JP2016131537A - Method for manufacturing cell structure into which nucleic acid is introduced - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for manufacturing a cell structure into which nucleic acid is introduced at high nucleic acid introducing efficiency in a simple manner.SOLUTION: A method for manufacturing a cell structure into which nucleic acid is introduced includes: a preparation step of preparing a low cloud point-type temperature-responsive polymer composition containing 2-(N,N-dimethylaminoethyl) methacrylate and/or a polymer of a derivative thereof, and nucleic acid; an arranging step of covering a culture surface of a cell culture vessel by the low cloud point-type temperature-responsive polymer composition to arrange a covered cell culture vessel; a first seeding step of seeding cells on a culture surface of the covered cell culture vessel at a cell density of 100 pieces./mmor more; a first culture step of culturing the cells seeded on the first seeding step until they reach a cell density of 200 pieces./mmor more; a second seeding step of seeding on the seeded cells of the covered cell culture vessel at a cell density of 3000 pieces./mmor more; and a second culture step of culturing the cells seeded in the second seeding step.SELECTED DRAWING: Figure 2

Description

  The present invention particularly relates to a method for producing a cell structure into which a nucleic acid is introduced, which has a high nucleic acid introduction efficiency and is simple.

As techniques for introducing genes into cells, many techniques such as synthetic polymers, micelles (liposomes), viruses, electroporation, colloidal gold guns, and microinjections (direct injection with fine needles) are known.
In particular, reverse transfection, in which a nucleic acid complex or the like is coated on a culture dish in advance, and a gene is introduced from the adhesive substrate side by using sustained release of nucleic acid from the coating layer or cell uptake, is a promising technique. It is known (see Patent Document 1).

  Techniques for producing cell agglomerates (also called embryoid bodies) include a technique using a non-adhesive coated round bottom culture container, a centrifuge / shaking culture technique, a technique of culturing in a micro-pattern printed flat bottom culture container, etc. It has been known.

International Publication No. 2013/099924

Here, when trying to prepare a cell aggregate (embryoplast) using the above-described conventional method for introducing a nucleic acid, there are the following problems.
In other words, embryoid body culture is a three-dimensional culture of cells that builds agglomerates (the number of cells is large). When cells are seeded at the density used for gene transfer, they are allowed to adhere to the bottom of the culture vessel. May result in a monolayer culture (two-dimensional). On the other hand, if a cell into which a gene has been introduced in advance is collected as a cell suspension by an enzyme treatment according to a conventional method and then attempts to form an aggregate again, there is a restriction on the time axis. That is, there is a problem that the time for which the introduced gene is expressed is transient and short, and the gene expression cannot be maintained until an aggregate is formed. Moreover, even if it tries to introduce a gene after producing a cell agglomerate, this is restricted by the cell cycle. In other words, since the cells in the aggregate are in the resting phase, gene transfer is impossible because the nucleus is protected by the nuclear membrane.

  Accordingly, an object of the present invention is to provide a method for producing a cell structure into which a nucleic acid has been introduced simply and with high nucleic acid introduction efficiency.

The gist of the present invention is as follows.
The method for producing a cell structure into which a nucleic acid of the present invention has been introduced comprises a low cloud point temperature-responsive polymer composition comprising a polymer of 2- (N, N-dimethylaminoethyl) methacrylate and / or a derivative thereof and a nucleic acid. A preparation step for preparing a coated product, a preparation step for preparing a coated cell culture device by coating a culture surface of a cell culture device with the low cloud point temperature-responsive polymer composition, and 100 cells / mm ^2. The first seeding step of seeding on the culture surface of the coated cell incubator at the above cell density, and culturing the cells seeded in the first seeding step until the cell density reaches 200 cells / mm ² or more. The first culturing step, the second seeding step of seeding the cells on the seeded cells of the coated cell culture device at a cell density of 3000 cells / mm ² or more, and the cells seeded in the second seeding step Second incubator Characterized in that it comprises a and.

In the present invention, in the second seeding step, the cell density is preferably 4000 cells / mm ² or more.

In the present invention, in the preparation step, in the coated cell culture device, the culture surface of the coated cell culture device preferably has an amount of the nucleic acid per unit area of 0.5 to 50 ng / mm ^2. .

