WO2021029241A1 - Cell culture substrate - Google Patents

Abstract

Provided are a cell culture substrate that can be prepared easily and also permits cell culture to be carried out efficiently, a method for producing the substrate, a method for culturing cells using the substrate, and a method for producing spheroids using the substrate. A cell culture substrate having a layer that has a cell adhesive surface, and a fine pattern of a cell non-adhesive material provided on the surface of the layer, the cell adhesive surface being the cell culture surface. Additionally, a method for producing a cell culture substrate, the method including a step for printing a cell non-adhesive material to form a fine pattern on a layer having a cell adhesive surface.

Description

Substrate for cell culture

The present invention relates to a base material for cell culture. More specifically, the present invention relates to a cell culture base material, a method for producing the same, a method for culturing cells using the base material, and a method for producing a spheroid using the base material.

In recent years, a technique for culturing cells on a substrate having various processed surfaces has attracted attention.

For example, in Patent Document 1, a hydrophilic crosslinked body is formed by curing a photosensitive composition containing a branched polyalkylene glycol derivative having a specific structure on a hydrophobic substrate, while the substrate is formed. It is described that a plurality of hydrophobic regions are exposed on the surface, and spheroids having a uniform size can be obtained in the hydrophobic regions. Further, in Patent Document 2, in a technique for forming a resin film of a photosensitive resin composition on a substrate, a resin composition containing a water-soluble polymer having an azide group as a photosensitive group is used to form the azide group. A cultured base material is prepared in which cells adhere only to the exposed portion of the base material by binding to an amino group on the surface of the base material and exposing it to a desired pattern as needed.

In Patent Document 3, a small circular, polygonal, or elliptical region that can be a cell culture bed is finely patterned on a support surface treated with a hydrophilic and cell-incompatible polymer by plasma treatment or the like. A culture substrate formed by exposing the surface of the substrate is disclosed.

JP-A-2009-278960 Patent No. 4373260 Patent No. 4336756

However, there has been no concept of forming a non-cell adhesive region on a cell adhesive surface to use as a culture medium.

Further, in the method of the prior art, for example, when a photosensitive composition is used, a catalyst is used for curing the photosensitive composition, and there is a fear that the remaining catalyst or the composition with an incomplete reaction affects the culture. is there. Further, patterning using plasma treatment is complicated because it is necessary to remove the polymer layer after equipment preparation and plasma treatment. Therefore, further improvement is desired.

An object of the present invention is to provide a novel base material for cell culture.

In addition, the present invention can be easily prepared and cell culture can be efficiently performed, a cell culture substrate, a method for producing the same, a method for culturing cells using the substrate, and the substrate. It is an object of the present invention to provide a method for producing a spheroid using.

As a result of diligent studies to achieve the above object, the present inventors used a cell adhesion layer as a base to be a cell culture surface without using a special device such as a photosensitive composition or a plasma generator. By forming a fine pattern on the surface of such a layer with a non-cell adhesive substance, a new cell culture substrate can be provided, and such a cell culture substrate can be selected for its composition, manufacturing method, etc. In some cases, it was found that not only the preparation of the culture substrate can be simplified but also the uniformity of the obtained cells and spheroids can be improved, and further studies have been carried out to complete the present invention.

That is, the present invention relates to the following [1] to [16].
[1] A cell culture substrate having a layer having a cell adhesive surface and a fine pattern of a non-cell adhesive substance provided on the surface of the layer, and the cell adhesive surface is a cell culture surface.
[2] The base material according to the above [1], wherein the layer having a cell adhesion surface is composed of a substance exhibiting cell adhesion.
[3] The base material according to the above [1] or [2], wherein the cell adhesion surface contains a polyimide resin.
[4] The cell non-adhesive substance is ethylene glycol, 2-methacryloyloxyethyl phosphorylcholine, hydroxyethyl methacrylate and derivatives thereof, or polymers thereof [polymers containing them at least as a polymerization component (for example, alone or together). Polymer)], and segmented polyurethane and derivatives thereof; the substrate according to any one of [1] to [3] above, which is selected from albumin, agarose, and cellulose.
[5] The base material according to any one of [1] to [4] above, wherein the fine pattern is a pattern composed of a plurality of unit shapes or a pattern composed of gaps between adjacent unit shapes. ..
[6] The base material according to any one of [1] to [5] above, wherein the fine pattern is a pattern selected from a mesh shape, a dot shape, a stripe shape, a honeycomb shape, and an inverse pattern thereof.
[7] The base material according to the above [5] or [6], wherein the shortest distance of the gap is 1 μm or more.
[8] The base material according to any one of [5] to [7] above, wherein the average circle-equivalent diameter of the unit shape is 1000 μm or less.
[9] The base material according to any one of [5] to [8] above, wherein the distance between the centers of perfect circles corresponding to adjacent unit shapes is 1500 μm or less.
[10] The base material according to any one of [1] to [9] above, wherein the thickness of the fine pattern is 10 μm or less.
[11] The fine pattern is microcontact printing method, spin coating method, casting method, roll coating method, die coating method, gravure coating method, spray coating method, bar coating method, flexographic printing method, dip coating method, inkjet method, [1] to the above, which are formed by a method selected from the group consisting of a patterning method formed by injecting a desired substance into the gap by the capillary force generated in the gap of the uneven structure formed on the surface of the base material. The base material according to any one of [10].
[12] The substrate according to any one of [1] to [11] above, wherein the fine pattern occupies 5% or more of the surface of the substrate.
[13] The base material according to any one of [1] to [12] above, which has a thickness of 1 to 1000 μm.
[14] A method for producing a base material for cell culture, which comprises a step of printing a non-cell adhesive substance on a layer having a cell adhesive surface to form a fine pattern.
[15] A method for culturing cells, which comprises a step of culturing cells using the substrate according to any one of [1] to [13] above.
[16] A method for producing a spheroid, which comprises a step of culturing cells using the substrate according to any one of [1] to [13] above.

The present invention can provide a novel cell culture substrate.
In such an aspect of the cell culture substrate of the present invention, it can be easily prepared and the cell culture work itself can be efficiently performed.

Further, in another aspect of the cell culture substrate of the present invention, it can be used for cell culture without prior defoaming treatment.
Further, in another aspect of the present invention, it is possible to provide a cell culture substrate capable of easily recovering the cultured cells even though they are adhered and cultured.
Moreover, in still another aspect of the cell culture substrate of the present invention, cells (spheroids) of a desired size can be efficiently produced.
In the cell culture substrate of the present invention, since the cell culture site is divided by a fine pattern, it is easy to efficiently obtain cells of a desired size according to this division. In particular, by appropriately selecting the shape (rule) of the pattern, it is possible to efficiently obtain cells having a desired size and uniform (size or particle size). By making the cell size the desired size, it is possible to suppress the production of defective cells (for example, those that have grown too large, those that lack oxygen in the center of spheroids, etc.), and to homogenize the functions according to the cell type (for example, those that are deficient in oxygen at the center of the spheroid). For example, it is advantageous in terms of (equalizing the medicinal effects, etc.).
Moreover, as described above, the cells can be cultured without undergoing the defoaming treatment, and the obtained cells can be easily collected, so that the cell culture can be efficiently performed as a whole. Therefore, according to the cell culture substrate of the present invention, it is possible to achieve both efficient cell culture, which is originally incompatible, and acquisition of cells of a desired size (further, a uniform size).

Further, by using the cell culture substrate of still another aspect of the present invention, cells and spheroids having a desired size and high uniformity can be safely prepared, and thus cell quality control becomes easy. It can produce an excellent effect.

