JP6035972B2 - Method for forming cell aggregate and method for producing substrate for cell aggregate formation - Google Patents

Description

  The present invention relates to a method for forming a cell aggregate, a method for producing a cell aggregate forming substrate, and a cell aggregate forming substrate.

  Even when a body tissue or organ is lost due to an accident or illness, research on regenerative medicine is being actively conducted to regenerate the tissue using the remaining cells and restore the function. In regenerative medicine, the mainstream is to replace the function by regenerating the smallest unit of tissue and transplanting it in large quantities. As a method for realizing this, there is a technique for forming a cell aggregate (spheroid) as a minimum unit of the tissue.

  As a method of forming such cell aggregates, there is a rubbing method that uses a film having a main component of biocompatible polyimide or a precursor thereof, for example, by rubbing the surface to form cell aggregates. (See Membrane, 32 (5), 266-270 (2007)). This rubbing method forms fine irregularities by rubbing the surface of a film such as polyimide using a cloth such as nylon or cotton, and such a surface has no specific interaction with specific cells. Therefore, there is an advantage that it can be applied to any cell as long as it is a cell type capable of forming multiple layers.

  However, in the rubbing method as described above, since there is a concern about the influence on the quality of the cell aggregate due to the adhesion of the dust generated by rubbing to the film surface, a new film forming process that can prevent the generation of dust is provided. A method for forming cell aggregates is desired.

Membrane, 32 (5), 266-270 (2007)

  The present invention has been made based on the above circumstances, and its object is to form a cell aggregate capable of preventing the generation of dust when forming fine irregularities on the surface of a film such as polyimide. Is to provide.

The invention made to solve the above problems is
(A1) a step of irradiating polarized ultraviolet rays to a film (hereinafter also referred to as “film (α)”) whose main component is polyimide or a precursor thereof (hereinafter, polyimide precursor is also simply referred to as “precursor”);
(A2) A method for forming a cell aggregate, comprising a step of seeding cells on the surface of the membrane irradiated with polarized ultraviolet light, and (a3) a step of forming a cell aggregate by culturing the seeded cells.

Moreover, another invention made in order to solve the said subject is:
(B1) A method for producing a cell aggregate-forming substrate, comprising a step of forming a film containing polyimide or a precursor thereof as a main component, and (b2) irradiating the film with polarized ultraviolet rays.

Furthermore, another invention made to solve the above problems is
It is a base material for cell aggregate formation provided with a base material and the film | membrane which is formed in the surface of this base material, and has a polyimide or its precursor as a main component, and the said film | membrane was irradiated with polarized ultraviolet rays.

  In addition, in this invention, a main component refers to a component with the largest mass ratio among the components to contain. The mass ratio of the main component is preferably 50% by mass or more, and more preferably 70% by mass or more.

  This invention can prevent generation | occurrence | production of dust in the case of fine uneven | corrugated formation to film surfaces, such as a polyimide. Therefore, since the method for forming a cell aggregate, the method for producing a substrate for forming a cell aggregate, and the substrate for forming a cell aggregate of the present invention can prevent the generation of dust, in the process of forming a cell aggregate, It can contribute to the improvement of the quality of the formed cell aggregate and the improvement of productivity by eliminating the need for dust removal.

In the formation method of the cell aggregate of this invention, it is a schematic diagram which shows an example before a film | membrane ((alpha)) irradiates polarized ultraviolet rays. In the formation method of the cell aggregate of this invention, it is a schematic diagram which shows an example of the state after irradiating a film | membrane ((alpha)) with polarized ultraviolet rays. This figure is also a schematic view showing an example of the cell aggregate-forming substrate of the present invention. In the cell aggregate formation method of this invention, it is a schematic diagram which shows an example of the state at the time of seed | inoculating a cell. In the cell aggregate formation method of this invention, it is a schematic diagram which shows an example of the state after forming a cell aggregate by culture | cultivation of a cell. It is a schematic diagram which shows an example of the state after peeling the cell aggregate of FIG. 4 from a film | membrane ((alpha)).

<Method of forming cell aggregate>
The method for forming a cell aggregate of the present invention comprises:
(A1) A step of irradiating polarized ultraviolet rays onto a film (α) whose main component is polyimide or a precursor thereof (hereinafter, polyimide or a precursor thereof is also referred to as “polyimide or the like”) (hereinafter also referred to as “step (a1)”). ),
(A2) a step of seeding cells on the surface of the membrane (α) irradiated with polarized ultraviolet light (hereinafter also referred to as “step (a2)”), and (a3) a cell aggregate by culturing the seeded cells. Forming step (hereinafter also referred to as “step (a3)”)
Have
Since the method for forming cell aggregates includes a step of irradiating polarized ultraviolet rays to a film (α) whose main component is polyimide or the like, the method for forming cell aggregates is to form fine irregularities on the surface of the film (α). In this case, generation of dust can be prevented, and the quality of the cell aggregate formed thereby can be improved. The fine uneven shape on the surface of the film (α) is presumed to be formed by polymer molecules such as polyimide being locally cut or anisotropically isomerized or dimerized by polarized ultraviolet rays. . Hereinafter, each step will be described in detail with reference to FIGS.

