and the boundary with the biotube became indistinguishable by observation using a stereomicroscope. When this biotube was observed by cross section along XX ′ of the biotube using a confocal laser microscope, it was found that GFP-positive cells were infiltrated into the biotube and were engrafted. (FIG. 5 (b). The above phenomenon is thought to be due to rapid adhesion of the bleed cells from the pellet-like cell structure.
From these results, the pellet-like cell structure can be easily co-cultured with different types of cells by (1) placing it on the cultured cell layer, and (2) the cell structure can be brought into contact with living tissue. It has been shown that the cells forming the cell can be infiltrated and adhered to the tissue, and (3) the cell can be filled in the defect site by covering or covering the defect site of the living tissue.

  Note that the biotube (also referred to as an artificial blood vessel) used in this example has collagen as a main component, and was created by the method described in the example of Japanese Patent Application Laid-Open No. 2004-261260. However, the same phenomenon occurs when a tube made by simply winding a collagen film is used.

  The results of Test Example A and Comparative Test Example A are shown in Table 1. FIG. 12 shows the correlation between the structure of cells cultured using the cell culture composition and 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 based on the method described in (A1a-3) (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-3) of 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, 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.

  FIG. 15 shows the result. 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 zeta potential on cell culture vessel surface (Test C1) Measurement of zeta potential on cell culture vessel surface (solid surface) (Test C1-1) Coating of cell culture composition on cell culture vessel Made of polystyrene Cell culture plates (Nunc, Nunclon® Δ Surface, 100 mm diameter) were cut into small pieces of size 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 A3) 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 A1) (A1a-7) of A), each polymer / Tris mixture was coated on the cut cell culture plate (Test Example C1, Test Example C2, Test Example C3, respectively). Test Example C4, Test 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. FIG. 16 shows the result. 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 the zeta potential of fine particles on the surface of a cell incubator Polymer / anionic substance mixture, that is, the cell of the present invention in accordance with the description in (A1a-5) 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. 17 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 of cell culture vessel (Test D-1) Surface structure analysis by atomic force microscope (AFM) FIG. 18 (a) shows an uncoated or cell culture plate coated with a composition for cell culture. The image which measured the surface in the dynamic 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. 18B shows an image of the surface of a cell culture plate that is 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 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 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. 19 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. 20 shows the observation results. The upper part of FIG. 20 shows an image in the dynamic tapping mode, and the lower part of FIG. 20 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 cell culture composition and / or cell culture device of the present invention makes it possible to reproduce a cell structure having a structure similar to the structure of a differentiated cell in the cell culture device and is expressed in the differentiated cell. It provides useful tools for obtaining information such as DNA and proteins.
For example, when the present invention is applied to vascular endothelial cells, luminal vascular cells 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 (3)

2-N, N-a polymer of dimethylaminoethyl methacrylate click relay DOO, and 2-amino-2-hydroxymethyl-1,3-propanediol, nucleic acid, heparin, polyacrylic acid, polymethacrylic acid, the alkali metal see containing and one or more anionic material selected from the group consisting of salts,
The ratio of the 2-amino-2-hydroxymethyl-1,3-propanediol to the polymer is 1.0 or less,
A composition for cell culture , wherein the C / A ratio is 0.5 to 16 . A cell culture vessel, wherein a culture surface is coated with the composition according to claim 1 . The culture surface, 2-N having per unit area, the amount of N- dimethylaminoethyl methacrylate click relay bets polymer is 5~200ng / mm ^2, a cell culture of claim 2.