JP5879942B2 - Cell culture substrate manufacturing method, cell culture substrate, and cell sheet manufacturing method using the same - Google Patents

Description

  The present invention relates to a method for producing a cell culture substrate capable of easily forming a cell culture substrate having a pattern of a cell adhesion region and a cell adhesion inhibition region.

As a cell culture substrate for culturing cells in a pattern, those having a pattern of a cell adhesion region and a cell adhesion inhibition region are disclosed. As such a cell culture substrate, for example, a substrate having a substrate and a layer formed on the substrate and formed of a pattern having a cell adhesion property or a cell adhesion inhibitory material is disclosed. (Patent Document 1).
However, in order to form a layer made of a material having cell adhesion or cell adhesion inhibition in a pattern, usually, a layer made of these materials is formed on the entire surface of the substrate, and a lithography method or the like is used. A patterning method is used.
For this reason, the problem that it is difficult to manufacture easily and in large quantities, and the problem that it is difficult to stably form the above-described pattern when the substrate has an uneven structure, etc. was there.

JP 2011-50303 A

  The present invention has been made in view of the above problems, and provides a method for producing a cell culture substrate that can easily and in large numbers form a cell culture substrate having a pattern of cell adhesion regions and cell adhesion inhibition regions. The main purpose.

  In order to achieve the above object, the present invention performs energy irradiation on the surface of a polymer substrate having a cell adhesion-inhibiting surface, and modifies at least a part of the surface of the polymer substrate. There is provided a method for producing a cell culture substrate, comprising a cell adhesion region forming step of forming a cell adhesion region having cell adhesion.

  ADVANTAGE OF THE INVENTION According to this invention, the cell culture substrate which has a pattern of a cell adhesion area | region and a cell adhesion inhibition area | region can be formed easily and in large quantities by using the method which modifies the surface of a polymer base material.

  In the present invention, it is preferable to have a stimulus-responsive layer forming step of forming a stimulus-responsive layer on the surface of the polymer substrate after the cell adhesion region forming step. This is because, without patterning the stimulus-responsive layer, it is possible to easily have a pattern of a cell adhesion region and a cell adhesion-inhibiting region and have stimulus response.

  In this invention, it is preferable to have a fine uneven | corrugated shape shaping process which shapes the fine uneven | corrugated shape on the surface of the said polymer base material. It is because, for example, the orientation of the cells cultured on the cell culture substrate can be easily controlled by forming the fine uneven shape.

  In the present invention, it is preferable that the polymer base material is long and energy irradiation is performed while the polymer base material is conveyed. This is because the cell culture substrate can be formed easily and in large quantities.

  The present invention provides a cell culture substrate comprising a cell adhesion inhibiting region having cell adhesion inhibiting property and a polymer substrate having a cell adhesion region having cell adhesion property formed on a continuous surface.

  According to the present invention, by having a cell adhesion region and a cell adhesion inhibition region on the surface of the polymer base material itself, it can be formed easily and in large quantities.

  The present invention provides a cell culture step of producing a cell culture substrate using the above-described method for producing a cell culture substrate, adhering and culturing cells on the cell adhesion region of the cell culture substrate, and the cell adhesion region. And an extended cell adhesion region forming step of forming an extended cell adhesion region by irradiating at least a part of the cell adhesion inhibition region adjacent to the surface and modifying the surface of the polymer substrate. A method for producing a patterned cell sheet is provided.

  According to the present invention, it is possible to observe the state in which cells migrate and the orientation changes by having the expanded cell adhesion region forming step.

  INDUSTRIAL APPLICABILITY The present invention has an effect that it can provide a method for producing a cell culture substrate that can easily and in large numbers form a cell culture substrate having a pattern of cell adhesion regions and cell adhesion inhibition regions.

It is process drawing which shows an example of the manufacturing method of the cell culture substratum of this invention. It is explanatory drawing explaining the protective layer in this invention. It is explanatory drawing explaining the cell adhesion area | region formation process in this invention. It is explanatory drawing explaining the cell adhesion area | region formation process in this invention. It is process drawing which shows an example of the stimulus responsive layer formation process in this invention. It is process drawing which shows the other example of the stimulus responsive layer formation process in this invention. It is process drawing which shows the other example of the stimulus responsive layer formation process in this invention. It is a schematic plan view which shows an example of the fine uneven | corrugated shape in this invention. It is the sectional view on the AA line of FIG. It is process drawing which shows an example of the fine uneven | corrugated shape formation process in this invention. It is process drawing which shows an example of the manufacturing method of the pattern cell sheet | seat of this invention. It is a microscope picture which shows the result of Example 1-1. It is a microscope picture which shows the result of Example 1-2. It is a microscope picture which shows the result of Example 2-1. It is a microscope picture which shows the result of Example 2-2. It is a microscope picture which shows the result of Example 3-1. It is a microscope picture which shows the result of Example 3-2. 6 is a photomicrograph showing the results of Example 4. It is a microscope picture which shows the result of Example 5-Example 7.

The present invention relates to a method for producing a cell culture substrate, a cell culture substrate, and a method for producing a cell sheet using the same.
Hereinafter, the method for producing a cell culture substrate, the method for producing a cell culture substrate and a cell sheet of the present invention will be described in detail.

A. First, a method for producing a cell culture substrate of the present invention will be described.
In the method for producing a cell culture substrate of the present invention, the surface of a polymer substrate having a cell adhesion-inhibiting surface is irradiated with energy, and at least a part of the surface of the polymer substrate is modified, It has a cell adhesion region forming step of forming a cell adhesion region having cell adhesion.

Such a method for producing a cell culture substrate of the present invention will be described with reference to the drawings. FIG. 1 is a process diagram showing an example of a method for producing a cell culture substrate of the present invention. As illustrated in FIG. 1, in the method for producing a cell culture substrate of the present invention, a polymer substrate 1 containing polystyrene having a cell adhesion-inhibiting surface is prepared. A protective layer 3 having an opening at a location where a cell adhesion region is formed and capable of shielding energy is laminated (FIG. 1 (a)), and then the exposed polymer base material from above the protective layer 3 By selectively modifying the surface by irradiating energy to 1 (FIG. 1 (b)), the cell adhesion region 2a having cell adhesion was formed in a pattern, and the surface was not modified. A cell adhesion inhibiting region 2b having cell adhesion inhibiting property is formed in the region (FIG. 1 (c)). Next, as shown in FIG. 1 (d), by removing the protective layer 3, a cell culture substrate 10 including a polymer substrate 1 having a cell adhesion region 2a and a cell adhesion inhibition region 2b on the surface is obtained. (FIG. 1 (e)).
Here, FIGS. 1B to 1C show the cell adhesion region forming step. FIG. 1A shows a protective layer forming step.

According to the present invention, a cell culture substrate having a cell adhesion region and a cell adhesion inhibition region can be easily formed by using a method for modifying the surface of a polymer substrate.
Compared to conventional materials such as cell adhesion materials, cell adhesion inhibition materials, initiators, etc. formed on the base material by coating, etc., these materials are not used, so they have high safety. It can be. For example, since there is no influence of residues such as an initiator, and since patterning by a lithography method or the like can be made unnecessary, it can usually be free from the influence of a developer or the like that adversely affects cell culture. . Therefore, the cell culture substrate can be made capable of stably culturing cells.
Furthermore, since these materials and a separate process are unnecessary, a cell culture substrate can be easily formed from a roll-shaped polymer substrate by a continuous process such as roll-to-roll, roll-to-sheet.
Furthermore, the cell culture substrate produced by the production method of the present invention has a cell adhesion region (modified region) and a cell adhesion inhibition region (unmodified region) on the surface of the polymer substrate itself. Since it is formed, for example, it is possible to easily control the orientation of cells by providing a fine uneven shape, or to provide stimulus responsiveness by laminating a stimulus responsive layer.