In the present invention, in the preparation step, a polymer of 2-N, N-dimethylaminoethyl methacrylate and / or a derivative thereof that the culture surface of the coated cell culture vessel has per unit area in the coated cell culture vessel. Is preferably 5 to 200 ng / mm ² .

  ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of the cell structure into which the nucleic acid was introduce | transduced simply with high nucleic acid introduction | transduction efficiency can be provided.

(A) It is a photograph when the cell seed | inoculated and cultured in (Test C-1) of an Example is observed with a phase contrast microscope. (B) It is a photograph when the cell seed | inoculated and cultured in (Test C-1) of an Example is observed with the fluorescence phase-contrast microscope. It is a photograph when the cell structure obtained as a result of seeding | inoculating and culturing a cell in (Test C-2) of an Example is observed with a fluorescence phase contrast microscope.

  Hereinafter, embodiments of a method for producing a cell structure into which a nucleic acid of the present invention has been introduced will be described in detail with reference to the drawings.

A polymer of 2-N, N-dimethylaminoethyl methacrylate (also referred to herein as “DMAEMA”) is known as a temperature-responsive polymer, and its lower critical solution temperature (hereinafter referred to as “Lower Critical Solution Temperature”) (Also referred to as “cloud point”) is about 32 ° C.
In the specification of the present application, the “cloud point” of a composition is not necessarily a strict meaning, but only “the temperature at which it dissolves below a predetermined temperature but becomes insoluble and precipitates above a predetermined temperature”. It does not indicate, but 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 a condition lower than a predetermined temperature”.
As a result of extensive research, the inventors have recently conducted a low cloud point type temperature responsive polymer composition having a cloud point of about room temperature (25 ° C.) (hereinafter also simply referred to as “temperature responsive polymer composition”). Was successfully prepared. Examples of the low cloud point type temperature responsive polymer composition include an intramolecular ion complex type temperature responsive polymer composition, a mixed type temperature responsive polymer composition, and the like.
The intramolecular ionic complex-type temperature-responsive polymer composition includes a repeating unit (described later) of 2-N, N-dimethylaminoethyl methacrylate (DMAEMA) and a repeating unit (described later) of a hydrolyzate of the DMAEMA. The above-described molecule, which allows the inventors to continue to adhere cells in culture to a cell culture vessel even when operated in a clean bench at room temperature, and to recover the cultured cell structure as it is. A cell culture vessel coated with an inner ion complex type temperature-responsive polymer composition was prepared.
The mixed temperature-responsive polymer composition comprises (1) 2-N, N-dimethylaminoethyl methacrylate (DMAEMA) and / or a derivative polymer thereof, and (2) 2-amino-2-hydroxymethyl- 1,3-propanediol (Tris) and (3) a nucleic acid ((2) Tris is optionally included). Inventors can continue to adhere cells in culture to a cell incubator even if they are operated in a clean bench at room temperature, and the cultured cell structure can be recovered as it is. A cell culture vessel coated with a mold temperature-responsive polymer composition was prepared.

  In addition, by including a polymer of DMAEMA and / or a derivative thereof, and a nucleic acid, the cloud point is not higher than room temperature (25 ° C.), and the cells in culture can be removed even when operated in a clean bench at room temperature. A low cloud point type temperature responsive polymer composition capable of forming a low cloud point type temperature responsive polymer composition layer that can be continuously adhered to the cell structure and can be recovered as it is is obtained. .

(Method for producing cell structure into which nucleic acid is introduced)
A method for producing a cell structure into which a nucleic acid according to an embodiment of the present invention has been introduced,
A preparation step of preparing a low cloud point type temperature-responsive polymer composition comprising a polymer of 2- (N, N-dimethylaminoethyl) methacrylate and / or a derivative thereof and a nucleic acid;
A preparation step of preparing a coated cell culture device by coating a culture surface of a cell culture device with the low cloud point type temperature-responsive polymer composition;
A first seeding step of seeding cells on the culture surface of the coated cell culture device at a cell density of 100 cells / mm ² or more;
A first culture step of culturing the cells seeded in the first seeding step until a cell density of 200 cells / mm ² or more is obtained;
A second seeding step of seeding cells on the seeded cells of the coated cell culture device at a cell density of 3000 cells / mm ² or more;
A second culturing step of culturing the cells seeded in the second seeding step;
including.

  The cells used in the method for producing a cell structure into which the nucleic acid of the embodiment of the present invention has been introduced are preferably mesenchymal cells such as chondrocytes, fibroblasts, vascular endothelial cells, cardiomyocytes. 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.