FIG. 1 is a diagram showing a schematic diagram of an example of a fine pattern in the cell culture substrate of the present invention. FIG. 2 is a photograph of a fine pattern in the cell culture substrate of Example 1. FIG. 3 is a photograph of a cell morphology (culture period: 8 days) in which human adipose-derived stem cells were cultured using the cell culture substrate of Example 1. FIG. 4 is a photograph of a cell morphology (culture period: 14 days) in which human adipose-derived stem cells were cultured using the cell culture substrate of Example 1. FIG. 5 is a photograph of the cell morphology (culture period: 7 days) in which HCT116 cells (human Escherichia coli cancer cell line) were cultured using the cell culture substrate of Example 1. FIG. 6 is a photograph of the cell morphology (culture period: 7 days) in which primary rat hepatocytes were cultured using the cell culture substrate of Example 1.

The cell culture substrate of the present invention has a layer having a cell adhesive surface and a fine pattern of a cell non-adhesive substance provided on the surface of the layer, and the cell adhesive surface in which the fine pattern does not exist is a cell. It serves as a culture surface, and cells can be adhered and cultured on the cell culture surface. It can be said that the cell culture substrate of the present invention has a mask having a fine pattern of a non-cell adhesive substance on a layer having a cell adhesive surface.

The fine pattern in the present invention includes a case where it is composed of a plurality of unit shapes and a case where it is composed of a shape corresponding to a gap between adjacent unit shapes, and is formed of a cell non-adhesive substance. For example, when the fine pattern of the cell non-adhesive substance is composed of unit shapes, the cell adhesive surface is exposed in the gap between the unit shapes. When the fine pattern of the cell non-adhesive substance is composed of a shape corresponding to a gap between adjacent unit shapes, the cell adhesive surface is exposed at the portion of the unit shape. Further, FIG. 1 schematically shows an example of the cell culture substrate of the present invention (1 is a unit shape, 2 is a gap), 1 is a fine pattern of a non-cell adhesive substance, and 2 is a cell adhesive surface. One embodiment is exemplified by 1 being a cell-adhesive surface and 2 being a fine pattern of a non-cell-adhesive substance. The unit shape referred to here is also referred to as a unit pattern or a unit type.

The unit shape is not particularly limited, and includes those composed of straight lines, those composed of curved lines, those composed of surfaces, and those composed by combining them.

Specifically, for example, those composed of polygons, ellipses, circles, fans, etc. may be mentioned, and these may be combined with two or more types to form one unit type. Polygons include hexagons, pentagons, quadrilaterals (including, for example, squares, rectangles, parallelograms, diamonds, and trapezoids), triangles, and stars, and circles include circles as well as approximately circles. .. The unit shape may have a plurality of unit types having the same size or different sizes as long as a fine pattern can be formed.

For example, in the case of a unit shape having a region (section), the average circle equivalent diameter (maximum diameter) of the unit shape may be 1000 μm or less, and may be 900 μm or less or 800 μm or less. The lower limit can be appropriately set according to the type of cells as long as the cells can be cultured. For example, 30 μm can be mentioned. Further, for the reason of preventing the association of adjacent cells, the distance between the centers of the perfect circles corresponding to the adjacent unit shapes may be, for example, 1500 μm or less, preferably 1000 μm or less, and more preferably 800 μm or less.

When the unit shape is linear, its width is preferably 10 mm or less, more preferably 8 mm or less, and further 5 mm or less, for the reason that it allows the interaction of humoral factors produced by adjacent cells. preferable. The lower limit can be appropriately set according to the type of cells as long as the cells can be cultured, and examples thereof include 5 μm.

In addition, the gap between adjacent unit shapes may be 1 μm or more as the shortest distance due to the interaction between cells, and may be 5 μm or more or 10 μm or more. The upper limit can be appropriately set according to the type of cells as long as the cells can be cultured, and examples thereof include 100 mm.

The patterns formed by the fine patterns include, for example, a pattern in which circular dots and polygons are scattered, a honeycomb shape, a grid shape (mesh shape), a scale shape, a geometric pattern, a stripe shape, and a radial pattern. Alternatively, a shape selected from those reverse patterns is exemplified. The pattern may have continuity or symmetry, or may be any pattern without continuity or symmetry.

The average height (average thickness) of the fine pattern is preferably 10000 nm or less, more preferably 1000 nm or less, and further preferably 500 nm or less.
The lower limit of the average height (average thickness) of the fine pattern is not particularly limited as long as the fine pattern can be formed, but is, for example, 1 nm, 2 nm, 3 nm, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm and the like. May be good.
In addition, the ratio of the average height of the fine pattern to the average circle equivalent diameter of the unit shape (average height / average circle) regardless of whether the unit shape is a cell non-adhesive substance or an exposed cell adhesion surface. The equivalent diameter) is preferably 0.5 or less, more preferably 0.1 or less, and even more preferably 0.01 or less. The lower limit can be appropriately set according to the type of cells as long as the cells can be arranged and cultured. For example, 0.00001 can be mentioned.
In the cell culture substrate, the cell adhesion site [exposed cell adhesion site (the site of the cell adhesion surface where no fine pattern is formed)] is the cell culture site (for example, 1 in FIG. 1). In the case of a fine pattern of a non-cell adhesive substance, 2 is a cell adhesion site, and in the case of a fine pattern of a non-cell adhesion substance, 1 is a cell adhesion site).
Such a cell adhesion site can be said to be a portion where the cell adhesion surface is divided (partitioned) by a fine pattern.
Minimum width of cell adhesion site [For example, the minimum length (shortest length) of the gap between adjacent unit shapes when the unit shape is non-cell adhesion, and the unit shape when the unit shape is cell adhesion. The minimum diameter (shortest diameter)] can be selected according to the size of the cells to be cultured and the size of the cells (spheroids) to be obtained, but for example, 10 μm or more (for example, 15 μm or more), preferably 20 μm or more ( For example, it may be 30 μm or more), more preferably 50 μm or more (for example, 80 μm or more), or 100 μm or more.
The minimum width of the cell adhesion site may be, for example, 2000 μm or less, 1500 μm or less, 1200 μm or less, 1000 μm or less, 900 μm or less, 800 μm or less, 700 μm or less, and the like.

The proportion of the fine pattern of the cell non-adhesive substance on the surface of the base material is preferably 5% or more, more preferably 10% or more, still more preferably 15% or more. The upper limit of the proportion of the fine pattern of the cell non-adhesive substance on the surface of the substrate is not particularly limited, but may be, for example, 95%, 90%, 80%, 70% or the like.

The fine pattern may be formed by physically or chemically fixing the non-cell adhesive substance to the surface of the fine pattern, as long as the surface thereof is a non-cell adhesive surface, and the fine pattern itself may be formed. It may consist of a non-cell adhesive substance. The method for forming the fine pattern is not particularly limited, and for example, it can be formed by curing a photosensitive resin composition or by exposing it to a desired pattern by plasma treatment or the like, but the catalyst used for curing may be used. A plasma generator is required to remain and affect the cells, or to perform plasma treatment, and the pattern formed by plasma treatment changes over time and becomes unstable or cannot be completely removed. The following method is preferable from the viewpoint that the polymer may affect the cells and that the defoaming treatment is simple or unnecessary. Specifically, for example, micro contact printing method, spin coating method, casting method, roll coating method, die coating method, gravure coating method, spray coating method, bar coating method, flexographic printing method, dip coating method, nanoimprint method, In addition to the inkjet method, a patterning method in which a desired substance is injected into the gap by the capillary force generated in the gap of the uneven structure formed on the surface of the base material can be used. These can be carried out according to known conditions. Specifically, for example, a transfer mold having a desired fine pattern shape is prepared according to a known method, and a non-cell adhesive substance is brought into contact with the pattern surface of the transfer mold to form a surface of a layer having a cell adhesion surface. The pattern surface can be printed (transferred) to form the pattern surface. In addition to the above-mentioned effects, the cell culture substrate having a fine pattern produced according to such a method also has an effect of being excellent in stability over time.
As described above, the fine pattern can be formed without undergoing plasma treatment or using a photosensitive component.
Therefore, the fine pattern may be a pattern that has not been plasma-treated, or may be a pattern formed of a non-photosensitive component [for example, a non-photosensitive resin (resin composition)].