The method for producing a cell culture substrate of the present invention comprises at least a cell adhesion region forming step.
Hereinafter, each process of the manufacturing method of the cell culture substratum of this invention is demonstrated in detail.

1. Cell adhesion region forming step In the cell adhesion region forming step in the present invention, the surface of the polymer substrate having a cell adhesion-inhibiting surface is irradiated with energy to modify at least a part of the surface of the polymer substrate. This is a step of forming a cell adhesion region having cell adhesion.

(1) Polymer substrate The polymer substrate in this step has a cell adhesion-inhibiting surface, and the surface is modified by energy irradiation to form a cell adhesion region. There is no particular limitation as long as it is present.

  In this step, exhibiting cell adhesion inhibition means that cells are difficult to adhere and spread and have a low cell adhesion extension rate. In this step, specifically, such a state where the cell adhesion extension rate is low can be a state where the cell adhesion extension rate is 5% or less. In this step, the content is preferably 2% or less. This is because cell adhesion can be prevented stably.

The cell adhesion extension rate in this step is the ratio of the cells that have adhered and spread when seeded with cells to be cultured at a predetermined density and cultured for 14.5 hours under conditions suitable for the above-mentioned cell culture ({(adhesion Number of cells) / (number of cells seeded)} × 100 (%)).
Specifically, when the cells to be cultured are bovine vascular endothelial cells, the cells are seeded so that the seeding density is in the range of 4000 cells / cm ² or more and less than 30000 cells / cm ² , and in a 37 ° C. incubator (CO ₂ concentration 5 %), And the percentage of cells that have adhered and extended when cultured for 14.5 hours can be evaluated. In the case of bovine vascular endothelial cells, the cells were seeded by suspending in a DMEM medium containing 10% FBS (serum), and then seeding the cells so that the cells were distributed as uniformly as possible. This is done by shaking the substrate slowly.
Furthermore, the measurement of the cell adhesion extension rate is performed after exchanging the medium immediately before the measurement to remove the non-adhered cells. In addition, the cell adhesion extension rate is measured at a location where the cell density is likely to be specific (for example, the center of a predetermined area where the density is likely to be high and the periphery of the predetermined area where the density is likely to be low). To measure.

Moreover, having cell adhesiveness means that the cells are easy to adhere and spread and have a high cell adhesion extension rate. In this step, specifically, such a state where the cell adhesion extension rate is high can be a state where the cell adhesion extension rate is 60% or more.
In this step, it is preferably 80% or more. This is because cells can be stably adhered as the cell adhesion region.

The polymer substrate used in this step has a cell adhesion-inhibiting surface, and the degree of such cell adhesion inhibition is uniquely determined by the polymer material constituting the polymer substrate. It is not a thing, but is different for each cell. Further, even when the same material is used as the polymer material, there is a possibility that the cell adhesion inhibitory property is not exhibited due to the influence of surface roughness, impurities, and the like.
For this reason, the polymer substrate used in this step is subjected to the evaluation method using the above-mentioned cell adhesion extension rate for each cell, and has a desired cell adhesion inhibitory surface and the desired cell adhesion by energy irradiation. It is preferably selected after confirming that a cell adhesion region having a property can be formed.

The polymer material constituting the polymer substrate used in this step is not particularly limited as long as it is surface-modified by performing energy irradiation and can form a cell adhesion region. And those having the property that the water contact angle is lowered by energy irradiation. Specific examples of such a polymer material include polyester, polystyrene, polycarbonate, polyethylene terephthalate, and the like. Among these, polystyrene resin or polyethylene terephthalate resin is preferable. This is because, when a polymer substrate is formed using such a polymer material, it is easy to have a cell adhesion-inhibiting surface. Further, polystyrene is a material having low cytotoxicity. In addition, polyethylene terephthalate resin is available at a low cost and is a material suitable for mass production.
In this step, polystyrene can be particularly preferably used. This is because by using the above-described polymer material, a cell adhesion-inhibiting surface can be easily formed, and the cytotoxicity is low. Furthermore, since the glass transition point is low, thermal imprinting is easy, and it is easy to mold fine irregularities as described later.
The property that the water contact angle decreases in this step indicates that the water contact angle changes as a result of energy irradiation, and only the property that the water contact angle changes (change in hydrophilicity / hydrophobicity). As a result, it also includes the property that the water contact angle decreases, for example, the property that a functional group containing oxygen or the like is introduced by energy irradiation.
Therefore, when the cell adhesion region is formed using a material having a property of reducing the water contact angle, the cell adhesion region is formed with a reduced water contact angle, the above functional group is introduced, etc. As a result, the water contact angle is reduced, and those formed by mixing them are included.
In addition, the polymer material in this step may be a copolymer containing a component such as rubber so as to be easily handled in a roll form so as not to hinder cell culture. Further, other necessary additives may be included.

The water contact angle on the surface of the polymer substrate before energy irradiation in this step is not particularly limited as long as it shows a desired cell adhesion inhibitory state before energy irradiation, but is 80 ° or more. It is preferable that This is because, when the water contact angle is within the above range, the cell adhesion inhibitory property can be stably obtained. Moreover, it is because a cell adhesion area | region can be easily formed by energy irradiation.
In addition, the water contact angle can use the value measured using the contact angle measuring device 30 seconds after dripping water from a micro syringe under the temperature of 25 degreeC, 30% of humidity, and atmospheric pressure. Moreover, as a contact angle measuring device, Kyowa Interface Science Co., Ltd. CA-Z type can be used, for example.

  When the polymer substrate used in this step is irradiated with energy, that is, when this step is performed and the cell adhesion region is formed, the water contact angle is not particularly limited as long as it exhibits a desired cell adhesion. Although not particularly limited, it is preferably 60 ° or less. This is because, when the water contact angle upon energy irradiation is within the above range, cell adhesion can be stably achieved. Moreover, it is because the adhesion difference with the cell between the cell adhesion inhibition area | region formed by this process, ie, the area | region which is not irradiated with energy, can be enlarged.

  About the polymeric base material used for this process, it may be unstretched and what is formed by uniaxial stretching, successive biaxial stretching, and simultaneous biaxial stretching can be used.

  The shape of the polymer substrate used in this step is not particularly limited as long as it can form a pattern of a desired cell adhesion region and a cell adhesion inhibition region and can stabilize cell culture. It may be a shape or a shape having a step such as a container.

Regarding the thickness when the shape of the polymer substrate in this step is flat, the cell adhesion region and the cell adhesion inhibition region formed by this step stably exhibit cell adhesion and cell adhesion inhibition, respectively. It is not particularly limited as long as it can be used, and may be a plate shape, but a sheet shape that can be bent into a roll shape is preferable. This is because this step can be performed by a roll-to-roll process, that is, mass production is possible.
Specifically, such a sheet-like thickness is appropriately set according to the polymer material to be used, but is preferably in the range of 1 μm to 500 μm, and more preferably 50 μm to 200 μm. It is preferable to be within the range. This is because it is easy to form a roll.

In addition, the mode in which the polymer substrate in this step is a sheet is particularly limited as long as a desired cell adhesion region and cell adhesion inhibition region pattern can be formed and cell culture can be stabilized. It may be a single wafer shape, but is preferably a long shape.
Due to the long shape, it is possible to increase the degree of freedom in the manufacturing process of the cell culture substrate, such as storing in a roll shape, or performing this step while unwinding from the state stored in the roll shape. Because it can.
Moreover, even when forming a stimulus-responsive layer as described later or a concavo-convex shaped one, these can be formed by roll-to-roll and can be formed easily and in large quantities. Because it can be.
Furthermore, the final cell culture substrate can be stored in a roll shape as it is long, or the size can be determined according to the purpose of cell culture, etc. This is because the cell culture substrate can be cut out and used, and the degree of freedom in the process and method of using the cell culture substrate can be increased.
Here, the long shape means that the length is long enough to be wound up in a roll shape, and may be arbitrarily determined according to the weight that can be installed in the manufacturing apparatus. However, specifically, the length is preferably 10 m or more, particularly preferably in the range of 50 m to 5000 m, and particularly preferably in the range of 100 m to 1000 m.
Moreover, it is preferable that length is 10 times or more with respect to a width | variety. It is because it can be made excellent in handleability and the like.