  Examples of the nucleic acid include DNA (single-stranded or double-stranded DNA), RNA (single-stranded or double-stranded RNA), modified nucleic acid (for example, DNA or RNA with a morpholino ring added), and the like. And the like. The nucleic acid is, for example, in a form capable of expressing the function of the target gene (for example, a plasmid, a promoter region, a start codon, a target gene, a stop codon, which are arranged so that the gene is expressed in the introduced cell, It may be a plasmid or the like in which terminator regions are continuously arranged.

  The introduced nucleic acid may be used for the purpose of suppressing the expression of a specific gene such as siRNA, antisense, decoy, etc. in addition to the purpose of expressing the gene. The type of gene is not particularly limited. For example, herpes simplex virus thymidine kinase gene (HSV1-TK gene), p53 tumor suppressor gene, BRCA tumor suppressor gene, cytokine gene (for example, TNF-α gene, IL-2 gene, IL-4 gene, HLA-B7 / IL-2 gene, HLA-B7 / B2M gene, IL-7 gene, GM-CSF gene, IFN-γ gene, IL-12 gene, VEGF gene, HGF gene, FGF gene, etc. ), Cancer antigen peptide genes (eg, gp-100, MART-1, MAGE-1, etc.), c-myc antisense, c-myb antisense, cdc2 kinase antisense, PCNA antisense, E2F decoy, p21 (sdi -1) Gene, SOX2, c-Myc, OCT3, OCT4, K lf-4, NanoG and the like.

  The length of the nucleic acid is not particularly limited, but may be a length well known to those skilled in the art, for example, about 20 to 15,000 bp. In this regard, in the present invention, the length of the nucleic acid is not particularly limited as in the case of a conventional synthetic polymer gene introduction agent, and has advantages over the case of a viral vector.

  Details of each step will be described below.

In the method for producing a cell structure into which a nucleic acid according to an embodiment of the present invention is introduced, first, a low cloud point type temperature-responsive polymer composition (hereinafter, also referred to as “temperature-responsive polymer composition”) is prepared (preparation). Process).
-Manufacturing method of temperature-responsive polymer composition-
The temperature-responsive polymer composition can be produced, for example, by a method for producing a temperature-responsive polymer composition in one example and a method for producing a temperature-responsive polymer composition in another example as described below.

  Hereinafter, an example of a method for producing a low cloud point type temperature-responsive polymer composition will be described. In this example, the low cloud point type temperature-responsive polymer composition includes an intramolecular ion complex type temperature-responsive polymer and the nucleic acid.

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

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

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

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

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

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

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

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

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

Even if the production method is not the same as the production method of the temperature-responsive polymer composition of this example, as long as DMAEMA, the polymerization inhibitor, and water coexist in the reaction system at the time of ultraviolet irradiation, the temperature of this example The effect similar to the said effect of the manufacturing method of a responsive polymer composition can be acquired.
For example, a mixture containing DMAEMA and a polymerization inhibitor and water are prepared separately, and then an inert gas is bubbled into the mixture and water, and then the mixture and water are mixed under an inert atmosphere and at the same time UV The method for producing a temperature-responsive polymer composition in which the temperature is irradiated can also be included in the method for producing a temperature-responsive polymer composition of this example.

  As a 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. Further, a molecule having a ratio (Mw / Mn) of the weight average molecular weight (Mw) to the number average molecular weight (Mn) is preferably 1.1 to 10.0.

  The ratio of the number of amine nitrogen atoms in the polymer side chain of DMAEMA and / or its derivative to the number of phosphorus atoms in the phosphate residue in the nucleic acid in the low cloud point type temperature-responsive polymer composition of this example (N / P ratio) is not particularly limited. For example, by strongly aggregating the nucleic acid, the nucleic acid is protected from the action of degrading enzymes and the like, and the composition is stabilized. From the viewpoint of enhancing electrostatic action and efficiently delivering nucleic acid into cells, for example, it is preferably 2 or more, more preferably 5 or more, and also suppresses the toxicity of the composition to cells. From the viewpoint, it is preferably 20 or less, and more preferably 30 or less.

  Hereinafter, another method for producing a low cloud point type temperature-responsive polymer composition will be described. In this example, the low cloud point temperature responsive polymer composition is a mixed temperature responsive polymer composition comprising a temperature responsive polymer that is a polymer of DMAEMA.