The cell non-adhesive substance is not particularly limited as long as it does not adhere to the cells used for culturing or does not bind to cell surface molecules such as proteins and sugar chains present in the cell membrane of the cells used. , It may or may not have biocompatibility. Further, it may be hydrophobic or hydrophilic, and may be, for example, superhydrophobic (superhydrophobic) or superhydrophilic. From the viewpoint of cell non-adhesiveness, spheroid uniformity and formability, etc., it constitutes a hydrophobic (particularly superhydrophobic) [for example, a hydrophobic or hydrophilic (particularly hydrophobic) cell-adhesive surface or the surface thereof. More hydrophobic (particularly superhydrophobic) to the resin (and its contact angle)] is particularly preferred, but hydrophilic (particularly superhydrophilic) [eg, hydrophilic or hydrophobic (eg, hydrophobic)). A substance that is more hydrophilic (particularly superhydrophilic) to the cell-adhesive surface of the above or the resin (further, the contact angle thereof) constituting the surface is also preferable.
Examples of such substances include ethylene glycol and its derivatives, MPC (2-methacryloyloxyethyl phosphorylcholine) and its derivatives, HEMA (hydroxyethyl methacrylate) and its derivatives, compounds containing them, and polymers of these compounds, SPC. Compounds containing (segmented polyurethane) and its derivatives, proteins (albumin, etc.) obtained from living organisms, and sugar chains (agarose, cellulose, etc.) to which cells do not adhere are appropriately selected and used according to the type of cells. be able to. Among them, MPC and from the viewpoint of adhesion to the cell adhesion surface, from the viewpoint of simplifying the manufacturing process of the cell culture substrate, or from the viewpoint of improving the uniformity of the obtained cells or spheroids, etc. The derivatives or polymers thereof are preferred.
The substance may be appropriately modified according to the handleability, the desired degree of hydrophobicity (for example, superhydrophobicity), hydrophilicity (for example, superhydrophilicity), and the like. For example, hydrophilicity and low solubility in water may be achieved at the same time by cross-linking a hydrophilic substance. Further, the raw material (for example, hydrophobic or hydrophilic) is appropriately hydrophobized or hydrophilized (for example, introduction of a hydrophobic group or a hydrophilic group) to obtain the desired hydrophobic or hydrophilic property. The material may be obtained.
The cell non-adhesive substance (or fine pattern, non-adhesive molecule) may be a degradable component (eg, a component that decomposes in response to light and / or heat). When a cell non-adhesive substance (or a fine pattern) is formed from such a component, it may be possible to add a pattern step by step or change the pattern shape during the culture process.

Further, the fine pattern surface of the cell non-adhesive substance has, for example, a static water contact angle described later as an index as its surface characteristics from the viewpoint of making the size of the formed cell tissue uniform and improving the circularity. Can be judged as. For example, in the case of a hydrophobic surface formed of the above-mentioned substance, the static water contact angle is preferably 90 ° or more, more preferably 93 ° or more, still more preferably 95 ° or more. Further, it may be 150 ° or less, preferably 130 ° or less, and more preferably 120 ° or less.
On the other hand, in the case of a hydrophilic surface, the static water contact angle is preferably 65 ° or less, more preferably 55 ° or less, still more preferably 50 ° or less. Further, it may be 0 ° or more, preferably 5 ° or more, and more preferably 10 ° or more.
As an example, in a highly hydrophobic MPC (or a surface formed by the MPC), a static water contact angle such as 90 ° or more and 100 ° or more can be realized. ..
It should be noted that such a static water contact angle may be a value on a cell non-adhesive surface, or may be a value on a substance exhibiting cell non-adhesion (or a substance constituting a cell non-adhesive surface). Good.

The cell-adhesive surface is, for example, that when cells settle on the surface in a solution used for culturing, the cells adhere to each other with a certain adhesion point. Alternatively, it is a surface to be adhered so as to be fixed to such an extent that it can be peeled off by a liquid flow such as pipetting. In addition, a surface that adheres to the extent that it is possible to form a three-dimensional or three-dimensional tissue such as a layered or spheroid, rather than a surface on which cells adhere and maintain or proliferate two-dimensionally. Can be mentioned. Such a surface is formed, for example, by physically or chemically fixing or arranging a substance exhibiting cell adhesion on the surface of the base material, but the base material itself is composed of a substance exhibiting cell adhesion. May be good. Further, a smooth surface having no unevenness may be used so that a fine pattern can be easily formed by printing (transfer) or the like. The layer having a cell adhesion surface may be composed of a substance whose surface exhibits at least cell adhesion.

The substance exhibiting cell adhesion is not particularly limited as long as it is a substance that the cells used for culture adhere to or can bind to cell surface molecules such as proteins and sugar chains existing in the cell membrane of the cells used. Can be done. It may be hydrophilic or hydrophobic, but from the viewpoint of cell adhesion, spheroid formation, etc., it is hydrophilic (particularly hydrophilic, not superhydrophilic) or hydrophobic. (In particular, hydrophobic ones that are not superhydrophobic) are preferable, and those exhibiting hydrophobicity are more preferable. Further, the degree of adhesion of the substance exhibiting cell adhesion may be such that the cells do not come off from the cell culture surface. Examples of such substances include substances obtained or synthesized from living organisms, such as proteins (collagen, fibronectin, laminin, etc.) and synthetic resins (fluororesin, polyimide resin, polysulfone, polyethersulfone, etc.). , Polydimethylsiloxane, mixtures thereof, etc.). When a synthetic resin is selected, a cell culture substrate having excellent handleability can be obtained from the strength and heat resistance of the synthetic resin itself. In addition, from the viewpoint of biocompatibility, from the viewpoint of obtaining adherent cells and spheroids, from the viewpoint of improving the uniformity of the obtained cells and spheroids, or by appropriately adhering to various cells, the medium exchange work can be performed. From the viewpoint of facilitation, it is preferable to select a synthetic resin such as a polyimide resin. By selecting a non-living component such as a polyimide resin, the cells and spheroids obtained by using the cell culture substrate of the present invention containing the polyimide resin can be applied to fields such as regenerative medicine and drug discovery. It will be easy.

As the polyimide resin, a polyimide resin containing a structural unit represented by the following formula (I) can be exemplified. Further, from the viewpoint of good spheroid formation, a resin having a fluorine atom in the molecule is preferable, and a fluorine-containing polyimide (fluorine-containing polyimide resin) is more preferable. The polyimide resin used in the present invention is typically obtained by imidizing a polyamic acid obtained by polymerizing one or more kinds of acid dianhydride and diamine. The polyimide resin may contain polyamic acid as part of its chemical structure. As a method for producing the polyimide resin, it may be produced by a known method. As an example, the two-stage synthesis method can be used. The two-stage synthesis method of a polyimide resin is a method of synthesizing a polyamic acid as a precursor and converting the polyamic acid into a polyimide. The polyamic acid as a precursor may be a polyamic acid derivative. Examples of the polyamic acid derivative include a polyamic acid salt, a polyamic acid alkyl ester, a polyamic acid amide, a polyamic acid derivative from bismethylidene pyromeride, a polyamic acid silyl ester, and a polyamic acid isoimide. The polyimide consists of acid anhydrides such as pyromellitic dianhydride, biphenyltetracarboxylic dianhydride, and benzophenonetetracarboxylic dianhydride, and diamines such as oxydiamine, paraphenylenediamine, metaphenylenediamine, and benzophenonediamine. Polyimide can be exemplified. Examples of the resin having a fluorine atom include 4,4'-hexafluoroisopropyridene diphthalic acid anhydride (6FDA) / 1,4-bis (aminophenoxy) benzene (TPEQ) copolymer, 6FDA / 1,3. -Bis (4-aminophenoxy) benzene (TPER) copolymer, 6FDA / 4,4'-oxydiphthalic acid anhydride (ODPA) / TPEQ copolymer, 4,4'-(4,4'-isopropyridene Phenoxy) diphthalic acid (BPADA) / 2,2-bis [4- (4-aminophenoxy) phenyl] hexafluoropropane (HFBAPP), 6FDA / 2,2-bis (4- (4-aminophenoxy) phenyl) propane A fluorine-containing polyimide resin containing a structural unit represented by the following formula (I) such as a (BAPP) copolymer; an ethylene-tetrafluoroethylene copolymer or the like can be exemplified.