[Step (a1)]
In this step, the ultraviolet ray is irradiated to the film (α) whose main component is polyimide or its precursor.

  The polyimide or its precursor as a main component film (α) includes a film made of a polyimide component, a film made of a precursor component, a film containing polyimide as a main component and containing a precursor component, and a precursor as a main component. And a film containing a polyimide component and a film containing components other than polyimide and a precursor together with the above components.

  As shown in FIG. 1, the film (α) (film denoted by reference numeral 1 in FIG. 1) is usually formed on a substrate 100. Examples of the substrate 100 include substrates made of synthetic resin, TCPS (Tissue Culture Polystyrene), and glass. Examples of the method for forming the film (α) include a method of forming the film (α) by applying a solution such as polyimide on the substrate 100. Examples of the coating method include a spray method, an offset printing method, a roll coater method, an ink jet printing method, a solvent casting method, and a spin coating method. Although it does not specifically limit as film thickness, such as the said polyimide, From a viewpoint of the ease of formation of a film | membrane ((alpha)), 10 nm-300 nm are preferable.

The polarized ultraviolet light can be obtained by passing ultraviolet light having a predetermined wavelength through a polarizing plate, and the obtained polarized ultraviolet light is applied to the film (α) surface from a predetermined direction. The irradiation conditions of polarized ultraviolet rays are not particularly limited as long as a photocleavage reaction occurs, but the wavelength is usually 200 nm or more and 300 nm or less, preferably 220 nm or more and 280 nm or less, and more preferably 254 nm. The irradiation dose is usually 2,000 J / ^{m 2} or more, preferably 4,000J / ^{m 2} or more, more preferably 6,000J / ^{m 2} or more, more preferably 8,000J / ^{m 2} or more. The direction of irradiating the polarized ultraviolet light may be a fixed direction, a plurality of directions, or different directions for each region of the film (α) surface.

  The polarized ultraviolet light may be irradiated over the entire surface of the film (α) or may be irradiated in a pattern on a part of the surface of the film (α) through a photomask. When the pattern is irradiated, cell aggregates having a desired shape and size can be easily and reliably formed. This is presumed to be because the shape of the surface of the membrane (α) is different between the irradiation region of polarized ultraviolet rays and the other region, and this causes a difference in the adhesion between the seeded cells and the membrane (α). The By this pattern of irradiation, a cell aggregate having a desired shape that can match the living body at the time of transplantation can be obtained with high accuracy.

  The state after irradiating the polarized ultraviolet light is shown in FIG. As shown in FIG. 2, fine irregularities can be formed on the surface of the film (α) by irradiation with polarized ultraviolet rays.

  The polyimide can be produced by dehydrating and ring-closing and imidizing an amic acid structure possessed by a polyamic acid as described later. The polyimide may be a completely imidized product obtained by dehydrating and cyclizing all of the amic acid structure possessed by the polyamic acid that is the precursor, and only a part of the amic acid structure is dehydrated and cyclized, It may be a partially imidized product in which an imide ring structure coexists.

  When the main component of the film (α) is polyimide, the imidization ratio of this polyimide is preferably 40% or more, more preferably 50% or more, and further preferably 60% or more.

  Examples of the polyimide precursor include polyamic acid.

  As said polyimide and polyamic acid, what produces a photocleavage reaction by polarized ultraviolet rays and what has a photo-alignment group are preferable, and what produces a photocleavage reaction by polarized ultraviolet rays is more preferable. When the polyimide and polyamic acid have the above properties or groups, fine irregularities can be easily and reliably formed on the surface of the film (α).

  The polyimide that undergoes a photocleavage reaction by the polarized ultraviolet light preferably has a structural unit represented by the following formula (1). When the polyimide has the following structure, a part of the structure of the polymer main chain or side chain is cleaved by the photocleavage reaction, and fine irregularities at the molecular level are more easily and reliably formed on the surface of the film (α). be able to.

In the above formula (1), R ¹ is a tetravalent organic group represented by any of the following formulas (i-1) to (i-6). R ² is a divalent organic group having 1 to 20 carbon atoms.