Examples of the step in the shape of the polymer substrate having a step in this step include a concave cell culture region, a microchannel, and the like. In this step, since the cell adhesion region can be formed only by energy irradiation, the cell adhesion region can be formed with high accuracy even if it has such a step.
In this step, examples of the polymer substrate on which the concave cell culture region is formed include a container such as a microwell plate.

The polymer base material in this step may be porous. In addition, the thickness, pore size, pore density, and the like of the polymer base material in the case of a porous shape affect the passage time of the medium, and thus are appropriately set from a range suitable for the intended cell culture insert.
For example, when a porous polymer substrate is used for preparation of cell sheets, co-culture of two types of cells, drug permeation assay, etc., the pore size should be φ20 μm or less to the extent that cells do not migrate or infiltrate. Is preferred.

The polymer substrate used in this step may have a pressure-sensitive adhesive layer on the surface opposite to the surface modified by energy irradiation.
By having such a pressure-sensitive adhesive layer, the above-mentioned polymer base material is attached to a separately prepared support substrate, and this step is performed in the attached state, or the cell culture medium produced by the production method of the present invention. This is because the material can be easily attached to the support substrate.
The pressure-sensitive adhesive layer used in this step is not particularly limited as long as the desired adhesiveness can be exhibited, and a layer formed by applying a known pressure-sensitive adhesive can be used. Specific examples include those containing an adhesive such as polyester, acrylic ester, polyurethane, polyethyleneimine, silane coupling agent, perfluorooctane sulfonic acid (PFOS), and polyester, acrylic ester, polyurethane, among others. It is preferable that it contains adhesives, such as. This is because the polymer base material and the support substrate can be bonded with sufficient strength.

  Further, the support substrate used in this step is not particularly limited as long as it can support the polymer base material, and examples thereof include a container generally used for cell culture. Specifically, a plate such as a multiwell plate, a culture medium such as a dish, a petri dish, a flask, or a bottle can be stored, and a container in which cells can be cultured can be given.

(2) Cell adhesion region forming step This step involves irradiating the surface of the polymer substrate having a cell adhesion-inhibiting surface with energy, and modifying at least a part of the surface of the polymer substrate, This is a step of forming a cell adhesion region having cell adhesion.

The shape of the cell adhesion region formed by this step can be appropriately set according to the application, etc., for example, rectangular shape, circular shape, polygonal shape, line shape, branch shape, mesh shape, etc. Various shapes can be used.
In this step, when it is used for cell proliferation, it is preferably rectangular, circular, or polygonal, and in particular, when it is used for applications such as orienting cells, it should be linear. Is preferred.

  The energy irradiated in this step is not particularly limited as long as the cell adhesion region can be formed by performing it on the cell adhesion-inhibiting surface of the polymer substrate. Can include ultraviolet (UV) light, VUV light, plasma, and the like.

In this step, as a method of irradiating energy to a region that forms a cell adhesion region of a polymer substrate, that is, a method of irradiating energy only to a region that forms a cell adhesion region, A method of performing energy irradiation through a mask having an opening (mask method), a protective layer having an opening in a region where a cell adhesion region is formed on a polymer substrate, and energy irradiation through the protective layer (Protective layer method) can be used, and among these, the protective layer method can be preferably used. This is because by laminating the protective layer on the polymer base material, it becomes easy to irradiate energy immediately before seeding cells to form a cell adhesion region. In addition, as a result, it is possible to prevent problems such as changes in the pattern over time as compared to the case where the cell adhesion region is formed in advance, and it becomes possible to culture the cells with higher pattern accuracy. .
In addition, when this process is performed in the state in which the said polymer base material was affixed on the support substrate etc., the said protective layer can also be affixed on a support substrate etc. and can be used. Specifically, as illustrated in FIG. 2, when the support substrate 31 is a container 30 with a lid 34, this step is performed in a state where the protective layer 3 is attached to the lid 34 as a light shielding portion and the cell culture container 30 for pattern formation is formed. Can also be done. Further, the present step is not limited to the example shown in FIG. 2, and the present step may be performed by attaching the protective layer 3 as a light shielding portion inside the lid 34.

  The mask used in this step is not particularly limited as long as it can prevent the modification of the surface of the polymer base material from energy irradiation, and etching or laser processing of a metal substrate such as SUS. Alternatively, a light-shielding film made of an emulsion (silver salt) or chromium may be provided on a substrate patterned by electroforming, or on a substrate made of soda lime glass or quartz.

The protective layer used in this step is not particularly limited as long as it can prevent modification of the surface of the polymer substrate from energy irradiation and can be laminated on the surface of the polymer substrate. And those made of a light-shielding material.
As such a light-shielding material, it is preferable that the surface of the polymer substrate has adhesiveness until the end of the cell adhesion region forming step and can be easily peeled off during cell culture. This is because the use of a pressure-sensitive adhesive or the like can be avoided during lamination with the polymer base material or the like.
In this step, such a light-shielding material is appropriately selected according to the polymer material constituting the polymer base material. For example, a black colorant is dispersed in the binder resin. And metal thin films such as chromium and chromium oxide, and silicone resins such as PDMS (polydimethylsiloxane).
In this step, in particular, when the energy irradiation is UV irradiation or VUV irradiation, a black colorant dispersed in a binder resin, a metal thin film, or the like can be preferably used. In the case of irradiation, a silicone resin such as PDMS can be preferably used.

  The black colorant used in this step is not particularly limited as long as it can shield energy, and examples thereof include carbon fine particles and metal oxides.

  The binder resin used in this step is not particularly limited as long as it can stably disperse and retain the black colorant. For example, acrylate-based, methacrylate-based, polyvinyl cinnamate Negative photosensitive resin having a reactive vinyl group such as a cyclized rubber system, polymethyl methacrylate resin, polyacrylate resin, polycarbonate resin, polyvinyl alcohol resin, polyvinyl pyrrolidone resin, hydroxyethyl cellulose resin, carboxymethyl cellulose resin, Examples thereof include a polyvinyl chloride resin, a melamine resin, a phenol resin, an alkyd resin, an epoxy resin, a polyurethane resin, a polyester resin, a maleic acid resin, and a polyamide resin.

  The opening formed in the mask or protective layer in this step is not particularly limited as long as it is formed in the region where the cell adhesion region is formed and can form the cell adhesion region with high accuracy. When the irradiation is UV irradiation or VUV irradiation, it is preferably a through hole. When the energy irradiation is plasma irradiation, as shown in FIG. It may be a non-through hole that has a space at a site to be formed.

In this step, the method for irradiating the polymer substrate with energy is particularly limited as long as the polymer substrate can be stably irradiated with energy and can form a cell adhesion region with high accuracy. However, when the polymer base material is long, energy irradiation may be performed after cutting into a predetermined size from the state wound in a roll shape. It is preferable that the energy irradiation is performed while the polymer base material is unwound and conveyed from the state wound in a roll shape. Moreover, the polymer base material after energy irradiation may be wound up again in a roll shape, or may be cut into a single sheet.
Moreover, energy irradiation may be performed on the polymer base material alone, or energy irradiation may be performed after the polymer base material is bonded to a separately prepared support substrate.
In addition, about the code | symbol in FIG. 4, since it shows the same member as the thing of FIG. 1, description here is abbreviate | omitted.