In formula (I) above, X ⁰ represents either an oxygen atom, a sulfur atom, a carbonyl group, a sulfonyl group, or a divalent organic group;
Y represents a divalent organic group;
Z ¹ , Z ² , Z ³ , Z ⁴ , Z ⁵ , and Z ⁶ independently indicate either a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.
p is 0 or 1.
In the polyimide resin, the chemical structure represented by the formula (I) may be different or the same for each constituent unit of the resin. It is preferable that at least one of X ⁰ , Y, Z ¹ , Z ² , Z ³ , Z ⁴ , Z ⁵ , and Z ⁶ contains one or more fluorine atoms.

In the above formula (I), when p = 0, X ⁰ may not exist (in other words, the left and right benzene rings may be directly bonded), but when p = 1. the left and right benzene ring linked via X ^0.

Specific examples of the divalent organic group represented by X ⁰ include an alkylene group, an arylene group, an aryleneoxy group, an arylentio group and the like. Further, it may be a fused ring type divalent hydrocarbon group, a heterocyclic fused ring type divalent hydrocarbon group, and these oxy groups and thio groups. Among these, an alkylene group, an aryleneoxy group and an arylenthio group are preferable, an alkylene group and an aryleneoxy group are more preferable, and these may be substituted with a fluorine atom. The alkylene group has, for example, 1 to 12 carbon atoms, preferably 1 to 6 carbon atoms.

Examples of the alkylene group substituted with a fluorine atom, which is an example of X ⁰ , include -C (CF ₃ ) _2- , -C (CF ₃ ) _2- C (CF ₃ ) _2-, and the like. .. Among the above-mentioned alkylene groups which are examples of X ⁰ , −C (CF ₃ ) ₂₋ is preferable.

As the arylene group which is an example of X ⁰ , for example, the following can be exemplified.

Examples of the arylene oxy group, which is an example of X ⁰ , include the following.

As the arylentio group which is an example of X ⁰ , for example, the following can be exemplified.

From the viewpoint that spheroids can be formed well on the substrate, the divalent organic group represented by X ⁰ is selected from the group consisting of b-2 to b-10 and c-2 to c-10. It may be selected from the group consisting of b-7 to b-9 and c-7 to c-9, and may have a structure represented by b-8.

Above arylene group is an example of X ^0, arylene group and Arirenchio groups are each independently a halogen atom (e.g., fluorine atom, chlorine atom, bromine atom, iodine atom, preferably fluorine atom or chlorine It may be substituted with a group selected from the group consisting of an atom, more preferably a fluorine atom), a methyl group and a trifluoromethyl group. The number of these substituents may be plural, and in that case, the types of the substituents may be the same or different from each other. Suitable substituents substituted with an arylene group, an aryleneoxy group and an arylentio group are a fluorine atom and / or a trifluoromethyl group, preferably a fluorine atom. The arylene group, the aryleneoxy group and the arylentio group are preferably substituted with at least one fluorine atom when Y does not contain a fluorine atom.

In the above formula (I), the divalent organic group represented by Y is not particularly limited, and examples thereof include a divalent organic group having an aromatic ring. Specifically, a group consisting of one benzene ring or a group having a structure in which two or more benzene rings are directly bonded via a carbon atom (that is, a single bond or an alkylene group), an oxygen atom, or a sulfur atom. Can be mentioned. Specifically, the following groups can be exemplified.

The above-mentioned divalent organic group having an aromatic ring, which is an example of Y, is a halogen atom (for example, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, preferably a fluorine atom or a chlorine atom, if substitutable. , More preferably a fluorine atom), may be substituted with a group selected from the group consisting of a methyl group and a trifluoromethyl group. The number of these substituents may be plural, and in that case, the types of the substituents may be the same or different from each other. A suitable substituent substituting a divalent organic group having an aromatic ring is preferably a fluorine atom and / or a trifluoromethyl group, particularly when X ⁰ does not contain a fluorine atom, and is more preferable. Is a fluorine atom.

From the viewpoint of spheroid formation, Y is a structure selected from the group consisting of d-3, d-9, e-1 to e-4, f-6, and f-7 in the above formula (I). The structure is preferably e-1, e-3 or e-4.

In the above formula (I), Z ¹ , Z ² , Z ³ , Z ⁴ , Z ⁵ , and Z ⁶ may be the same or different from each other, and each of them independently has a hydrogen atom and a fluorine atom. , Chlorine atom, bromine atom or iodine atom, and if at least one of X ⁰ and Y does not contain a fluorine atom, at least one of Z ¹ , Z ² , Z ³ , Z ⁴ , Z ⁵ , and Z ⁶ . One is preferably a fluorine atom.

From the viewpoint of spheroid formation, in a preferred embodiment of the present invention, the divalent organic group represented by X ⁰ in the above formula (I) is −C (CF ₃ ) _2- , the above b-2-b. Selected from the group consisting of -10 and c-2 to c-10; and from the group consisting of d-3, d-9, e-1 to e-4, f-6, and f-7. Be selected. In a more preferred embodiment of the present invention, in the above formula (I), the divalent organic group represented by X ⁰ is −C (CF ₃ ) _2- , b-7 to b-9 and c-7 to. Selected from the group consisting of c-9; and Y is selected from the group consisting of e-1, e-3 and e-4.

The polyimide resin composed of the structural unit represented by the above formula (I) can be obtained by a method of calcining a polyamic acid obtained by polymerizing an acid dianhydride and a diamine. The imidization rate of the above-mentioned "polyimide resin composed of the structural unit represented by the formula (I)" does not have to be 100%. That is, the polyimide resin composed of the structural units represented by the formula (I) may be composed of only the structural units represented by the above formula (I), but as long as the objective effect of the present invention is not impaired. , A structural unit in which the cyclic imide structure remains as an amic acid without dehydration ring closure may be partially contained.

It is preferable that the polyamic acid synthesis reaction is carried out in an organic solvent. The organic solvent used in the polyamic acid synthesis reaction is not particularly limited as long as the reaction between the acid dianhydride as the raw material and the diamine can proceed efficiently and is inert to these raw materials. .. For example, N-methylpyrrolidone (NMP), N, N-dimethylacetamide, N, N-dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, sulfolane, methyl isobutyl ketone, acetonitrile, benzonitrile, nitrobenzene, nitromethane, acetone, methyl ethyl ketone, isobutyl ketone. , Polar solvents such as methanol; non-polar solvents such as toluene and xylene. Above all, it is preferable to use a polar solvent. These organic solvents may be used alone or as a mixture of two or more. The reaction mixture after the amidation reaction may be directly subjected to thermal imidization. The concentration of the polyamic acid in the solution is not particularly limited, but is preferably 5% by weight or more from the viewpoint of the polymerization reactivity of the obtained resin, the viscosity after the polymerization, and the ease of handling in the subsequent film formation and firing. It is more preferably 10% by weight or more, preferably 50% by weight or less, and more preferably 40% by weight or less. The viscosity of the resin composition is not particularly limited, but can be measured according to the method described in Examples described later, and is, for example, 1 to 20 Pa · s, preferably 3 to 15 Pa · s at 23 ° C. It is within the range.