In formula (i-1), formula (i-3) and formula (i-4), R ^A1 , R ^A3 and R ^A4 are each independently a monovalent organic group having 1 to 4 carbon atoms. is there. n1 is an integer of 0-4. n3 is an integer of 0-4. n4 is an integer of 0-8. R ^A1, when ^{R A3} and ^{R A4} is plural respective plurality of ^{R A1, R A3} and ^{R A4} may each be the same or different.
In the formula (i-2), R ^A21 is a monovalent organic group having 1 to 4 carbon atoms. n2 is an integer of 0-6. R ^A22 is a single bond, a methylene group or an alkylene group having 2 to 4 carbon atoms. When n2 is 2 or more, the plurality of R ^A21 may be the same or different.
In the above formula (i-5), R ^A5 is a fluorine atom or a monovalent organic group having 1 or 2 carbon atoms. n5 is an integer of 0-9. When n5 is 2 or more, the plurality of R ^A5 may be the same or different.
In the above formula (i-6), R ^A61 is a fluorine atom or a monovalent organic group having 1 or 2 carbon atoms. n61 is an integer of 0-5. R ^A62 is a fluorine atom or a monovalent organic group having 1 to 4 carbon atoms. n62 is an integer of 0-4. If R ^A61 and ^{R A62} is plural respective plurality of ^{R A61} and ^{R A62} may each be the same or different.
In formulas (i-1) to (i-6), * represents a bonding site with a carbon atom.

Examples of the monovalent organic group having 1 to 4 carbon atoms represented by R ^A1 , R ^A21 , R ^A3 , R ^A4 and R ^A62 include, for example, a chain hydrocarbon group having 1 to 4 carbon atoms and 3 carbon atoms. -4 cycloaliphatic hydrocarbon groups, groups containing a group having a hetero atom such as an oxygen atom or a nitrogen atom between carbon-carbons of these hydrocarbon groups.

  Examples of the chain hydrocarbon group having 1 to 4 carbon atoms include a methyl group, an ethyl group, an n-propyl group, an i-propyl group, an n-butyl group, an i-butyl group, and a t-butyl group. It is done.

  Examples of the alicyclic hydrocarbon group having 3 to 4 carbon atoms include a cyclopropyl group and a cyclobutyl group.

Examples of the group containing a group having a hetero atom such as an oxygen atom or a nitrogen atom between carbon and carbon of these hydrocarbon groups include -O-, -CO-, and -COO between the carbon and carbon of the hydrocarbon group. And a group containing a linking group having at least one heteroatom such as —, —OCO—, —NO—, —NH ₂ — and the like.

Examples of the alkylene group having 2 to 4 carbon atoms represented by R ^A22 include an ethylene group, a propylene group, and a butylene group.

Examples of the monovalent organic group having 1 or 2 carbon atoms represented by the above R ^A5 and R ^A61 include, among the monovalent organic groups having 1 to 4 carbon atoms exemplified as the above R ^{A1 and the} like, 1 carbon atom Or the thing of 2 etc. are mentioned.

Of those, ^{R 1,} the formula (i-1), the organic group is preferably represented by (i-2), the organic group represented by the formula (i-1) is more preferable. In the above (i-1) and (i-2), n1 and n2 are more preferably 0.

Examples of the divalent organic group having 1 to 20 carbon atoms represented by R ² include an aliphatic diamine compound having 1 to 20 carbon atoms, an alicyclic diamine compound having 1 to 20 carbon atoms, and 1 to 1 carbon atoms. 20 aromatic diamine compounds and divalent organic groups derived from diamine compounds such as diaminosiloxane compounds represented by the following formula (2).

  In said formula (2), m is an integer of 1-10.

  Examples of the aliphatic diamine compound having 1 to 20 carbon atoms include tetramethylene diamine and hexamethylene diamine.

  Examples of the alicyclic diamine compound having 1 to 20 carbon atoms include bis (4-aminocyclohexyl) methane, bis (4-amino-3-methylcyclohexyl) methane, and the like.

  Examples of the aromatic diamine compound having 1 to 20 carbon atoms include p-phenylenediamine, m-phenylenediamine, 2,5-diaminotoluene, 2,6-diaminotoluene, 4,4′-diaminobiphenyl, 3, 3'-dimethyl-4,4'-diaminobiphenyl, 3,3'-dimethoxy-4,4'-diaminobiphenyl, diaminodiphenylmethane, diaminodiphenyl ether, 2,2'-diaminodiphenylpropane, bis (3 5-diethyl 4-aminophenyl) methane, diaminodiphenylsulfone, diaminobenzophenone, diaminonaphthalene, 1,4-bis (4-aminophenoxy) benzene, 1,4-bis (4-aminophenyl) benzene, 9,10- Bis (4-aminophenyl) anthracene, 1,3-bis (4-amino Phenoxy) benzene, 4,4′-bis (4-aminophenoxy) diphenylsulfone, 2,2-bis [4- (4-aminophenoxy) phenyl] propane, 2,2-bis (4-aminophenyl) hexafluoro Examples include propane and 2,2-bis [4- (4-aminophenoxy) phenyl] hexafluoropropane.

Among these, the divalent organic group having 1 to 20 carbon atoms represented by R ² is preferably a group derived from a fluorinated diamine compound from the viewpoint of biocompatibility, and derived from a fluorinated aromatic diamine compound. And more preferably a group derived from 2,2-bis (4-aminophenyl) hexafluoropropane and 2,2-bis [4- (4-aminophenoxy) phenyl] hexafluoropropane, and 2,2 A group derived from -bis (4-aminophenyl) hexafluoropropane is particularly preferred. In addition, the said polyimide may contain the 1 type (s) or 2 or more types of group derived from the said diamine compound.