2. Method for Producing Cell Culture Substrate The method for producing the cell culture substrate of the present invention is not particularly limited as long as it has at least the cell adhesion region forming step, but other steps may be performed as necessary. You may have.
Examples of such other processes include a stimuli-responsive layer forming process for forming a stimulus-responsive layer on the surface of the polymer substrate after the cell adhesion region forming process, and a fine uneven shape on the surface of the polymer substrate. And the like. In addition, evaluation of cell adhesion inhibition and the like is performed on a substrate made of a polymer material, and it is confirmed that the cell has a desired cell adhesion inhibition property and formability of a cell adhesion region. Or a protective layer forming step of forming a protective layer having an opening in a region where the cell adhesion region is formed on the polymer substrate before the cell adhesion region forming step, and the polymer substrate is modified by energy irradiation. It may have a pressure-sensitive adhesive layer forming step for forming a pressure-sensitive adhesive layer on the surface opposite to the surface.

(1) Stimulus responsive layer forming step The stimulus responsive layer forming step in the present invention is a step of forming a stimulus responsive layer on the surface of the polymer substrate after the cell adhesion region forming step.
By having this step, it is possible to easily have a pattern of a cell adhesion region and a cell adhesion inhibition region without patterning the stimulus responsive layer and have stimulus responsiveness.

  The stimulus-responsive layer formed by this step is formed on the polymer substrate on which the cell adhesion region and the cell adhesion-inhibiting region are formed, and the stimulus-responsive layer on the cell adhesion region is provided by applying a stimulus. Is capable of exhibiting cell adhesion inhibitory properties. Specific examples of such a stimulus responsive layer include those containing a stimulus responsive material having stimulus swellability and cell non-adhesiveness.

By including such a stimulus-responsive material, the cell shows a contracted state before the stimulus is applied during cell culture, and cells are affected by the cell adhesion region on the surface of the polymer substrate exposed from the contracted stimulus-responsive material. After adhesion and stimulation, it shows a swollen state and peels cells from the polymer substrate and / or covers the surface of the polymer substrate, thereby affecting the influence of the cell adhesion region on the polymer substrate surface. Can be reduced and cells can be detached.
For this reason, when a substrate coated with a cell adhesive material such as collagen is brought into contact with the cells after applying such stimulation, the cells can be easily collected.

In this step, having the stimulus swelling property means having the property of changing the property of absorbing and swelling water after the application of the stimulus. Specifically, the volume change (after stimulation / before stimulation) during swelling before and after stimulation is greater than 1.
In addition, the volume change before and after stimulation by stimulation is the volume before stimulation of the volume of the stimulation-responsive layer after being left in a cell culture solution at 37 ° C. for 24 hours, and then suitable for each stimulation-responsive material. The volume of the stimulus-responsive layer after the stimulus is continuously applied for 24 hours can be determined as the volume after stimulation by the ratio of the volume after stimulation / the volume before stimulation.

  The stimulus-responsive material used in this step is not particularly limited as long as it has cell adhesion inhibition and stimulus-swelling properties. For example, a temperature-responsive material that swells due to a temperature change, or by light irradiation. Examples include a photoresponsive polymer that swells, a magnetically responsive polymer that swells due to magnetic force, and a potential responsive polymer that changes in potential.

Specific examples of the temperature-responsive material used in this step include poly-N-isopropylacrylamide (PIPAAm), poly-Nn-propylacrylamide, poly-Nn-propylmethacrylamide, poly-N- Examples thereof include ethoxyethyl acrylamide, poly-N-tetrahydrofurfuryl acrylamide, poly-N-tetrahydrofurfuryl methacrylamide, and poly-N, N-diethyl acrylamide. Among them, PIPAAm, poly-Nn- Propylmethacrylamide and poly-N, N-diethylacrylamide can be preferably used, and PIPAAm can be particularly preferably used. This is because it exhibits an aggregated state at a temperature suitable for culturing cells, exhibits cell adhesion, and swells within a temperature range with little damage to the cells and exhibits cell detachability. For this reason, the cell sheet can be formed and peeled off without adversely affecting the cells.
In this step, the temperature-responsive material may be composed of only one kind of compound or may contain two or more kinds.

  The photoresponsive material used in this step is not particularly limited as long as it swells with or without light irradiation. For example, azobenzene as disclosed in JP-A-2005-210936, Those containing photoresponsive components such as diarylethene, spiropyran, spirooxazine, fulgide and leuco dyes can be used.

  The potential responsive material used in this step is not particularly limited as long as it swells by application of a potential. For example, as disclosed in JP 2008-295382 A, There may be mentioned those having a cell-adhesive moiety such as a peptide containing an RGD sequence and a spacer substance such as alkanethiol, cysteine, and alkanedisulfide that binds to the electrode surface via thiolate.

  The magnetic force responsive material used in this step is not particularly limited as long as it swells by applying and removing magnetic force. For example, a ferrite as disclosed in JP-A-2005-312386 For example, positively charged liposomes encapsulating magnetic particles in which positively charged liposomes are encapsulated.

  In this step, among such stimulus-responsive materials, a temperature-responsive material is preferable, and polyisopropylacrylamide (PIPAAm), which is a polymer of isopropylacrylamide (IPAAm), is particularly preferable. This is because when the stimulus-responsive layer is a temperature-responsive layer containing a temperature-responsive material, it is easy to apply a stimulus. In addition, PIPAAm has a lower critical temperature of 32 ° C., and has less influence on the cells at a temperature at which the degree of cell adhesion is lowered to detach the cells.

The PIPAAm film as the temperature-responsive layer is considered to behave as follows.
That is, at a temperature higher than the lower critical temperature, the polymer in the membrane is dehydrated and the polymer contracts, while at a temperature lower than the lower critical temperature, the polymer in the membrane has an affinity for the surrounding water. It is considered that the polymer hydrates and the polymer swells. At this time, when the temperature is higher than the lower critical temperature, the cells adhere due to the influence of the cell adhesion region on the surface of the polymer substrate exposed from the contracted polymer. Cells are detached from the PIPAAm membrane with little influence from the cell adhesion area of the material. Regarding cell adhesiveness, the state of the surface of the polymer substrate is considered to have an effect. By forming a PIPAAm film on a polymer substrate on which a cell adhesion region and a cell adhesion inhibition region are formed in a pattern as in the present invention, the cell adhesion region has cell adhesion at a temperature higher than the lower critical temperature. In the cell adhesion inhibition region, cell adhesion inhibition can be imparted regardless of the lower critical temperature. Therefore, cells cultured by controlling the number and shape can be collected with minimal invasiveness. Furthermore, since the PIPAAm film is formed on the surface of the substantially flat polymer base material, the cell sheet cultured on the PIPAAm film can be easily contacted with the substrate on which collagen is formed on the surface. Recovery can be performed.

In this step, the method for forming the stimulus responsive layer on the surface of the polymer substrate is not particularly limited as long as the method can stably form the stimulus responsive layer on the surface of the polymer substrate. However, for example, a method of laminating a separately formed stimulus-responsive layer on the surface of the polymer substrate (lamination method) or a coating solution for a stimulus-responsive layer containing a stimulus-responsive material on the surface of the polymer substrate A method for applying, drying and curing (coating method), or applying a composition for a stimulus-responsive layer containing a monomer component capable of forming a stimulus-responsive material to the surface of the polymer substrate. Examples thereof include a method (polymerization method) of polymerizing monomer components using the surface as a polymerization initiation point. In this step, among these, a polymerization method or a coating method is preferable. This is because the use of an adhesive or the like can be avoided for adhesion between the stimulus-responsive layer and the polymer substrate. In this step, a polymerization method is particularly preferable. This is because the stimulus-responsive layer can be formed with high thickness accuracy, and can be made excellent in control of swelling property to stimuli, that is, controllability of cell adhesion inhibition expressed by the stimulus.
In this step, when the stimulus responsive layer is formed by a polymerization method in a continuous process such as roll-to-roll, the composition for the stimulus responsive layer preferably contains an oligomer or prepolymer of a monomer component. . By including such an oligomer or prepolymer that has been polymerized to some extent, graft polymerization can be performed with a smaller number of radiation irradiations, and production efficiency can be improved.
Further, when the stimulus responsive layer is formed by a polymerization method, the stimulus responsive layer composition may include an anchor agent such as a polymerization initiator or a silane coupling agent. In this step, in particular, when polymerization is initiated by ultraviolet irradiation, it is preferable to combine a polymerization initiator and an anchor agent.
In this step, as a specific example of forming the stimulus responsive layer by a polymerization method, for example, when the stimulus responsive layer is made of a temperature responsive material and PIPAAm is used as the temperature responsive material, a monomer N-isopropylacrylamide (NIPAAm) can be used as a component (stimulus responsive monomer).