The polyamic acid is imidized by either thermal imidization or chemical imidization to obtain a resin containing a fluorine-containing polyimide. In a specific embodiment, the polyamic acid is imidized (thermally imidized) by heat treatment to obtain a resin containing a fluorine-containing polyimide. The polyimide obtained by thermal imidization has no possibility of residual catalyst and is more preferable for cell culture applications.

When imidizing by thermal imidization, for example, the polyamic acid is placed in air, more preferably in an atmosphere of an inert gas such as nitrogen, helium, or argon, or in vacuum, preferably at a temperature of 50 to 400 ° C. , More preferably 100 to 380 ° C., preferably 0.1 to 10 hours, more preferably 0.2 to 5 hours, to carry out an imidization reaction to obtain a resin containing polyimide.

The polyamic acid to be subjected to the thermal imidization reaction is preferably in the form of being dissolved in a suitable solvent. The solvent may be any one that dissolves polyamic acid, and the above-mentioned solvent for the polyamic acid synthesis reaction can also be used.

In the case of imidization by chemical imidization, the polyamic acid can be directly imidized in a suitable solvent by using the dehydration cyclization reagent described later.

The dehydration cyclization reagent can be used without particular limitation as long as it has an action of chemically dehydrating and cyclizing polyamic acid to form polyimide. As such a dehydration cyclization reagent, the imidization can be efficiently promoted by using the tertiary amine compound alone or in combination with the tertiary amine compound and the carboxylic acid anhydride. Is preferable.

Examples of the tertiary amine compound include trimethylamine, triethylamine, tripropylamine, tributylamine, pyridine, 1,4-diazabicyclo [2.2.2] octane (DABCO), and 1,8-diazabicyclo [5.4. 0] Undec-7-ene, 1,5-diazabicyclo [4.3.0] Nona-5-ene, N, N, N', N'-tetramethyldiaminomethane, N, N, N', N' -Tetramethylethylenediamine, N, N, N', N'-Tetramethyl-1,3-propanediamine, N, N, N', N'-tetramethyl-1,4-phenylenediamine, N, N, N Examples thereof include', N'-tetramethyl-1,6-hexanediamine, N, N, N', N'-tetraethylmethylenediamine, N, N, N', N'-tetraethylethylenediamine and the like. Among these, pyridine, DABCO, N, N, N', N'-tetramethyldiaminomethane are preferable, and DABCO is more preferable. The tertiary amine may be only one kind or two or more kinds.

Examples of the carboxylic acid anhydride include acetic anhydride, trifluoroacetic anhydride, propionic anhydride, butyric anhydride, isobutyric anhydride, succinic anhydride, maleic anhydride and the like. Among these, acetic anhydride and trifluoroacetic anhydride are particularly preferable, and acetic anhydride is more preferable. The carboxylic acid anhydride may be only one kind or two or more kinds.

As the solvent that dissolves polyamic acid in chemical imidization, a polar solvent having excellent solubility is preferable. For example, tetrahydrofuran, N, N-dimethylacetamide, N, N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide and the like can be mentioned, and among these, N, N-dimethylacetamide, N, N-dimethylformamide and N are particularly mentioned. -It is preferable that at least one selected from the group consisting of methylpyrrolidone is used from the viewpoint of uniform reaction. When these solvents are used as the solvent for the amidation reaction, the polyamic acid can be used as it is for chemical imidization without being separated from the reaction mixture after the amidation reaction.

The weight average molecular weight of the polyimide resin is, for example, 5000 to 2000000, preferably 8000 to 1000000, and more preferably 20000 to 500000. In this specification, the weight average molecular weight of the resin can be measured according to the method described in Examples described later, and when the weight average molecular weight is within the above range, the synthesis and handling of the polyimide resin, film formation, and spheroids The formability becomes better.

The cell-adhesive surface may further contain additive components such as a plasticizer and an antioxidant in addition to the above-mentioned substance exhibiting cell adhesion.

As for the layer having the cell adhesive surface, the cell adhesive surface may be all or a part of the layer as long as the surface in contact with the cells is the cell adhesive surface. For example, a substance exhibiting cell adhesion may be formed by being physically or chemically fixed or arranged on the surface of a layer, or the layer itself may be composed of a substance exhibiting cell adhesion. ..

The immobilization of the substance on the surface is a method of drying the solution containing these on the surface of the base material, a method of melting and crimping the substance, and curing the substance applied to the base material with an energy ray such as UV. Method, a method of causing a chemical reaction (for example, a condensation reaction between functional groups such as a carboxyl group and an amino group) between a functional group of the substance and a functional group on a base material to form a covalent bond. Alternatively, it can be immobilized on the surface of the base material by a method of bonding the thiol group of the substance and a thin metal (platinum, gold, etc.) formed in advance on the base material. The thickness at the time of immobilization is not particularly limited, and 0.01 to 1000 μm is exemplified. Further, for example, the substance is cast, spray coated, dip coated, spin coated, rolled on a release sheet (for example, an organic polymer film such as a polyethylene base material, ceramics, metal, glass, etc.) whose surface has been peeled off. By coating to an appropriate thickness by a method such as coating and heating, a layer made of such a substance can be formed into a sheet.
The cell adhesion surface (layer having the cell adhesion surface) can be formed without undergoing plasma treatment or using a photosensitive component.
Therefore, the cell adhesion surface (layer having a cell adhesion surface) may be a surface (layer) that has not been plasma-treated, and is formed of a non-photosensitive component [for example, a non-photosensitive resin (resin composition)]. It may be a surface (layer).

The cell-adhesive substance or cell-adhesive surface [hydrophobic (particularly non-superhydrophobic hydrophobic) cell-adhesive substance or cell-adhesive surface] preferably has a static water contact angle of 70 ° or more. The roll angle may be 15 ° or more, and the static water contact angle may be 70 ° or more and the fall angle may be 15 ° or more.
When the cell adhesive substance or the cell adhesive surface satisfies such a condition, spheroid formation is further promoted.
From the viewpoint of spheroid adhesion and spheroid formation, the static water contact angle is more preferably 75 ° or more (for example, more than 75 °), still more preferably 77 ° or more, still more preferably 79 ° or more, and more. More preferably, it is 80 ° or more (for example, more than 80 °), and the upper limit of the static water contact angle is, for example, less than 150 °, preferably 120 ° or less (for example, less than 120 °), and more preferably. It is 110 ° C. or lower, more preferably 100 ° C. or lower (for example, less than 99 ° C., 98 ° C. or lower, 97 ° C. or lower, 95 ° C. or lower, etc.).
On the other hand, the cell adhesion substance or cell adhesion surface [hydrophilic (particularly hydrophilic (particularly non-superhydrophilic) hydrophilic substance or cell adhesion surface] has a static water contact angle of 65 ° or less, more preferably 55. It may have ° or less, more preferably 50 ° or less. The lower limit value may be 0 ° or more, preferably 5 ° or more, and more preferably 10 ° or more.
From the viewpoint of spheroid formation, it is preferable that the falling angle is higher in the order of 18 ° or more, 19 ° or more, 20 ° or more, 22 ° or more, 24 ° or more, 26 ° or more, 28 ° or more, and 30 ° or more. The upper limit of the fall angle is, for example, less than 80 °, preferably 70 ° or less (for example, less than 70 °), more preferably 60 ° or less (for example, less than 60 °), and further preferably 50 °. The following (for example, less than 50 °). The static water contact angle and the fall angle may be values measured by the following method.