  That is, in the method for producing a temperature-responsive polymer composition of this example, first, a mixed temperature-responsive polymer composition is prepared (mixture preparation step). Specifically, (1) 2-N, N-dimethylaminoethyl methacrylate (DMAEMA) and / or a polymer thereof and (2) 2-amino-2-hydroxymethyl-1,3-propanediol ( (Tris) and (3) nucleic acid ((2) Tris may be optional).

  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. (3) It is presumed that the nucleic acid plays a role of enabling migration and orientation of cells to be cultured and a role of suppressing cytotoxicity.

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

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

  Examples of (1) DMAEMA derivatives include, for example, derivatives in which a hydrogen atom of a methyl group of a methacryl group is substituted with a halogen, derivatives in which a methyl group of a methacryl group is substituted with a lower alkyl group, hydrogen of a methyl group of a dimethylamino group Derivatives in which atoms are substituted with halogen, and derivatives in which the methyl group of the dimethylamino group is substituted with a lower alkyl group are exemplified.

  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.

  Said (3) nucleic acid is as above-mentioned.

The nucleic acid (3) preferably has a certain size, for example, a molecular weight (M) of 1 kDa to 5,000 kDa.
If the molecular weight is within the above range, the nucleic acid can ionically bond with the cationic substance to play a role of capturing the cationic substance for a long time, and stable ionic complex fine particles can be formed. 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.

  The low cloud point type temperature-responsive polymer composition may further contain an anionic substance other than nucleic acid. Examples of the anionic substance other than the nucleic acid include heparin, hyaluronic acid, dextran sulfate, polystyrene sulfonic acid, polyacrylic acid, polymethacrylic acid, polyphosphoric acid, polyvinyl benzoic acid, sulfated polysaccharide, curdlan and polyalginic acid, and These alkali metal salts are exemplified. In addition, anionic functional groups are introduced by dehydrating and condensing dicarboxylic acid such as oxalic acid to the 4-position amino group of poly (4-aminostyrene) which is a cationic polymer. Polymer derivatives that function as substances can also be used.

  Two or more types of anionic substances other than the nucleic acid may be included.

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

  When the mixed temperature-responsive polymer composition in the above ratio is used, a cell structure can be easily formed in the culture step described later. 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.

  Ratio of the number of amine nitrogen atoms in the polymer side chain of DMAEMA and / or its derivative to the number of phosphorus atoms in the phosphate residue in the nucleic acid in the temperature-responsive polymer composition of another example above (N / The P ratio is not particularly limited. For example, by strongly aggregating the nucleic acid, the nucleic acid is protected from the action of degrading enzymes and the like, and the composition is stabilized. From the viewpoint of strengthening electric action and efficiently delivering nucleic acid into cells, for example, it is preferably 2 or more, more preferably 5 or more, and the viewpoint of suppressing toxicity of the composition to cells. Therefore, it is preferably 20 or less, and more preferably 30 or less.

  In addition, in the method for producing a temperature-responsive polymer composition of another example described above, (2) 2-amino-2-hydroxymethyl-1,3-propanediol (Tris) may not necessarily be used. The low cloud point type temperature-responsive polymer composition according to (2) may not contain (2) Tris.

(Preparation process)
Next, the prepared low cloud point type temperature-responsive polymer composition is coated on the culture surface of the cell culture vessel to prepare a coated cell culture vessel (preparation step).
That is, the low cloud point type temperature responsive polymer composition is coated on the culture surface of a cell culture vessel to form a low cloud point type temperature responsive polymer composition layer to prepare a coated cell culture vessel. In the present specification, a cell culture vessel in which the low cloud point type temperature-responsive polymer composition layer is formed on the culture surface may be referred to as a “coated cell culture vessel”.

In the above coated cell culture vessel, the amount of DMAEMA and / or a derivative thereof contained in the low cloud point type temperature-responsive polymer composition that the culture surface has per unit area is not particularly limited. From the viewpoint that the point-type temperature-responsive polymer composition can be uniformly applied to the cell culture vessel and the cell structure can be easily formed, for example, 5 to 200 ng / mm ² is preferable. That is, it is preferable to apply the low cloud point type temperature-responsive polymer composition on the culture surface of the cell culture vessel so that the polymer of DMAEMA and / or a derivative thereof is 5 to 200 ng / mm ² . The low cloud point type temperature-responsive polymer composition can stably carry a sufficient amount of nucleic acid by having cationic DMAEMA.