  FIG. 5 is an explanatory diagram showing an example of a method for forming a stimulus-responsive layer by a lamination method. FIG. 5 shows a method of laminating a separately formed stimulus-responsive layer 4 on the surface of the polymer substrate 1. Moreover, FIG. 6 is explanatory drawing which shows an example of the method of forming a stimulus responsive layer by the apply | coating method. In FIG. 6, the stimulus-responsive layer coating solution is applied to the surface of the polymer substrate 1 (FIG. 6A), and the coating film 4 ′ is dried and cured (FIG. 6B). The stimulus responsive layer 4 is formed (FIG. 6C). FIG. 7 is an explanatory diagram showing an example of a method for forming a stimulus-responsive layer by a polymerization method. In FIG. 7, a composition for a stimulus-responsive layer containing a monomer component capable of forming a stimulus-responsive material is applied to the surface of the polymer substrate 1 (FIG. 7A), and applied to the coating film 4 ″. On the other hand, by irradiating radiation such as ultraviolet rays or electron beams (FIG. 7B), a method of forming the stimulus-responsive layer 4 by polymerizing the monomer component using the surface of the polymer substrate 1 as a polymerization start point. (FIG. 7C).

  The composition for a stimulus-responsive layer used in this step contains the monomer component, but usually contains a photopolymerization initiator and a solvent.

  The photopolymerization initiator contained in the composition for the stimulus-responsive layer used in this step is particularly limited as long as it can polymerize the stimulus-responsive monomer by light irradiation to form a stimulus-responsive material. However, it is possible to use, for example, 2-hydroxy-2-methyl-propiophenone, benzophenone, Michler's ketone, 4,4'-bisdiethylaminobenzophenone, 4-methoxy-4'-dimethylamino Aromatic ketones such as benzophenone, 2-ethylanthraquinone and phenanthrene, benzoin ethers such as benzoin methyl ether, benzoin ethyl ether and benzoin phenyl ether, benzoin such as methyl benzoin and ethyl benzoin, 2- (o-chlorophenyl) -4, 5-Phenylimidazole dimer 2- (o-chlorophenyl) -4,5-di (m-methoxyphenyl) imidazole dimer, 2- (o-fluorophenyl) -4,5-diphenylimidazole dimer, 2- (o-methoxy) Phenyl) -4,5-diphenylimidazole dimer, 2,4,5-triarylimidazole dimer, 2- (o-chlorophenyl) -4,5-di (m-methylphenyl) imidazole dimer, 2-benzyl-2-dimethylamino-1- (4-morpholinophenyl) -butanone, 2-trichloromethyl-5-styryl-1,3,4-oxadiazole, 2-trichloromethyl-5- (p- Halomethylthiayl such as cyanostyryl) -1,3,4-oxadiazole, 2-trichloromethyl-5- (p-methoxystyryl) -1,3,4-oxadiazole Compound, 2,4-bis (trichloromethyl) -6-p-methoxystyryl-S-triazine, 2,4-bis (trichloromethyl) -6- (1-p-dimethylaminophenyl-1,3- Butadienyl) -S-triazine, 2-trichloromethyl-4-amino-6-p-methoxystyryl-S-triazine, 2- (naphth-1-yl) -4,6-bis-trichloromethyl-S-triazine, 2- (4-Ethoxy-naphth-1-yl) -4,6-bis-trichloromethyl-S-triazine, 2- (4-butoxy-naphth-1-yl) -4,6-bis-trichloromethyl- Halomethyl-S-triazine compounds such as S-triazine, 2,2-dimethoxy-1,2-diphenylethane-1-one, 2-methyl-1- [4- (methylthio) phenyl 2-morpholinopropanone, 1,2-benzyl-2-dimethylamino-1- (4-morpholinophenyl) -butanone-1,1-hydroxy-cyclohexyl-ketone, benzyl, benzoylbenzoic acid, benzoylbenzoic acid Methyl, 4-benzoyl-4'-methyldiphenyl sulfide, benzylmethyl ketal, dimethylaminobenzoate, isoamyl P-dimethylaminobenzoate, 2-n-butoxyethyl-4-dimethylaminobenzoate, 2-chlorothioxanthone, 2,4 diethyl Thioxanthone, 2,4 dimethylthioxanthone, isopropylthioxanthone, ethanone, 1- [9-ethyl-6- (2-methylbenzoyl) -9H-carbazol-3-yl] -1- (o-acetyloxime), 4-benzoyl Methyldiphenyl sulfide, 1-hydroxy-cyclohexyl-phenyl ketone, 2-benzyl-2- (dimethylamino) -1- [4- (4-morpholinyl) phenyl] -1-butanone, 2- (dimethylamino) -2- [(4-Methylphenyl) methyl] -1- [4- (4-morpholinyl) phenyl] -1-butanone, α-dimethoxy-α-phenylacetophenone, phenylbis (2,4,6-trimethylbenzoyl) phosphine Oxide, 2-methyl-1- [4- (methylthio) phenyl] -2- (4-morpholinyl) -1-propanone, 1,2-octadione, 1- [4- (phenylthio) phenyl]-, 2- ( o-benzoyloxime)] and the like. In this step, these photopolymerization initiators can be used alone or in admixture of two or more.

  The solvent used in this step is not particularly limited as long as it can uniformly disperse or dissolve the above components. For example, diethyl ether, tetrahydrofuran, dioxane, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, propylene Ethers such as glycol dimethyl ether and propylene glycol diethyl ether; glycol monoethers such as ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether (so-called Cellosolves); methyl ethyl ketone, acetone, methyl isobutyl ketone Ketones such as ethyl, cyclopentanone, cyclohexanone; ethyl acetate, butyl acetate, n-propyl acetate, i-propyl acetate, n-butyl acetate, i-butyl acetate, acetate esters of the above glycol monoethers (for example, methyl Cellosolve acetate, ethyl cellosolve acetate), esters such as methoxypropyl acetate, ethoxypropyl acetate, dimethyl oxalate, methyl lactate, ethyl lactate; alcohols such as ethanol, propanol, butanol, hexanol, cyclohexanol, ethylene glycol, diethylene glycol, glycerin Methylene chloride, 1,1-dichloroethane, 1,2-dichloroethylene, 1-chloropropane, 1-chlorobutane, 1-chloropentane, chlorobenzene, bromobenzene, o- Halogenated hydrocarbons such as chlorobenzene and m-dichlorobenzene; Amides such as N, N-dimethylformamide, N, N-diethylformamide, N, N-dimethylacetamide and N, N-diethylacetamide; N-methylpyrrolidone Pyrrolidones such as lactones; lactones such as γ-butyrolactone; sulfoxides such as dimethyl sulfoxide; other organic polar solvents; and further, aromatic hydrocarbons such as benzene, toluene, xylene, and others And organic nonpolar solvents. These solvents are used alone or in combination.

  Examples of the silane coupling agent used in this step include methacryloxy silane, vinyl silane, amino silane, and epoxy silane. Among them, methacryloxy silane can be preferably used.