(Measurement method of static water contact angle)
Equipment: Automatic contact angle meter (Kyowa Interface Science: DM-500)
Measuring method: Measure the adhesion angle of the droplet immediately after dropping 2 μL of water on the surface (non-cell adhesive surface or cell adhesive surface) or film (film formed of non-cell adhesive or cell adhesive substance). (Measurement temperature: 25 ° C).

(Measuring method of falling angle)
Equipment: Automatic contact angle meter (Kyowa Interface Science: DM-500)
Measuring method: After dropping 25 μL of water on a surface (non-cell adhesive surface or cell adhesive surface) or a film (film formed of non-cell adhesive or cell adhesive substance), the substrate is continuously tilted. The fall angle is defined as the angle at which the cells flow down (measurement temperature: 25 ° C).

In addition, in terms of improving the size uniformity and circularity of the obtained spheroids, it is also important to balance the degree of adhesion between the cell-adhesive surface and the fine pattern surface of the cell non-adhesive substance. Therefore, even if the fine pattern surface of the cell non-adhesive substance exhibits adhesiveness, it may be less adhesive than the cell adhesive surface. For example, when the above static water contact angle is used as an index, the static water contact angle on the cell adhesive surface (or cell adhesive substance) and the fine pattern surface (or cell non-adhesive substance) of the cell non-adhesive substance. The difference (or its absolute value) is preferably 3 ° or more (for example, 5 ° or more), more preferably 10 ° or more (for example, 12 ° or more), and further preferably 15 ° or more. preferable. The upper limit can be appropriately selected depending on the combination of hydrophobicity and hydrophilicity of the cell non-adhesive substance (surface) and the cell adhesive substance (surface), and is not particularly limited, but for example, 100 °, 90 °, It may be 80 °, 70 °, 60 °, 50 °, 40 °, 30 ° and the like.

The resin constituting the cell adhesive surface may further contain additive components such as a plasticizer and an antioxidant.

The thickness of the cell culture substrate of the present invention is not particularly limited, but is preferably 1 to 1000 μm, more preferably 10 to 1000 μm from the viewpoint of handleability. The sheet area is also not particularly limited, and examples thereof include 0.01 to 10000 cm ² , preferably 0.03 to 5000 cm ² .

The cell culture substrate thus obtained can be used as it is, but from the viewpoint of being installed and used as it is in a known cell culture device, it may be appropriately sized according to the size of the target device. A medium containing cells may be placed on one of the surfaces to carry out cell culture, and the cell culture substrate may be used as a culture plate, each well of the plate, a culture chalet (culture dish), a flask, a culture bag, or the like. It can be housed and fixed in a housing, and cell culture can be carried out on a part or the entire surface of the fixed sheet.

Since the cell culture substrate of the present invention has the above-mentioned structure, it is not necessary to perform a filling operation or a defoaming operation for each section at the time of cell seeding, and cell culture can be efficiently performed. In addition, when the cell culture substrate is vibrated to the extent that it is taken out from the culture chamber, for example, the cultured cells on the substrate are adhered to the portion where the fine pattern of the cell non-adhesive substance is not formed, so that the observation during culture is performed. On the other hand, it is also easy to collect cultured cells by giving vibrations such as liquid flow and liquid feeding generated by tilting the container.

Further, since the cell culture substrate of the present invention can form a fine pattern (furthermore, a cell adhesive surface) regardless of a reaction such as hardening, it can be obtained by using such a cell substrate. The safety of cultured cells is high. Further, since the surface of the base material for cell culture has a predetermined pattern, it is possible that the portion to which the cells can adhere is exposed in a uniform shape, so that a uniform culture can be obtained. For example, a culture having a variation (SD) in size of 0.35 times or less, preferably 0.33 times or less of the average value can be obtained. Furthermore, since the cell culture substrate of the present invention does not change with time and can be made stable, the quality of the culture obtained by using such a cell culture substrate is also high. It will be good.

As a preferable method for producing the cell culture substrate of the present invention, a method including a step of printing a non-cell adhesive substance on a layer having a cell adhesive surface to form a fine pattern can be mentioned.

A known printing method can be used to form a fine pattern. Examples of the printing method include microcontact printing method, spin coating method, casting method, roll coating method, die coating method, gravure coating method, spray coating method, bar coating method, flexographic printing method, dip coating method, nanoimprint method, and the like. In addition to the inkjet method, a patterning method in which a desired substance is injected into the gap by the capillary force generated in the gap of the uneven structure formed on the surface of the base material can be used.

For example, in order to form a fine pattern by the micro contact printing method, a transfer mold having a fine pattern may be used for printing. There is no particular limitation on the method for preparing the transfer type. For example, a method of preparing a known transfer type such as an elastomer stamp can be mentioned. Specifically, the desired shape as described above is prepared on an elastomer sheet such as a silicone material by using a known processing machine and directly microfabricated, and the master substrate subjected to microfabrication is molded with an elastomer. There is a method to take. In the case of molding, first, a substrate having a desired shape as described above prepared by using a known processing machine is used as a master mold, and an elastomer resin solution is introduced therein and cured, and then the master mold is used. The one obtained by peeling off is used as a duplicate type. Here, the duplicate type will have an inverted shape of a desired shape. Next, the duplicated mold is subjected to surface treatment if necessary, the resin solution is introduced again and cured, and then the duplicated mold is peeled off to prepare a transfer mold having a desired shape. ..

There is no particular limitation on the substrate used. For example, olefin resins (eg polyethylene, polypropylene), polyester resins (eg polyethylene terephthalates), polycarbonate resins, acrylic resins (eg polymethyl (meth) acrylates, styrene resins (eg polystyrene), polyethers. Resins such as ketones, polyetheretherketones, and silicone-based resins; natural rubbers, rubbers such as EPDM; glass; metal materials such as stainless steel; ceramics, etc. These include mechanical strength and dimensional stability. Since it has transparency and the like, it can be preferably used.

The processing machine used for shape processing on a substrate is not particularly limited, and for example, an apparatus used for cutting with a drill, photolithography, inkjet printing, screen printing, and laser patterning method is used. Specific examples thereof include a fine excavation machine and a laser machine.

As the elastomer resin solution, any known one can be used without particular limitation, and for example, a silicone material can be preferably used from the viewpoint of transfer mold releasability. As the silicone material, a curable silicone material such as silicone rubber, a curable silicone rubber oligomer or silicone monomer that becomes a silicone resin, or a curable silicone resin oligomer or monomer is preferable, and a curable polysiloxane is more preferable. As the curable silicone material, a material called liquid silicone is usually used, and from the viewpoint of excellent peelability and mechanical strength, a two-component mixed type used in combination with a curing agent is preferable. If a low-viscosity curable silicone material is used, it is possible to perform processability such as removal of bubbles entrained during production and fine molding of a transfer pattern. Further, the curable polysiloxane may be a one-component curing type or a two-component curing type, and may be a thermosetting type or a room temperature curing type. Specific examples of the curable silicone material include, for example, alkylsiloxane, alkenylsiloxane, alkylalkenylsiloxane, and polyalkylhydroxanesiloxane, and among them, a two-component mixed system of alkylalkenylsiloxane and polyalkylhydroxanesiloxane is low. Those that cure at room temperature are preferable in terms of viscosity and processability.

When the elastomer resin solution is introduced into the mold, defoaming treatment or the like may be performed according to a known method, if necessary.

Examples of the surface treatment for taking the transfer duplication mold include a treatment of forming a non-adhesive surface so as to facilitate mold release and improve transfer uniformity. For example, in addition to the mold release treatment using a fluorine-based solvent or the like, a treatment in which a metal (Au, Pt, Ag, Ti, Al, Cr, Pd, etc.), carbon, or the like is deposited on the surface and then coated can be mentioned. .. In addition, a treatment for reducing the remaining reactive functional groups may be performed.