In the coated cell culture vessel, the amount of the nucleic acid contained in the low cloud point type temperature-responsive polymer composition that the culture surface has per unit area is not particularly limited, but, for example, 0.5 to 50 ng / mm ² is preferable, and 2.5 to 7.5 ng / mm ² is more preferable. When the ratio of the nucleic acid is within the above range, the probability of introducing (delivering) the nucleic acid into a large number of cells seeded at a high density can be increased, and as a result, the efficiency of introducing the nucleic acid into the cell is improved. be able to.

  According to the coated cell culture device, for example, even when operating in a clean bench at room temperature, the low cloud point type temperature-responsive polymer composition layer is a certain amount of time, specifically 5 to 40 minutes, Since it has been solidified, the low cloud point type temperature-responsive polymer composition layer, and thus the cells in culture can continue to adhere to the cell culture vessel.

In particular, when a mixed type temperature responsive polymer composition containing (2) Tris is used as the low cloud point type temperature responsive polymer composition, the following effects are exhibited.
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 nucleic acid (3) contained in the composition suppresses cytotoxicity by optimizing the balance between positive and negative charges in the composition in the culturing step (the surface of the cell membrane of mammalian cells is Cationic substances are often cytotoxic because they are negatively charged), and the balance between the hydrophilicity and hydrophobicity of the composition is preferred to allow cell migration and orientation Estimated.
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.

The method for forming the low cloud point type temperature responsive polymer composition layer is not particularly limited. For example, an aqueous solution of the low cloud point type temperature responsive polymer composition cooled to a temperature below the cloud point is used as a cell incubator. And a method of allowing the low cloud point type temperature-responsive polymer composition to settle on the bottom surface of the culture dish.
The coated cell culture vessel can also be used after removing the water (supernatant) used for coating the composition, and after removing the water, the composition is dried. It is also possible to use it afterwards.

  The low cloud point type temperature-responsive polymer composition layer may be further dried. Although it does not specifically limit as a drying method, For example, the method etc. which leave still for a fixed time (for example, about 24 hours) in the cell incubator which stopped humidification etc. (drying process) are mentioned.

  When the low cloud point type temperature-responsive polymer composition contains a polymer, nucleic acid, and tris of DMAEMA and / or a derivative thereof, DMAEMA and / or a derivative thereof are used in the coated cell culture vessel in the culturing step. It is presumed that the hydrophobic side chain of this polymer and the amino group of Tris give some stimulation to the cells while interacting with each other. In addition, the nucleic acid suppresses cytotoxicity in the culturing process by optimizing the balance between the positive charge and the negative charge in the above composition (since the surface of the cell membrane of mammalian cells is negatively charged, It is presumed that the active substance often has cytotoxicity) and that the balance between the hydrophilicity and the hydrophobicity of the composition is suitable to enable cell migration and orientation.

(First sowing step)
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 100 cells / mm ² or more (first 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.

Regarding the cell density, for example, in order to seed at a cell density of 100 cells / mm ² or more when seeding in a 24-well plate having a culture surface area of 200 mm ² and a medium volume of 1 mL, 2 × It is necessary to use a cell suspension having a cell density of 10 ⁴ cells / mL or more.

In addition, the cell density is preferably 300 cells / mm ² or less in order to prevent problems related to the cell cycle such as growth arrest caused by contact between cells in culture.

The cell density is more preferably 100 cells / mm ² to 200 cells / mm ² . When the cell density is within the above range, it becomes possible to deliver a sufficient amount of nucleic acid to the cells that grow while adhering to and spreading on the culture dish. In the culturing step, the above effect of facilitating formation of a cell structure having a pellet-like structure is easily obtained.

  The cells seeded in the first seeding step are considered to adhere to the low cloud point type temperature-responsive polymer composition layer and take up the polymer composition or the nucleic acid described above by endocytosis. At this time, since the probability and opportunity of incorporation are continuously supplied for a relatively long period (10 days at the longest) for culturing cells, it is considered that more nucleic acid is incorporated. In this respect, it is greatly different from the prior art in which the time for contacting the nucleic acid with the cells is a relatively short period of several seconds to 3 hours.