  The content of the monomer component and photopolymerization initiator used in this step in the composition layer for the stimulus-responsive layer is particularly limited as long as it can form a stimulus-responsive material having a desired degree of polymerization. Instead, it is appropriately set according to the polymerization conditions and the like.

  The thickness of the composition layer for the stimulus-responsive layer in this step is not particularly limited as long as the stimulus-responsive monomer can form a stimulus-responsive material polymerized at a desired degree of polymerization. It is appropriately set according to the composition of the composition for the sex layer and the cells to be cultured.

  The method for applying the composition for stimuli-responsive layer in this step is not particularly limited as long as it can form a coating film having a desired thickness by applying the above composition. Spin coating or die coating A known coating method such as, for example, can be used. Moreover, when apply | coating with respect to a polymeric substrate of a large area, it is preferable to use application methods, such as blade coating, gravure coating, and offset gravure coating.

The polymerization method of the monomer component in this step is not particularly limited as long as it is a method capable of stably forming the stimulus-responsive material. For example, for the coating film of the stimulus-responsive layer composition, Thus, a method of irradiating with an electron beam or ultraviolet rays can be used.
In this step, after the polymerization of the monomer components, a removal treatment for removing unreacted monomer components and the like by washing may be performed.

  As the shape of the stimulus-responsive layer formed by this step, one that covers the entire surface of the polymer substrate is usually used, but it is formed in a pattern on the polymer substrate. May be.

  The film thickness of the stimulus responsive layer formed by this step is not particularly limited as long as it can exhibit stimulus responsiveness, and specifically, within a range of 0.5 nm to 300 nm. It is preferable that it is in the range of 1 nm-100 nm especially.

(2) Fine uneven shape shaping step The fine uneven shape shaping step in the present invention is a step of shaping the fine uneven shape on the surface of the polymer substrate.
By having this step, for example, orientation control of cells cultured on the cell culture substrate can be easily performed.

The fine uneven shape formed on the surface of the polymer substrate by this step is not particularly limited as long as it is recognized by cells and affects the function of the cells.
Specific examples of such fine concavo-convex shapes include those that control cell orientation (fine concavo-convex shape for orientation control), those that control differentiation (fine concavo-convex shape for differentiation control), and function maintenance control. Examples thereof include those having fine concavo-convex shape for function maintenance control, and those performing form / proliferation control (fine concavo-convex shape for form / proliferation control).

The fine uneven shape for orientation control in this step is not particularly limited as long as the cells can be oriented in a desired direction. For example, a recognizable concave portion is arranged in a line shape. An uneven structure can be mentioned.
When cells are seeded on a cell culture substrate on which a concavo-convex structure in which concave portions having a predetermined width are arranged in a line is formed, many cells can be oriented because the cells recognize the concavo-convex structure.

  FIG. 8 is a schematic plan view showing an example when cell culture is performed on a cell culture substrate on which fine irregularities for orientation control are formed in this step. FIG. 9 is a sectional view taken along line AA in FIG. As shown in FIG. 8 to FIG. 9, when an uneven structure having a width that allows one cell to enter is formed as the fine uneven shape for orientation control, the cells are aligned along the concave portion of the fine uneven shape. The major axis direction can be oriented.

Specifically, the fine uneven shape for orientation control in this step is appropriately set according to the type of cells to be cultured, but when the cells to be seeded are cardiomyocytes, The shape is preferably a shape capable of recognizing the groove and maintaining the orientation even when moved by pulsation.
Specifically, the width of the concave portion in the concavo-convex shape capable of recognizing the groove and maintaining the orientation even when moved by such pulsation is preferably within a range of 5 μm to 50 μm, and in particular, 5 μm to 30 μm. It is preferable to be within the range. Further, the depth of the recess is preferably in the range of 0.2 μm to 10 μm, and more preferably in the range of 1 μm to 7 μm.

The fine concavo-convex shape for differentiation control in this step is not particularly limited as long as it can affect cell differentiation. For example, mechanical stress that contributes to at least one cell differentiation control is applied. An example of such an uneven structure is given.
Specifically, such a concavo-convex structure is appropriately set according to the type of cell and the differentiation state to be controlled. For example, in the literature (Control of stem cell fates by physical interactions with the extracellular matrix. Cell Stem Cell., 2009, 5, 17-26), a human mesenchymal stem cell seeded on an island-like fibronectin scaffold, It has been reported that differentiation is induced to osteoblasts when cells adhere to and spread on the scaffold, and adipocytes when cells adhere to the scaffold but do not spread. That is, as the surface to which human mesenchymal stem cells can adhere is reduced, the rate of differentiation into osteoblasts decreases, and conversely, the rate of differentiation into adipocytes increases.
In this step, for example, in order to induce differentiation of human mesenchymal stem cells into osteoblasts, the shape and / or size that allows cells to adhere and extend as the above-described fine uneven shape for differentiation control, Specifically, the area is three or more times the cell size, and more specifically, a square of 100 μm × 100 μm or the like is preferable.
On the other hand, when inducing the differentiation of human mesenchymal stem cells into adipocytes, a part of the cells adhere, but the shape and / or size that cannot be extended, specifically, the cell size 1 The area is preferably less than double, and more specifically, a square of 10 μm × 10 μm is preferable.
Note that the above is an example, and the planar shape of the fine unevenness may be appropriately set according to the type and characteristics of the cells.

The method for shaping the fine uneven shape in this step is not particularly limited as long as it is a method capable of forming a desired fine uneven shape. For example, in the heated state of the polymer substrate, the fine uneven shape A thermal imprinting method in which a mold having a fine irregular shape of the inverted shape is pressed against a polymer base material can be preferably used.
The degree of heating in this step is not particularly limited as long as the fine uneven shape can be transferred by pressing the mold, but usually the glass transition point of the polymer material constituting the polymer substrate. This can be done.
In addition, the heating method in this step is not particularly limited as long as a desired fine uneven shape can be shaped. For example, a method using a heated mold or a heating apparatus such as an oven is used. A method can be mentioned.

FIG. 10 is an explanatory diagram for explaining an example of a method for forming a fine uneven shape by a thermal imprint method. As shown in FIG. 10, the heated mold 21 is pressed against the polymer base material 1 before the cell adhesion region forming step (FIG. 10 (a)) and molded (FIG. 10 (b)). To (c)).
In this example, a roll-shaped mold is used as the mold, and a method of continuously shaping is shown.

The shape of the mold used in this step is not particularly limited as long as a desired fine uneven shape can be formed on the surface of the polymer substrate, and may be plate-shaped or roll-shaped. There may be.
In this step, when the polymer base material is long, it is preferably a roll. This is because a fine irregular shape can be continuously formed on the surface of the polymer substrate, and this step can be performed in a short time.

  The timing of performing this step may be before or after the cell adhesion region forming step, before or after the stimulus responsive layer forming step, but preferably before the cell adhesion region forming step. . This is because the cell adhesion region having the fine uneven shape can be stably formed.

(3) Polymer substrate selection step As the polymer substrate selection step in the present invention, cell adhesion inhibition and the like are evaluated on a polymer substrate made of a polymer material, and desired cell adhesion and cell This is a step of confirming and selecting the adhesive region forming ability.
By having this step, it is possible to stably form a cell adhesion region and a cell adhesion inhibition region suitable for cells.
The evaluation method for cell adhesion inhibition and the like in this step can be the same as the evaluation method using the adhesion extension rate described in the above section “1. Cell adhesion region forming step”. Description is omitted.

(4) Protective layer formation process The protective layer formation process in this invention forms the protective layer which has an opening part in the area | region which forms a cell adhesion area | region on the said polymer base material before the said cell adhesion area | region formation process. It is a process.
By having this step, the execution timing of the cell adhesion region forming step can be easily performed immediately before cell seeding. Moreover, the area | region (cell adhesion inhibition area | region) which has not received energy irradiation can be prevented from being exposed by maintaining the state which laminated | stacked the said protective layer after the said cell adhesion area | region formation process. Therefore, it is possible to prevent the pattern of the cell adhesion region from changing over time from the cell adhesion region formation step to the seeding of the cells, and to cultivate the cells in a desired pattern.