  Examples of the polyamic acid that causes a photocleavage reaction by the polarized ultraviolet light include a polyamic acid having a structural unit represented by the following formula (3).

In said formula (3), R < ¹ > and R < ² > are synonymous with said formula (1), respectively.

(Method for synthesizing polyamic acid)
The polyamic acid is, for example, a tetracarboxylic dianhydride (hereinafter referred to as “specific tetracarboxylic acid”) containing an alicyclic structure of a tetravalent organic group represented by the above formulas (i-1) to (i-6). It can be synthesized by reacting a diamine compound with an acid dianhydride ”.

As the specific tetracarboxylic dianhydride, for example,
Examples of the organic group represented by the above formula (i-1) include 1,2,3,4-cyclobutanetetracarboxylic dianhydride;
Examples of the organic group represented by the above formula (i-2) include 1,2,3,4-cyclopentanetetracarboxylic acid, 2,3,5-tricarboxycyclopentylacetic acid dianhydride and the like;
Examples of the organic group represented by the above formula (i-3) include 2,3,4,5-tetrahydrofurantetracarboxylic dianhydride;
Examples of the organic group represented by the above formula (i-4) include 1,2,4,5-cyclohexanetetracarboxylic dianhydride;
As what gives the organic group represented by the said Formula (i-5), for example, 3, 4- dicarboxy- 1-cyclohexyl succinic dianhydride etc .;
Examples of the organic group represented by the above formula (i-6) include alicyclic tetracarboxylic acids such as 3,4-dicarboxy-1,2,3,4-tetrahydro-1-naphthalene succinic acid And dianhydrides. Among these, 1,2,3,4-cyclobutanetetracarboxylic dianhydride and 2,3,5-tricarboxycyclopentylacetic acid dianhydride are preferable, and 1,2,3,4-cyclobutanetetracarboxylic dianhydride is preferable. Anhydrides are more preferred. In addition, you may use this specific tetracarboxylic dianhydride individually or in combination of 2 or more types.

  Moreover, you may use together other tetracarboxylic dianhydrides other than the said specific tetracarboxylic dianhydride in the range which does not impair the effect of this invention.

As said other tetracarboxylic dianhydride, for example,
As the aliphatic tetracarboxylic dianhydride, 1,2,3,4-butanetetracarboxylic dianhydride and the like;
As aromatic tetracarboxylic dianhydrides, pyromellitic acid, 2,3,6,7-naphthalenetetracarboxylic acid, 1,2,5,6-naphthalenetetracarboxylic acid, 1,4,5,8-naphthalenetetra Carboxylic acid, 2,3,6,7-anthracenetetracarboxylic acid, 1,2,5,6-anthracenetetracarboxylic acid, 3,3 ′, 4,4′-biphenyltetracarboxylic acid, 2,3,3 ′ , 4-biphenyltetracarboxylic acid, bis (3,4-dicarboxyphenyl) ether, 3,3 ′, 4,4′-benzophenonetetracarboxylic acid, bis (3,4-dicarboxyphenyl) sulfone, bis (3,4-dicarboxyphenyl) methane, 2,2-bis (3,4-dicarboxyphenyl) propane, 1,1,1,3,3,3-hexafluoro-2,2-bis (3 -Dicarboxyphenyl) propane, bis (3,4-dicarboxyphenyl) dimethylsilane, bis (3,4-dicarboxyphenyl) diphenylsilane, 2,3,4,5, -pyridinetetracarboxylic acid, 2,6 And dianhydrides such as bis (3,4-dicarboxyphenyl) pyridine. ;
In addition, you may use another tetracarboxylic dianhydride individually or in combination of 2 or more types.

  Examples of the diamine compound used for synthesizing the polyamic acid include an aliphatic diamine compound, an alicyclic diamine compound, an aromatic diamine compound, a diaminosiloxane compound, and the like.

Examples of the aliphatic diamine compound, alicyclic diamine compound, and aromatic diamine compound include compounds similar to the respective compounds exemplified as the diamine compound that gives the divalent organic group represented by R ^2. .

  The ratio of the tetracarboxylic dianhydride and the diamine compound used for the synthesis of the polyamic acid is such that the acid anhydride group of the tetracarboxylic dianhydride is 0.2 with respect to 1 equivalent of the amino group contained in the diamine compound. A ratio of equivalent to 2 equivalents is preferable, and a ratio of 0.3 equivalent to 1.2 equivalents is more preferable.

  The polyamic acid synthesis reaction is preferably performed in an organic solvent. The reaction temperature is preferably −20 ° C. to 150 ° C., more preferably 0 ° C. to 100 ° C., and further preferably 0 ° C. to 50 ° C. The reaction time is preferably 0.5 hours to 24 hours, more preferably 2 hours to 15 hours, and even more preferably 5 hours to 12 hours.