After the above-mentioned non-cell adhesive substance is attached to the transfer-type fine pattern thus obtained, the fine pattern of the non-cell adhesive substance is contacted (adhered) to a layer having a cell adhesive surface for printing. By such a method, a fine pattern can be formed with a thin film with high accuracy. Further, it is also possible to obtain a pattern by placing an elastomer pattern on a cell culture substrate in the state of being inverted in the above step and injecting and forming a non-cell adhesive substance from the side surface by utilizing the capillary phenomenon. ..

After the printing step, a known post-treatment step such as drying the obtained cell culture substrate may be included for the purpose of fixing the non-cell adhesive substance to the layer having the cell adhesive surface.

The present invention also provides a method for culturing cells, which comprises a step of culturing cells using the cell culture substrate of the present invention.

There are no particular restrictions on the applicable cells. For example, any organ or tissue (brain, liver, pancreas, spleen, heart, lung, intestine, cartilage, bone, fat, kidney, nerve) of humans or non-human animals (monkeys, pigs, dogs, rats, mice, etc.) , Skin, bone marrow, embryo, etc.), established established cells, or cells subjected to genetic manipulation or the like can be used. Further, it may be an insect cell, a plant cell, a microorganism, or the like.

More specifically, for example, pluripotent stem cells (ES cells, iPS cells), somatic stem cells (nerve stem cells, mesenchymal stem cells, hematopoietic stem cells, cancer stem cells and other undifferentiated stem cells) or progenitor cells thereof are used. be able to. In addition, for example, cells derived from digestive system organs such as liver cells and pancreatic cells, cells derived from cardiovascular organs such as kidney cells, nervous system cells, and myocardial cells, and binding tissues such as fat cells and skin dermis. Differentiation of derived fibroblasts, epithelial cells derived from epithelial tissues such as skin epidermis, bone cells, chondrogenic cells, cells derived from eye tissues such as retina, vascular cells, hematogenous cells, germline cells, etc. Cells can also be used. In addition, cancerous cells can also be used. Yeast, Escherichia coli, Bacillus subtilis, coryneform bacteria, lactic acid bacteria and the like can also be used. As such cells, one type of cell can be used alone, or two or more types of cells can be mixed and used in an arbitrary ratio.

The conditions for cell culture are not particularly limited as long as the base material for cell culture of the present invention is used. For example, the medium to be used may be appropriately selected according to the cells. For example, an arbitrary cell culture basal medium, a special medium developed for the target cells, a differentiation medium, a primary culture medium, or the like can be used. Specifically, Eagle's Small Essential Medium (EMEM), Dulbecco's Modified Eagle's Medium (DMEM), α-MEM, Grassgo MEM (GMEM), IMDM, RPMI1640, Ham F-12, MCDB Medium, Williams Medium E, Hepatocyte thick medium , And a mixed medium thereof and the like. Any medium containing the components necessary for cell proliferation and differentiation can be used. Further, a medium to which serum, various growth factors, differentiation-inducing factors, antibiotics, hormones, amino acids, sugars, salts and the like are added may be used. The culture temperature is not particularly limited, but it is usually about 25 to 40 ° C.

Since cells may form spheroids by the cell culture, one aspect of the present invention is a method for producing spheroids, which comprises a step of culturing cells using the cell culture substrate of the present invention. it can.

Since the obtained spheroid contains a large amount of cells, it can be used for research on development and regeneration of various tissues / organs, application to regenerative medicine, toxicity test in the field of drug discovery, etc. The diameter of the obtained spheroid is not particularly limited, and is, for example, 10 to 1000 μm, preferably 10 to 800 μm.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto. In the following examples, room temperature means 20 to 30 ° C.

Example 1
<Base material> Preparation of 6FDA / TPEQ polyamic acid In a 500 mL volume three-necked flask, 14.882 g (50.9 mmol) of 1,4-bis (aminophenoxy) benzene, 4,4'-hexafluoroisopropyridene diphthalic anhydride 22.618 g (50.9 mmol) and 212.5 g of N-methylpyrrolidone were charged. A fluoropolyamic acid resin composition (solid content concentration: 15.0% by mass) was obtained by aging for 6 days after stirring at room temperature for 2 hours in a nitrogen atmosphere. The weight average molecular weight of the polyamic acid was 157,000 and the viscosity (23 ° C.) was 7 Pa · s. The weight average molecular weight of the polyamic acid and the weight average molecular weight of the fluorine-containing polyimide after firing are substantially the same.

The fluorine-containing polyamic acid resin composition obtained above was applied onto a glass substrate using a die coater so that the thickness of the fluorine-containing polyimide film after firing was 40 μm to form a coating film. Then, the coating film was calcined at 360 ° C. for 1 hour in a nitrogen atmosphere. Then, the fired product was peeled off from the glass substrate to obtain a fluorine-containing polyimide film. The static water contact angle of this fluorine-containing polyimide film was 83.4 °, and the fall angle was 20.2 °.

The method for measuring the physical properties in the above is as follows.
(Measurement of weight average molecular weight)
Equipment: HCL-8220GPC manufactured by Tosoh Corporation
Column: TSKgel Super AWM-H
Eluent (LiBr · H2O, NMP containing phosphoric acid): 0.01 mol / L
Measuring method: A 0.5% by weight solution is prepared with an eluent, and the molecular weight is calculated based on a calibration curve prepared with polystyrene.
(Measurement of viscosity)
Equipment: AS ONE Viscometer VISCOMETER TV-22
Setting: VI RANGE: H ROTOR No.6 SPEED: 10rpm
Standard solution for viscometer calibration: Nippon Grease Co., Ltd. JS 14000
Measuring method: After calibrating with a standard solution for viscometer calibration, measure with 0.3 g of varnish. (Measurement temperature: 23 ° C)
(Measurement of static water contact angle)
Equipment: Automatic contact angle meter (Kyowa Interface Science: DM-500)
Measuring method: Measure the adhesion angle of the droplet immediately after dropping 2 μL of water on the film (measurement temperature: 25 ° C.).
(Measurement of falling angle)
Equipment: Automatic contact angle meter (Kyowa Interface Science: DM-500)
Measuring method: After 25 μL of water is dropped on the film, the base material is continuously tilted, and the angle at which it flows down is defined as the falling angle (measurement temperature: 25 ° C.).

<Making a pattern transfer type>
Using a micro excavation machine (manufactured by PMT Co., Ltd. (Fukuoka)), a 0.7 mm thick polymethylmethacrylate (PMMA) plate with a diameter of 300 μm [corresponding to the width (minimum width) of the cell adhesion site in this example. ], A master mold was made by excavating a fine pattern structure so that the circles would be staggered at a pitch (the shortest distance between the center of the circle and the center of the adjacent circle) of 500 μm. The shortest distance between the gaps in the pattern is 200 μm.

Next, in order to obtain a replica type, PDMS (polydimethylsiloxane) room temperature curable resin solution (SILPOT: manufactured by Toray Dow Corning) was weighed in a predetermined ratio (main agent: initiator = 10: 1) to a 100 mm dish. The mixture was stirred and mixed for 5 minutes. Then, in order to remove the air bubbles of PDMS, vacuum degassing was performed for 1 hour. The PMMA excavation master mold was submerged in the degassed PDMS resin solution to the bottom of the dish with the surface facing up, and degassed in vacuum for about 1 hour until the air bubbles were removed again. After degassing, the PMMA master type dish was left on a horizontal table under normal air and at room temperature for 3 days to cure PDMS. After curing of PDMS, the PMMA master type was peeled off to obtain a PDMS replica type.