(First culture step)
Next, the cells seeded in the first seeding step are cultured until the cell density becomes 200 cells / mm ² or more (first culture step).
In addition, the numerical range of the cell density of 200 cells / mm ² or more is meant to indicate a range substantially corresponding to the confluent state determined by those skilled in the art, and this numerical range varies depending on the cell type and culture conditions. Can result.

The seeded cells are preferably cultured by standing.
The culture temperature is not particularly limited, but is preferably 30 ° C. or higher, more preferably 35 ° C. or higher, more preferably 38 ° C. or lower, and particularly preferably 37 ° C. .
The culture time varies depending on the cell type, and the growth rate varies depending on the state and the number of passages even for the same type of cells, but is not particularly limited, but the cell group exceeds the confluent state determined by those skilled in the art. For example, in the case of mesenchymal stem cells derived from subcutaneous fat, it is usually about 1 to 10 days.

  In addition, culture conditions, such as a culture medium, can be appropriately determined by those skilled in the art based on the cell type and the purpose of the experiment.

  In this step, the seeded cells are introduced with the nucleic acid contained in the low cloud point temperature-responsive polymer composition while growing.

  In addition, the projection area of the cell with respect to the total surface area (total surface area of one surface of the low cloud point type temperature-responsive polymer composition layer) (100%) of the culture surface of the cell incubator after the first culture step The ratio of (surface area occupied by cells) is not particularly limited, but is preferably 90 to 100% (90 to 100% confluent), more preferably 95 to 100% (95 to 100% confluent), and particularly preferably 100. %.

(Second sowing step)
Then, the cells are seeded on the seeded cells of the coated cell culture vessel at a cell density of 3000 cells / mm ² or more (second seeding step).
This step can be performed, for example, using a general 37 ° C. cell incubator.

  If cells are seeded at the above cell density, the detailed mechanism is unknown, but a cell structure having a pellet-like structure can be formed in the following culture step. This cell structure has living cells in the pellet-like structure.

The cell density is preferably 6000 cells / mm ² or less in order to make it difficult to cause problems related to the cell cycle, such as growth arrest caused by contact between cells in culture.

The cell density is more preferably 3000 cells / mm ^{2 to} 5000 cells / mm ² . If the cell density is within the above range, it becomes possible to deliver a sufficient amount of nucleic acid to the cells that grow while adhering to and spreading on the culture dish. As a result, the subsequent second culture step described below is performed. Thus, the above effect of facilitating formation of a cell structure having a pellet-like structure is easily obtained.

(Second culture step)
Next, the cells seeded in the second seeding step are cultured (second culture step).
This step can be performed, for example, using a general 37 ° C. cell incubator.

The seeded cells are preferably statically cultured.
The culture temperature is not particularly limited, but is preferably 30 ° C. or higher, more preferably 35 ° C. or higher, more preferably 38 ° C. or lower, and particularly preferably 37 ° C. .
The culture time is not particularly limited because the growth rate differs depending on the cell type, and the growth rate varies depending on the state and the number of passages even in the same type of cells, but the cell group is determined by those skilled in the art. It is preferable that the time does not exceed the state of the felt. For example, in the case of mesenchymal stem cells derived from subcutaneous fat, it is usually about 1 to 10 days.

  In addition, culture conditions, such as a culture medium, can be appropriately determined by those skilled in the art based on the cell type and the purpose of the experiment.

The cells seeded in the second seeding step form one cell mass together with the cells seeded in the first seeding step and introduced with the nucleic acid contained in the low cloud point type temperature-responsive polymer composition.
The cell mass causes a phenomenon in which all the cells are detached from the end of the culture dish at one time, and the cells are aggregated at one place as if the sheet is contracted while being rounded. Cells that gather together spontaneously assemble as if to form some kind of tissue, forming a three-dimensional pellet structure.

Here, surprisingly, in the cell mass, not only the cells seeded in the first seeding step, but also the second seeding step that is not in direct contact with the low cloud point type temperature-responsive polymer composition. It was found that the seeded cells also received nucleic acid introduction.
This is because the low-cloud-point temperature-responsive polymer composition microparticles, introduced nucleic acid molecules, or mRNA can be delivered from the cells into which nucleic acid was introduced in the first seeding step via the molecule itself or exosomes. Although sex is speculated, it is thought that it will be elucidated by future research.