The organic solvent is not particularly limited as long as it can dissolve the synthesized polyamic acid. For example,
As aprotic polar solvents, N-methyl-2-pyrrolidone (NMP), N, N-dimethylacetamide, N, N-dimethylformamide, N, N-dimethylimidazolidinone, dimethyl sulfoxide, γ-butyrolactone, tetramethyl Urea, hexamethylphosphortriamide, etc .;
Examples of the phenol solvent include m-cresol, xylenol, phenol, halogenated phenol and the like.

  Among these, the organic solvent is preferably an aprotic polar solvent, and more preferably N-methyl-2-pyrrolidone (NMP).

  In addition, you may use the said organic solvent individually or in combination of 2 or more types. Moreover, even if it is a solvent which does not melt | dissolve a polyamic acid, or a solvent which is hard to melt | dissolve, this solvent may be added to the said organic solvent in the range by which the uniform solubility of a polyamic acid is not lost.

  The amount of the organic solvent used (a) is such that the total amount (b) of the tetracarboxylic dianhydride and the diamine compound is 0.1% by mass to 50% by mass with respect to the total amount (a + b) of the reaction solution. Is preferable, and the amount of 5% by mass to 30% by mass is more preferable.

  The polyamic acid solution obtained as described above may be used as it is for forming the film (α), or may be used for forming the film (α) by isolating the polyamic acid contained in the solution. The released polyamic acid may be further purified and used to form the film (α).

  As a method for isolating the polyamic acid, for example, after pouring the reaction solution into a large amount of poor solvent to obtain a precipitate, the precipitate is dried under reduced pressure, and the organic solvent in the reaction solution is reduced with an evaporator. The method of distilling off is mentioned. Examples of the method for purifying the polyamic acid include a method in which the isolated polyamic acid is dissolved again in an organic solvent and then precipitated in a poor solvent, and a method in which the step of distilling under reduced pressure with an evaporator is performed once or a plurality of times. Can be mentioned.

  In addition, the obtained polyamic acid can be used for the synthesis | combination of the below-mentioned polyimide by spin-drying | dehydrating and ring-closing this. In this case, the reaction solution of the polyamic acid may be subjected to a dehydration ring-closing reaction as it is, or the polyamic acid contained in the reaction solution may be isolated as described above and subjected to a dehydration ring-closing reaction. May be purified as described above and subjected to a dehydration cyclization reaction.

(Polyimide synthesis method)
The polyimide can be synthesized by dehydrating and ring-closing the amic acid structure of the polyamic acid obtained as described above. At this time, all of the amic acid structure may be imidized to be a completely imidized product, or a part of the amic acid structure may be imidized to be a partially imidized product. Examples of the method for dehydrating and cyclizing the polyamic acid include, for example, (i) a method of heating the polyamic acid, (ii) dissolving the polyamic acid in an organic solvent, adding a dehydrating agent and a dehydrating ring-closing catalyst to this solution as necessary. And heating method.

  In the method of heating the polyamic acid (i) above, the reaction temperature is preferably 150 ° C to 450 ° C, more preferably 170 ° C to 350 ° C. When the reaction temperature is less than 150 ° C., the dehydration ring-closing reaction does not proceed sufficiently. The reaction time is preferably 30 seconds to 10 hours, and more preferably 5 minutes to 5 hours.

  In the method (ii) of adding a dehydrating agent and a dehydrating ring-closing catalyst to the polyamic acid solution, for example, an acid anhydride such as acetic anhydride, propionic anhydride, or trifluoroacetic anhydride is used as the dehydrating agent. it can. As a usage-amount of a dehydrating agent, 0.01-20 mol is preferable with respect to 1 mol of a polyamic acid structural unit. As the dehydration ring closure catalyst, for example, tertiary amines such as pyridine, collidine, lutidine, triethylamine and the like can be used. However, it is not limited to these. The amount of the dehydration ring closure catalyst used is preferably 0.01 mol to 10 mol with respect to 1 mol of the dehydrating agent used. Examples of the organic solvent used in the dehydration ring-closing reaction include the same organic solvents as those exemplified as the organic solvent used in the synthesis of polyamic acid. The reaction temperature for the dehydration ring closure reaction is preferably 0 ° C to 180 ° C, more preferably 10 ° C to 150 ° C. The reaction time is preferably 0.5 hour to 20 hours, and more preferably 1 hour to 8 hours.

  The polyimide obtained in the above method (i) may be used as it is for the formation of the film (α), or the polyimide may be purified and used for the formation of the film (α). On the other hand, in the above method (ii), a reaction solution containing polyimide is obtained. This reaction solution may be used as it is for the formation of the membrane (α), or may be used for the formation of the membrane (α) by removing the dehydrating agent and the dehydration ring-closing catalyst from the reaction solution. You may use for formation of ((alpha)), You may refine | purify the isolated polyimide and may use it for formation of a film | membrane ((alpha)). As a method for removing the dehydrating agent and the dehydrating ring closure catalyst from the reaction solution, for example, a method such as solvent substitution can be applied. Examples of the method for isolating and purifying polyimide include the same methods as those exemplified as the method for isolating and purifying polyamic acid.