The method for forming the protective layer in this step is not particularly limited as long as it can stably laminate a protective layer having a desired thickness. For example, the protective layer is made of a light shielding material. In some cases, a protective layer-forming coating solution containing the light-shielding material is applied to form a protective layer-forming layer, or a protective layer-forming layer that is separately formed is laminated on the polymer substrate, and then opened by patterning. And a method of preparing a mesh sheet-like protective layer in which an opening is formed in advance and laminating on a polymer substrate.
As the patterning method, a known method such as a photolithography method can be used.
In this step, in particular, when the polymer base material is long, a method of laminating the protective layer forming layer and using a photolithography method using a photomask can be preferably used. . This is because the protective layer can be formed by roll-to-roll, and mass production can be facilitated. In the case of patterning by a photolithography method, the protective layer forming layer preferably has a negative or positive photosensitivity. This is because a separate layer of a resist or the like is not required and a patterned protective layer can be easily formed.

(5) Pressure-sensitive adhesive layer forming step The pressure-sensitive adhesive layer forming step in the present invention is a step of forming a pressure-sensitive adhesive layer on the surface opposite to the surface modified by energy irradiation of the polymer substrate.
The pressure-sensitive adhesive layer formed by this step can be the same as that described in the above section “1. Polymer substrate”.
In this step, the pressure-sensitive adhesive layer can be formed by applying a pressure-sensitive adhesive layer-forming coating solution containing the pressure-sensitive adhesive contained in the pressure-sensitive adhesive layer or by separately forming a pressure-sensitive adhesive layer on the polymer substrate. It is possible to use a method of laminating.
The coating method is not particularly limited as long as it can form a pressure-sensitive adhesive layer having a uniform thickness, and a known coating method can be used. Specifically, the method described in the section “(1) Stimulus responsive layer forming step” can be used.

(6) Others The method for producing a cell culture substrate of the present invention includes the cell adhesion region forming step and the like. When the polymer substrate is elongated, these steps are independently performed. What is performed, i.e., the long polymer base material may be unwound from a state wound in a roll shape for each step, and may be wound after performing a predetermined treatment. It is preferable to carry out continuously, that is, to carry out by roll-to-roll until the cell culture substrate which is the final product. This is because mass production is possible.

B. Cell culture substrate Next, the cell culture substrate of the present invention will be described.
The cell culture substrate of the present invention includes a cell adhesion inhibiting region having cell adhesion inhibiting property and a polymer substrate having a cell adhesion region having cell adhesion property formed on a continuous surface. .

  Examples of the cell culture substrate of the present invention include those already shown in FIG. 1 (d).

  According to the present invention, it is a polymer substrate in which the cell adhesion inhibiting region and the cell adhesion region are formed on a continuous surface, that is, the cell adhesion inhibiting region and the cell adhesion inhibiting region are provided on the surface of the polymer substrate itself. By doing so, it can be formed easily and in large quantities.

The cell culture substrate of the present invention includes at least the polymer substrate.
Hereinafter, each configuration of the cell culture substrate of the present invention will be described.

1. Polymer substrate The polymer substrate used in the present invention comprises a cell adhesion-inhibiting region and a cell adhesion region formed on a continuous surface, that is, an unmodified region of the surface of the polymer substrate itself. The cell adhesion region is included, and the modified region is included as the cell adhesion region.
Such a polymer substrate, a cell adhesion region and a cell adhesion inhibition region can be the same as the contents described in the above section “A. Method for producing cell culture substrate”. Is omitted.

  Moreover, the polymer base material used for this invention may have a fine uneven | corrugated shape on the surface. Such a fine concavo-convex shape can be the same as the content described in the above-mentioned section “A. Method for producing cell culture substrate”, and thus description thereof is omitted here.

2. Cell culture substrate The cell culture substrate of the present invention includes at least the polymer substrate, and if necessary, has a stimulus-responsive layer, a protective layer, an adhesive layer, and the like on the polymer substrate. It may be a thing. Such a stimuli-responsive layer, protective layer, and adhesive layer can be the same as those described in the above section “A. Method for producing cell culture substrate”, and thus the description thereof is omitted here. To do.

C. Pattern Cell Sheet Manufacturing Method Next, the pattern cell sheet manufacturing method of the present invention will be described.
The method for producing a patterned cell sheet of the present invention is a cell culture in which a cell culture substrate is produced using the above-described method for producing a cell culture substrate, and cells are adhered and cultured on the cell adhesion region of the cell culture substrate. Extended cell adhesion that forms an expanded cell adhesion region by irradiating energy to at least a part of the cell adhesion inhibition region adjacent to the cell adhesion region and modifying the surface of the polymer substrate And a region forming step.

Such a method for producing a patterned cell sheet of the present invention will be described with reference to the drawings. FIG. 11 is a process diagram showing an example of the method for producing a patterned cell sheet of the present invention. As illustrated in FIG. 11, in the method for producing a patterned cell sheet of the present invention, first, the cell culture substrate 10 is produced using the method for producing a cell culture substrate, and the cell adhesion region of the cell culture substrate 10 is produced. Cells are adhered to and cultured on 2a (FIG. 11 (a)), and energy irradiation is performed on the cell adhesion-inhibiting region 2b adjacent to the cell adhesion region 2a (FIG. 11 (b)). The expanded cell adhesion region 12a is formed by modifying the surface of 1 (FIG. 11 (c)). Further, by continuing the culture on the cell culture substrate 10 on which such an extended cell adhesion region is formed, as shown in FIG. 11D, the cells are grown and oriented up to the extended cell adhesion region 12a. be able to.
In addition, Fig.11 (a) is a cell culture process, FIG.11 (b)-(c) is an extended cell adhesion area | region formation process.

According to the present invention, by having the expanded cell adhesion region forming step, it is possible to easily form a cell sheet in which the morphology and orientation state of the cells are accurately controlled.
The cell sheet is a sheet-like cell aggregate in which cells are connected to each other in at least a single layer by intercellular bonding.

The manufacturing method of the pattern cell sheet of this invention has the said cell culture process and an extended cell adhesion area | region formation process.
Hereinafter, each process of the manufacturing method of the pattern cell sheet of this invention is demonstrated in detail.

1. Cell culture step The cell culture step in the present invention is a step of culturing with cells adhered on the cell adhesion region of the cell culture substrate produced by the above-described method for producing a cell culture substrate.
The cell culture substrate used in this step and the method for producing the cell culture substrate are the same as those described in the above section “A. Method for producing cell culture substrate”, and thus description thereof is omitted here. To do.

The method for adhering cells on the cell adhesion region in this step is not particularly limited as long as cells can be seeded so as to have a desired density, and a general seeding method can be used. For example, the method of apply | coating the suspension which suspended the cell in the culture medium selected according to the kind of cell can be mentioned.
Moreover, a general culture method can be used also about the method of culture | cultivating the cell adhere | attached on the cell adhesion area | region.

  As cells used in this step, various cells can be used as long as they are anchorage-dependent, for example, epithelial cells and endothelial cells constituting each tissue and organ in a living body, skeletal muscle cells and smooth muscle cells exhibiting contractility. Cardiomyocytes, neurons constituting the nervous system, glial cells, fibroblasts, liver parenchymal cells involved in biological metabolism, non-hepatic parenchymal cells and adipocytes, stem cells present in various tissues as cells having differentiation ability, Furthermore, bone marrow cells, ES cells, iPS cells, stem cells, various cells induced by differentiation from ES cells or iPS cells, and the like can be used.