Pt vapor deposition was performed on the surface of the obtained duplicate mold so that the thickness was 6 nm. Then, apply PEG-SH solution (2 mM PTE-200SH, manufactured by Nichiyu) so as to cover the Pt surface, react for 30 minutes in a light-shielded state, and then immerse the PDMS replica type in 40-50% EtOH solution. Unreacted PEG-SH was removed, immersed in milliQ water for washing, and then dried to PEG-modify to form a non-adhesive surface. The obtained PDMS type was washed and dried to remove unreacted PEG-SH, and a PDMS replication type (PEG-modified PDMS replication type) was completed.

By repeating the above molding operation using the above PEG-modified PDMS replication type and further producing an inverted type of PDMS, a PDMS transfer type having the same fine pattern as the PMMA master type was completed.

<Base material for cell culture with pattern>
After immersing the PDMS transfer type pattern surface prepared above in an MPC polymer solution (1% ethanol solution (hydrophobic MPC polymer)), the excess liquid was removed by air blowing, and the fluorine-containing polyimide obtained above was obtained by the microcontact printing method. The pattern was transferred onto the film culture substrate. The transferred film was dried in a dryer at 50 ° C. for 2 hours and observed under a microscope. As a result, it was confirmed that the MPC polymer pattern was transferred onto the film. (Fig. 2). The static water contact angle of the transferred MPC polymer was 107.5 ° on the film (cell non-adhesive surface). The obtained patterned cell culture substrate was contained in the culture substrate. The fine pattern area of the MPC polymer was 61%, the exposed area of the fluorine-containing polyimide film was 39%, and the thickness of the fine pattern was 0.04 μm (manufactured by Keyence: laser microscope). The ratio of the average circle equivalent diameter (average height / average circle equivalent diameter) of the circles exposed on the fluorine-containing polyimide film was 1.3 × 10 ^-4 .

Test Example 1
Human adipose derived stem cells (ADSC, Ronza: PT-5006) were cultured in KBM-ADSC-2 medium (manufactured by Kojin Bio) containing 5% FBS and 1% antibiotics, and 2.5 A cell suspension was prepared to a concentration of × 10 ⁵ cells / mL.

<Making spheroids>
The patterned cell culture substrate prepared above was placed on the cell culture surface of a 35 mm petri dish as it was without defoaming, and 0.2 mL of the cell suspension prepared above was seeded (5.0 × 10 ⁴ cells / petri dish). ), 1 hour later, 3.5 mL of KBM-ADSC-2 medium was added, and the cells were placed in a 5% (v / v) CO ₂ incubator at 37 ° C. and cultured (day 0 of culture). By injecting the suspension into a petri dish, it was confirmed that the cells could be uniformly seeded in each pattern without dispensing. Culturing was carried out until the 14th day without changing the medium.
The seeded cells adhered and proliferated in the cell adhesion region (fluorine-containing polyimide region) on the film culture substrate, and as the culture progressed, spheroids retained (adhered) to the patterned cell culture substrate were formed. The appearance was confirmed by observation using an optical microscope (Fig. 3: 8th day of culture, Fig. 4: 14th day of culture).

<Spheroid morphology evaluation>
The spheroids were photographed on the 8th and 14th days of the culture, and the circle-equivalent diameter of the spheroids was analyzed using the image analysis software WinROOF (manufactured by Mitani Corporation).
The obtained spheroids had high size uniformity (culture day 8: average circle equivalent diameter 125.9 μm, SD 33.5 μm, culture day 14: average circle equivalent diameter 126.1 μm, SD 29.4 μm). Therefore, by using the cell culture substrate of the present invention, a uniform and large amount of spheroids could be stably and easily obtained. Further, after the imaging, the spheroid could be easily desorbed from the base material by sending the culture solution to the petri dish, and it was easy to recover it.

Test Example 2
Using HCT116 cells (human colon cancer cell line, RIKEN: RCB2979), spheroids were prepared in the same manner as in Example 1. As the culture medium, a serum medium in which 10% FBS was added to DMEM basal medium (manufactured by Gibco) was used.

As a result, as shown in FIG. 5, spheroids (7th day of culture: average circle equivalent diameter 239 μm, SD 21 μm) retained (adhered) on the patterned cell culture substrate may appear as the culture progresses. It was observed that it was confirmed that the cells could be cultured on the cell culture substrate of the present invention even if the cell types were different.

Test Example 3
Spheroids were prepared in the same manner as in Test Example 1 except that primary rat hepatocytes (seed density: 1.0 × 10 ⁵ cells / petri dish) were used as cells and DMEM + supplement medium (serum-free) was used as the medium.

As a result, as shown in FIG. 6, spheroids (7 days of culture: average circle equivalent diameter 160 μm, SD 32 μm) retained (adhered) to the patterned cell culture substrate may appear as the culture progresses. It was observed that it was confirmed that the cells could be cultured on the cell culture substrate of the present invention even if the cell types were different.

Since the cell culture substrate of the present invention can be easily prepared and the cell culture work itself can be efficiently performed, it is suitably used in the field of cell preparations such as spheroid-containing preparations, for example. be able to.

1 Unit shape 2 Gap between adjacent unit shapes

Claims (16)

1. A cell culture substrate having a layer having a cell adhesive surface and a fine pattern of non-cell adhesive substances provided on the surface of the layer, and the cell adhesive surface is a cell culture surface.
2. The base material according to claim 1, wherein the layer having a cell adhesive surface is composed of a substance exhibiting cell adhesion.
3. The base material according to claim 1 or 2, wherein the cell adhesive surface contains a polyimide resin.
4. Cell non-adhesive substances are selected from ethylene glycol, 2-methacryloyloxyethyl phosphorylcholine, hydroxyethyl methacrylate and derivatives thereof, or polymers thereof, and segmented polyurethanes and derivatives thereof; albumin, agarose, and cellulose. , The base material according to any one of claims 1 to 3.
5. The base material according to any one of claims 1 to 4, wherein the fine pattern is a pattern composed of a plurality of unit shapes or a pattern composed of gaps between adjacent unit shapes.
6. The base material according to any one of claims 1 to 5, wherein the fine pattern is a pattern selected from a mesh shape, a dot shape, a stripe shape, a honeycomb shape, and an inverse pattern thereof.
7. The base material according to claim 5 or 6, wherein the shortest distance between the gaps is 1 μm or more.
8. The base material according to any one of claims 5 to 7, wherein the average circle equivalent diameter of the unit shape is 1000 μm or less.
9. The base material according to any one of claims 5 to 8, wherein the distance between the centers of perfect circles corresponding to adjacent unit shapes is 1500 μm or less.
10. The base material according to any one of claims 1 to 9, wherein the thickness of the fine pattern is 10 μm or less.
11. Fine patterns include microcontact printing method, spin coating method, casting method, roll coating method, die coating method, gravure coating method, spray coating method, bar coating method, flexographic printing method, dip coating method, inkjet method, and base. Any of claims 1 to 10, which is formed by a method selected from the group consisting of a patterning method formed by injecting a desired substance into a gap by a capillary force generated in a gap of an uneven structure formed on a material surface. The base material described in.
12. The base material according to any one of claims 1 to 11, wherein the fine pattern occupies 5% or more of the surface of the base material.
13. The base material according to any one of claims 1 to 12, which has a thickness of 1 to 1000 μm.
14. A method for producing a base material for cell culture, which comprises a step of printing a non-cell adhesive substance on a layer having a cell adhesive surface to form a fine pattern.
15. A method for culturing cells, which comprises a step of culturing cells using the substrate according to any one of claims 1 to 13.
16. A method for producing a spheroid, which comprises a step of culturing cells using the substrate according to any one of claims 1 to 13.

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