  Examples of the polyimide having a photoalignable group and a precursor thereof include polyimide having a photoalignable group in a side chain, and polyamic acid having a photoalignable group in a side chain. Here, the photo-alignment group is a functional group that can impart anisotropy to the film by light irradiation. This anisotropy is imparted by a photoisomerization reaction or a photodimerization reaction. Since the polyimide and its precursor have a photo-alignment group, the growth of the seeded cells can be promoted on the film (α) irradiated with polarized ultraviolet rays. This is presumably because fine irregularities are formed on the surface of the film (α) as in the case of polyimide that causes a photocleavage reaction even when it has a photo-alignment group.

  As the photo-alignment group, groups derived from various compounds exhibiting photo-alignment can be employed. For example, a group having an azobenzene structure containing azobenzene or a derivative thereof as a basic skeleton, cinnamic acid or a derivative thereof is basically used. A group containing a cinnamic acid structure contained as a skeleton, a group containing a chalcone structure containing chalcone or a derivative thereof as a basic skeleton, a group containing a benzophenone structure containing benzophenone or a derivative thereof as a basic skeleton, a coumarin or a derivative thereof as a basic skeleton And a group containing a coumarin structure, a group containing a polyimide structure containing polyimide or a derivative thereof as a basic skeleton, and the like. Among these, a group including a cinnamic acid structure containing cinnamic acid or a derivative thereof as a basic skeleton is preferable from the viewpoint of excellent photoalignment and ease of introduction of the above group.

  The polyimide in the polyimide having the photoalignable group is not particularly limited as long as it has an imide structure in the structural unit constituting the main chain. In addition, the polyamic acid in the polyamic acid having the photoalignable group is not particularly limited as long as the structural unit constituting the main chain has an amic acid structure. However, this polyamic acid excludes those containing the imide structure.

  As a method for synthesizing the polyamic acid having a photoalignable group, for example, tetracarboxylic dianhydride and a diamine compound having an epoxy group are polymerized in an appropriate solvent, and then the resulting polymer is photoaligned. And a method of reacting a carboxylic acid compound having a functional group.

  Examples of the method for synthesizing the polyimide having the photoalignable group include a method of dehydrating and cyclizing the polyamic acid obtained in the method for synthesizing the polyamic acid having the photoalignable group by heating or the like.

  The polyimide or the like preferably contains a fluorine atom. When the polyimide and its precursor contain fluorine atoms, the membrane (α) has excellent biocompatibility and can promote the formation of cell aggregates from the seeded cells.

[Step (a2)]
In this step, as shown in FIG. 3, cells C are seeded on the surface of the film (α) irradiated with the polarized ultraviolet light (the film represented by reference numeral 2 in FIG. 3). Examples of cells that can be seeded include cells of various organs such as liver and kidney, skin cells, and the like, but are not limited to these and include all cells that can form cell aggregates. The cell C to be seeded is preferably a cell derived from the same species as the species to be treated. For example, when used for human therapy, human-derived cells are preferred, and the same human-derived cells are more preferred.

  It is preferable that the cells C to be seeded are preliminarily cultured in advance by various culture methods and the number of cells is sufficiently increased. Examples of the preculture method include known culture methods such as monolayer culture, coat dish culture, gel culture, and suspension culture. Pre-cultured cells are collected from the medium and seeded on the surface of the membrane (α) irradiated with polarized ultraviolet rays.

The concentration of the cells C to be seeded on the surface of the membrane (α) is preferably 1 × 10 ⁴ to 1 × 10 ⁷ cells / ml regardless of whether or not pre-culture is performed, and 1 × 10 ⁵ to 1 × 10 ⁶ cells / ml. More preferred is ml.

[Step (a3)]
In this step, as shown in FIG. 4, cell aggregates CM are formed by culturing the seeded cells C. Cell C is usually cultured in a medium. As this medium, a conventionally known medium can be appropriately selected according to the type of cells C to be seeded. Examples of the medium include MEM medium, BME medium, DME medium, αMEM medium, IMEM medium, ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium, RPMI medium, and mixed media thereof. . In addition, as a medium for angiogenic cells, a medium obtained by adding serum components such as fetal calf serum to the above medium can also be used. The type of the medium may be changed for each passage, or the same medium may be used.

  As culture | cultivation temperature, 35 to 38 degreeC is preferable and 36 to 37 degreeC is more preferable. The culture time is preferably 2 days to 7 days, more preferably 2 days to 4 days. The size of the formed cell aggregate is preferably 300 μm or less, more preferably 100 μm to 200 μm, from the viewpoint of cell activity.

  The obtained cell aggregate CM can be peeled off from the membrane (α) by chelate (EDTA) treatment or pipetting (see FIG. 5), and can be collected by centrifugation.