(Cell structure with nucleic acid introduced)
The cell structure into which the nucleic acid according to the embodiment of the present invention has been introduced can be produced by the method for producing a cell structure into which the nucleic acid according to the embodiment of the present invention has been introduced.

The method for producing a cell structure into which a nucleic acid is introduced according to an embodiment of the present invention is a culture that forms a three-dimensional structure (three-dimensional culture), unlike a culture that forms a single-layer structure (single-layer culture). As a result, cell aggregates can be obtained (embryoplast culture), and cells that are in a state close to cells existing in the living body and that have the same function can be prepared in a test tube.
Conventionally, studies on genes, proteins, cell functions, etc. that have been performed by introducing genes into cells that have been cultured in a single layer are expected to be conducted in the future using cells that have been three-dimensionally cultured. In addition, research that has been conventionally performed by introducing genes into cells existing in a living body (that is, research by animal experiments) is expected to be performed in a test tube in the future. This point is also a point which responds to social needs from the viewpoint of animal welfare, from the viewpoint of reducing the difficulty of experiments and improving reproducibility.

  In addition to the above-described knowledge regarding the cell structure into which the pellet-like nucleic acid has been introduced, the inventors have placed the cell structure in a predetermined site in the living body, or similar to the environment of the predetermined site in the living body. It was found that the cells have a function of differentiating into cells suitable for the tissue of the site (cells of the desired type at the site).

The prepared cell structure has a property of firmly adhering to collagen and fibronectin.
In particular, in the method for producing a cell structure into which a nucleic acid has been introduced, the nucleic acid contained in the low cloud point type temperature-responsive polymer composition is a gene encoding an anticoagulant protein, and the nucleic acid is introduced in an anastomosis of a blood vessel. When the cell structure is affixed to the anastomosis site, the cell structure physically closes the needle hole to prevent blood leakage, and the anticoagulant protein expressed in the cell structure The effect of suppressing the coagulation of blood passing through is obtained. In this case, the cell structure can be used as a thrombolytic agent.
Examples of the anticoagulant protein include natto kinase, thromboplastin, and VEGF.

Moreover, the prepared cell structure has a property of firmly adhering to a cancer lesion.
In particular, in the transplant material, the low cloud point type temperature responsive polymer composition is a mixed temperature responsive polymer composition, and the nucleic acid contained in the mixed temperature responsive polymer composition encodes an antitumor protein. When a transplant material is injected into a cancer lesion in the healing of the cancer lesion, an effect of continuing to secrete the antitumor protein expressed in the cell structure into the cancer lesion for a predetermined period is obtained. In this case, the cell structure can be used as a prodrug sustained-release agent.
Examples of the antitumor protein include TNF-α and Maitake fraction IV.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to the following Example at all.

  In the following tests, commercially available reagents were used without further purification unless otherwise specified.

(Test A) Preparation of low cloud point type temperature responsive polymer composition (mixed type temperature responsive polymer composition) (Test A1) Preparation of temperature responsive polymer In a transparent vial made of soft glass having a capacity of 50 mL, 2 -7.0 g of (N, N-dimethylaminoethyl) methacrylate was added and stirred using a magnetic stirrer. The mixture (liquid) was deoxygenated by purging nitrogen gas of G1 grade high purity (purity: 99.99995%) for 10 minutes (flow rate: 2.0 L / min).
Thereafter, the reaction product 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- )

(Test A2) Preparation of polymer / nucleic acid Hereinafter, PBS (Gibco, DPBS (1 ×)) was used.
Using DNA encoding RFP (pTurbo604-N1) as a nucleic acid, this DNA was dissolved in PBS to prepare a DNA solution (final concentration: 500 μg / mL).
A polymer solution is prepared by dissolving the temperature-responsive polymer synthesized in (Test A1) in PBS, and the solution is cooled to 4 ° C. so as not to form a hydrophobic cohesive cluster due to the globule phase transition. (Final concentration was polymer: 400 μg / mL).
40 μL of the polymer solution was added dropwise to 8 μL of the DNA solution and mixed. This mixture was diluted by adding 832 μL of PBS to obtain a mixed temperature-responsive polymer composition (final concentration, polymer: 16 μg / mL, DNA: 4 μg / mL).
The N / P ratio in the mixed temperature-responsive polymer composition was 8.

(Test B) Coating of cell culture composition on cell culture vessel PBS solution of low cloud point type temperature-responsive polymer composition prepared in (Test A) and cooled to 4 ° C. 200 μL of this solution is added to each well of a culture plate (Iwaki Co., Ltd., microplate, model number: 3815-024, bottom area per well: 200 mm ² ) at room temperature, and quickly applied to the entire bottom surface of the hole. Cast. The cell culture dish was then incubated for 3 hours in a cell culture incubator (37 ° C., 5% CO ₂ ).
In this way, a 24-well cell culture plate (cell culture vessel) was obtained in which the well as the culture surface was coated with the low cloud point type temperature-responsive polymer composition.

(Test C-1) Seeding and culturing cells At room temperature, in each well of the 24-well cell culture plate prepared in (Test B), mesenchymal stem cells derived from beagle subcutaneous fat (primary cultured cells, ADSC) Is suspended in a complete medium (Dulbecco's modified Eagle medium (DMEM) + 5% fetal calf serum (FCS) solution) (DMEM: Gibco, model number: 11965), (FCS: Invitrogen, lot number: 852546). Then, 1 mL of a culture solution adjusted to a cell density of 200 cells / mm ² (4 × 10 ⁴ cells / mL) was added. FIG. 1 (a) shows a photograph of the cells observed with a phase contrast microscope.
The cells were cultured in a cell culture incubator at 37 ° C. for 10 days. When the cells were observed using a fluorescence phase contrast microscope (Olympus, model number: IX-70) (wavelength: 699 nm), the expression of RFP was confirmed in ADSC (FIG. 1 (b)).

(Test C-2) Cell seeding and culture Next, primary ADSC was added at a cell density of 3500 cells / mm ² on the cultured cells at room temperature.
The cells were cultured in a cell culture incubator at 37 ° C. for 24 hours.
After 6 hours of culturing, the cells that have been formed into a circular sheet-like structure on the culture dish are detached from the outer edge of the culture dish at one time, and the sheet shrinks as if the sheet is curled. The movement of the cells led to a phenomenon in which cells aggregated in one place. The above phenomenon was sufficiently visible with the naked eye. Cells that gathered together formed a three-dimensional pellet-like structure. When the cell structure was observed using a fluorescence phase contrast microscope, red fluorescence derived from RFP could be confirmed in almost the entire cell mass (FIG. 2).

  The cell structure having a pellet-like structure thus obtained was detached from the culture dish by trypsin treatment, and a cell suspension was prepared using the cells forming the cell structure. When FACS analysis was performed on this cell suspension, RFP-derived fluorescence was confirmed in 50% to 90% of cells. Considering the number of cells seeded in (Test C-1) and (Test C-2), not only the cells seeded on the culture surface in (Test C-1) but also seeded in (Test C-2). It was confirmed that the nucleic acid was also introduced into the cells further seeded on the cells.

  ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of the cell structure into which the nucleic acid was introduce | transduced simply with high nucleic acid introduction | transduction efficiency can be provided.

Claims (4)

A preparation step of preparing a low cloud point type temperature-responsive polymer composition comprising a polymer of 2- (N, N-dimethylaminoethyl) methacrylate and / or a derivative thereof and a nucleic acid;
A preparation step of preparing a coated cell culture device by coating a culture surface of a cell culture device with the low cloud point type temperature-responsive polymer composition;
A first seeding step of seeding cells on the culture surface of the coated cell culture device at a cell density of 100 cells / mm ² or more;
A first culture step of culturing the cells seeded in the first seeding step until a cell density of 200 cells / mm ² or more is obtained;
A second seeding step of seeding cells on the seeded cells of the coated cell culture device at a cell density of 3000 cells / mm ² or more;
A second culturing step of culturing the cells seeded in the second seeding step;
A method for producing a cell structure into which a nucleic acid has been introduced, comprising: 2. The production method according to claim 1, wherein in the second seeding step, the cell density is 4000 cells / mm ² or more. The said preparation process WHEREIN: The quantity of the said nucleic acid which the culture surface of this coated cell culture device has per unit area is 0.5-50ng / mm < ² > in the said coated cell culture device. the method of. In the preparation step, in the coated cell culture vessel, the amount of the 2-N, N-dimethylaminoethyl methacrylate and / or its derivative polymer per unit area on the culture surface of the coated cell culture vessel is 5 The implant material of claim 1, which is ˜200 ng / mm ² .