<Method for producing substrate for cell aggregate formation>
The method for producing a cell aggregate-forming substrate of the present invention comprises:
(B1) a step of forming a film mainly composed of polyimide or a precursor thereof (hereinafter also referred to as “step (b1)”), and (b2) a step of irradiating the film with polarized ultraviolet rays (hereinafter referred to as “step (b2)”. ) ")
Have
Since the method for producing the cell aggregate base material has a step of irradiating polarized ultraviolet light onto a film containing polyimide or the like as a main component, it is finely applied to the surface of the film (α) irrespective of conventional rubbing. An uneven shape can be formed, and as a result, generation of dust can be prevented.

[Step (b1)]
In this step, a film (α) whose main component is polyimide or a precursor thereof is formed. This film (α) is usually formed on a substrate. As said polyimide etc. and a board | substrate, each description in the above-mentioned <the formation method of a cell aggregate> is applicable, for example.

  Examples of the method for forming the film (α) include a method in which a solution in which polyimide or polyamic acid is dissolved is applied on the substrate. In addition, when the main component of the film (α) is polyimide, a method of forming a polyimide film (α) by applying a polyamic acid solution on a substrate and then heating and dehydrating and closing the film is also included. . As the heating conditions in this case, the same conditions as those described in the above section (Method of synthesizing polyimide) can be applied.

[Step (b2)]
In this step, the film (α) is irradiated with polarized ultraviolet rays. As the polarized ultraviolet light and its irradiation method, those described in the step (a1) in the method for forming a cell aggregate can be applied.

<Base material for cell aggregate formation>
As shown in FIG. 2, the base material A for forming cell aggregates of the present invention is formed on a substrate 100 and a film (α) formed on the surface of the substrate 100 and containing polyimide or a precursor thereof as a main component (FIG. 2). The film (α) is irradiated with polarized ultraviolet rays. The cell aggregate-forming substrate A is used for forming cell aggregates. Specifically, a cell aggregate can be formed by seeding cells on the surface of the membrane (α) irradiated with polarized ultraviolet rays of the cell aggregate-forming substrate A and culturing the cells.
Since the substrate A for forming cell aggregates includes a film (α) whose main component is polyimide or the like irradiated with polarized ultraviolet rays, high-quality cell aggregates are formed by the substrate in which the adhesion of dust is suppressed. Can be formed.

  As a manufacturing method of the said board | substrate 100, a polyimide, etc. and the said base material A for cell aggregate formation, each in the above-mentioned <Method for forming cell aggregate> and <Method for manufacturing base material for cell aggregate formation> The explanation can be applied.

  EXAMPLES Hereinafter, although this invention is demonstrated concretely based on an Example, this invention is not limited to these Examples. The measuring method of each physical property value is shown below.

<Synthesis of polyimide>
[Synthesis Example 1]
19.2 g (0.98 mol) of 1,2,3,4-cyclobutanetetracarboxylic dianhydride as a specific tetracarboxylic dianhydride and 2,2-bis (4-aminophenyl) hexa as a diamine compound 41.0 g (0.1 mol) of fluoropropane was put into 343.5 g of NMP and reacted at room temperature for 10 hours to obtain a polyimide precursor (polyamic acid) solution.

<Polyimide film formation>
The solution of polyamic acid synthesized in Synthesis Example 1 was spin-coated on a glass substrate, and then pre-baked on a hot plate at 80 ° C. for 1 minute to evaporate the solvent. Thereafter, post-baking was performed on a hot plate at 230 ° C. for 10 minutes to cause dehydration and ring closure, and a polyimide film (α) having a film thickness of 100 nm was formed.

<Formation of cell aggregate-forming substrate and cell aggregate>
[Example 1]
The surface of the polyimide film irradiated with the polarized ultraviolet light was prepared by irradiating the entire surface of the polyimide film prepared as described above with 10,000 J / m ² of polarized ultraviolet light having a wavelength of 254 nm to produce a cell aggregate-forming substrate. Rat skin-derived fibroblasts (FR cells) were seeded at a concentration of 2.7 × 10 ⁵ cells / ml, and the cells were cultured at 37 ° C. for 4 days using a medium.

[Comparative Example 1]
FR cells were cultured in the same manner as in Example 1 except that polarized ultraviolet rays were not irradiated.

  As a result of the above culture, the seeded FR cells were able to proliferate and form cell aggregates in the examples, whereas in the comparative example, cell proliferation was not observed and cell aggregates could not be formed. It was. In the examples, no dust was observed on the surface of the polyimide film after irradiation with polarized ultraviolet rays.

  This invention can prevent generation | occurrence | production of dust in the case of fine uneven | corrugated formation to film surfaces, such as a polyimide. Therefore, this invention can provide the formation method of the cell aggregate which can prevent generation | occurrence | production of dust, the manufacturing method of the base material for cell aggregate formation, and the base material for cell aggregate formation. Therefore, the cell aggregate formation method, the cell aggregate formation base material, and the production method thereof can improve the quality of the formed cell aggregate, and further improve the productivity by eliminating the need for dust removal. The present invention can be suitably applied to the aggregate formation process.

A A cell aggregate-forming substrate C Cell CM Cell aggregate 1 Film before irradiation with polarized ultraviolet light (α)
2 Film after irradiation with polarized UV light (α)
100 substrates

Claims (3)

(A1) A step of irradiating polarized ultraviolet rays to a film mainly composed of polyimide or a precursor thereof , polyamic acid ,
(A2) a step of seeding cells on the surface of the membrane irradiated with polarized ultraviolet light, and (a3) a step of forming a cell aggregate by culturing the seeded cells,
Polyamic acid is the polyimide or its precursor, which produces a light cleavage reaction by polarized UV, or all SANYO having photoalignable group,
The polyimide that causes a photocleavage reaction by the polarized ultraviolet light has a structural unit represented by the following formula (1),
Method of forming Ah Ru cell aggregates group the optical alignment group comprises cinnamic acid structure.

(In the formula (1), R ¹ is a tetravalent organic group represented by any of the following formulas (i-1) to (i-6). R ² is 2 having 1 to 20 carbon atoms. Valent organic group.)

(In Formula (i-1), Formula (i-3) and Formula (i-4), R ^A1 , R ^A3 and R ^A4 are each independently a monovalent organic group having 1 to 4 carbon atoms. N1 is an integer of 0 to 4. n3 is an integer of 0 to 4. n4 is an integer of 0 to 8. When R ^A1 , R ^A3, and R ^A4 are each plural, plural R ^A1 , R ^A3 and R ^A4 may be the same or different.
In formula (i-2), R ^A21 is a monovalent organic group having 1 to 4 carbon atoms. n2 is an integer of 0-6. R ^A22 is a single bond, a methylene group or an alkylene group having 2 to 4 carbon atoms. When n2 is 2 or more, the plurality of R ^A21 may be the same or different.
In formula (i-5), R ^A5 represents a fluorine atom or a monovalent organic group having 1 or 2 carbon atoms. n5 is an integer of 0-9. When n5 is 2 or more, the plurality of R ^A5 may be the same or different.
In formula (i-6), R ^A61 represents a fluorine atom or a monovalent organic group having 1 or 2 carbon atoms. n61 is an integer of 0-5. R ^A62 is a fluorine atom or a monovalent organic group having 1 to 4 carbon atoms. n62 is an integer of 0-4. If R ^A61 and ^{R A62} is plural respective plurality of ^{R A61} and ^{R A62} may each be the same or different.
In formulas (i-1) to (i-6), * represents a bonding site with a carbon atom. ) The method for forming a cell aggregate according to claim 1 , wherein the polyimide or a precursor thereof contains a fluorine atom. (B1) a step of forming a film mainly composed of polyimide or a precursor thereof polyamic acid , and (b2) a step of irradiating the film with polarized ultraviolet rays,
Polyamic acid is the polyimide or its precursor, which produces a light cleavage reaction by polarized UV, or all SANYO having photoalignable group,
The polyimide that causes a photocleavage reaction by the polarized ultraviolet light has a structural unit represented by the following formula (1),
The optical alignment group is, the production method of Ah Ru cell aggregate formation base materials by a group containing a cinnamic acid structure.

(In the formula (1), R ¹ is a tetravalent organic group represented by any of the following formulas (i-1) to (i-6). R ² is 2 having 1 to 20 carbon atoms. Valent organic group.)

(In Formula (i-1), Formula (i-3) and Formula (i-4), R ^A1 , R ^A3 and R ^A4 are each independently a monovalent organic group having 1 to 4 carbon atoms. N1 is an integer of 0 to 4. n3 is an integer of 0 to 4. n4 is an integer of 0 to 8. When R ^A1 , R ^A3, and R ^A4 are each plural, plural R ^A1 , R ^A3 and R ^A4 may be the same or different.
In formula (i-2), R ^A21 is a monovalent organic group having 1 to 4 carbon atoms. n2 is an integer of 0-6. R ^A22 is a single bond, a methylene group or an alkylene group having 2 to 4 carbon atoms. When n2 is 2 or more, the plurality of R ^A21 may be the same or different.
In formula (i-5), R ^A5 represents a fluorine atom or a monovalent organic group having 1 or 2 carbon atoms. n5 is an integer of 0-9. When n5 is 2 or more, the plurality of R ^A5 may be the same or different.
In formula (i-6), R ^A61 represents a fluorine atom or a monovalent organic group having 1 or 2 carbon atoms. n61 is an integer of 0-5. R ^A62 is a fluorine atom or a monovalent organic group having 1 to 4 carbon atoms. n62 is an integer of 0-4. If R ^A61 and ^{R A62} is plural respective plurality of ^{R A61} and ^{R A62} may each be the same or different.
In formulas (i-1) to (i-6), * represents a bonding site with a carbon atom. )

Images