2. Extended cell adhesion region forming step In the expanded cell adhesion region forming step in the present invention, at least a part of the cell adhesion inhibition region adjacent to the cell adhesion region is irradiated with energy to modify the surface of the polymer substrate. This is a step of forming an expanded cell adhesion region by polishing.

  In this step, a method for forming an extended cell adhesion region by irradiating energy to at least a part of the cell adhesion inhibition region adjacent to the cell adhesion region and modifying the surface of the polymer substrate; That is, for the method of irradiating energy only to the region forming the expanded cell adhesion region, the energy to be irradiated and the shape of the expanded cell adhesion region, the contents described in the above section “A. Method for producing cell culture substrate” The description here is omitted.

The location where the expanded cell adhesion region is formed in this step is a location that was a cell adhesion inhibition region before the implementation of this step, and is not particularly limited as long as it is a location adjacent to the cell adhesion region. Are appropriately set according to the purpose. For example, an extended cell adhesion region is formed so as to connect a plurality of cell adhesion regions, and the state of cell migration can be grasped by observing cells that move between the cell adhesion regions. In addition, for example, an extended cell adhesion region that improves or decreases cell orientation is formed adjacent to the cell adhesion region with respect to a cell adhesion region in which cells can be cultured in an oriented state or a non-oriented state. It is possible to grasp how the orientation of cells changes by observing.
Here, adjoining the cell adhesion region is not particularly limited as long as it is not separated by other members in plan view, and may be formed so as to be in contact with the cell adhesion region. It may be formed so as not to contact the cell adhesion region. Moreover, when formed so that it may not contact | connect a cell adhesion area | region, the space | interval of the grade which can move a cell toward a cell extension area | region from a cell adhesion area | region may be provided. In addition, the space | interval which can move a cell usually means the space | interval for 1-2 cells, for example, can be in the range of 10 micrometers-60 micrometers.

  In this step, the state of irradiating energy to the cell adhesion-inhibiting region may be a state in which the polymer substrate is immersed in the medium, but in the atmosphere, that is, after collecting the medium once, It is preferably in a state exposed to the atmosphere. This is because there is an advantage that the labor of subculture can be saved.

The number of times this step is performed may be one time or a plurality of times.
Here, as an aspect performed a plurality of times, for example, an extended cell adhesion region formed so as to be in contact with the cell adhesion region can be sequentially expanded.

3. Method for Producing Pattern Cell Sheet The method for producing a pattern cell sheet of the present invention includes the cell culture step and the extended cell adhesion region forming step, but may have other steps as appropriate. good.
For example, after the extended cell adhesion region forming step, a cell culture step for adhering cells of a different type from the cells adhered in the cell culture step to the expanded cell adhesion region, or after the extended cell adhesion region formation step, The peeling process which peels the cell or cell sheet which has adhere | attached on the adhesion | attachment area | region and an extended cell adhesion | attachment area | region can be mentioned.

The method for detaching cells or cell sheets in the detaching step in the present invention is not particularly limited as long as it is a method capable of stably detaching cell sheets and the like. For example, an enzyme such as trypsin is used. A method of peeling a cell sheet or the like can be used.
In addition, when using a cell culture substrate having a stimulus-responsive layer formed on a polymer substrate, use a method in which a stimulus is applied in accordance with the nature of the stimulus-responsive layer and then peeled off. Can do.
Specifically, when the stimulus-responsive layer is a temperature-responsive layer containing poly-N-isopropylacrylamide (PIPAAm), which is a temperature-responsive material, the cells are cultured by culturing the cells at around 37 ° C. A method of peeling a cell sheet or the like by forming a sheet or the like and lowering the temperature to 32 ° C. or lower can be used.

  The present invention is not limited to the above embodiment. The above-described embodiment is an exemplification, and the present invention has substantially the same configuration as the technical idea described in the claims of the present invention, and any device that exhibits the same function and effect is the present invention. It is included in the technical scope of the invention.

  Hereinafter, the present invention will be specifically described using examples and comparative examples.

[Example 1-1]
An OPS sheet (polymer base material) manufactured by SUNDIC was irradiated with 26 J / cm ^{2 of} 254 nm UV light through a photomask having an opening of 100 μm square to form a cell adhesion region. When bovine vascular endothelial cells were seeded at 3 × 10 ⁴ cells / cm ² , the cells adhered to the region corresponding to the photomask opening, and a cell pattern was obtained. The cell pattern was maintained for at least 3 weeks. In addition, the microscope picture which image | photographed the cell of the cell culture substrate surface after 3 weeks is shown in FIG.

[Example 1-2]
A cell adhesion region was formed and cells were seeded in the same manner as in Example 1-1 except that the shape of the opening was changed to 300 μm square. As a result, as in Example 1-1, cells adhered to the region corresponding to the photomask opening, and a cell pattern was obtained. The cell pattern was maintained for at least 3 weeks. In addition, the microscope picture which image | photographed the cell of the cell culture substrate surface after 3 weeks is shown in FIG.

[Example 2-1]
A cell line-bonded region is formed by closely contacting a rectangular line pattern PDMS with 10 μm line-shaped concave portions formed at intervals of 10 μm on an OPS sheet (polymer substrate) manufactured by SUNDIC, and irradiating with plasma (400 W, 3 minutes). Formed. When bovine vascular endothelial cells were seeded at 3 × 10 ⁴ cells / cm ² , the cells adhered to the depression-corresponding region of PDMS, and a cell pattern was obtained. In addition, the microscope picture which image | photographed the cell of the cell culture substrate surface after 3 weeks is shown in FIG.

[Example 2-2]
A cell adhesion region was formed and cells were seeded in the same manner as in Example 2-1, except that PDMS having a line-shaped recess width of 70 μm and an interval of 70 μm was used. As a result, as in Example 2-1, cells adhered to the region corresponding to the concave portion of PDMS, and a cell pattern was obtained. In addition, the microscope picture which image | photographed the cell of the cell culture substrate surface after 3 weeks is shown in FIG.

[Example 3-1]
A fine uneven shape having a width of 10 μm, a depth of 5 μm, and a period of 20 μm was applied to an OPS sheet (polymer base material) manufactured by SUNDIC by thermal imprinting. The OPS sheet with fine irregularities was irradiated with 26 J / cm ^{2 of} 254 nm UV light through a photomask having the same shape as in Example 1-1 to form a cell adhesion region. When bovine vascular endothelial cells were seeded at 3 × 10 ⁴ cells / cm ² , the cells adhered to the region corresponding to the photomask opening, and a cell pattern was obtained. In addition, the microscope picture which image | photographed the cell on the cell culture substrate surface after 3 weeks is shown in FIG.

[Example 3-2]
A cell culture substrate was formed and cells were seeded in the same manner as in Example 3-1, except that a photomask having the same shape as in Example 1-2 was used. A photomicrograph of the cells on the surface of the cell culture substrate after 3 weeks is shown in FIG.

[Example 4]
An OPS sheet (polymer base material) manufactured by SUNDIC was irradiated with 144 mJ / cm ² of UV light of 172 nm through a photomask having an opening of 300 μm width and a light shielding portion of 60 μm width to form a cell adhesion region. Subsequently, an N-isopropylacrylamide 10 w / w% IPA solution was applied and irradiated with an electron beam of 50 kGy (150 kV) to form a cell culture substrate having a stimulus-responsive layer. After washing with water, bovine vascular endothelial cells were seeded at 3 × 10 ⁴ cells / cm ² , and the cells adhered in a pattern. After culturing for one week, the cell culture substrate was taken out of the thermostatic chamber at 37 ° C., and subjected to a low temperature treatment that was allowed to stand at room temperature (23 ° C.), the cells were detached from the pattern. A photomicrograph of the situation is shown in FIG. In FIG. 18, (a) is a photograph immediately after standing at a room temperature of 23 ° C., (b) is a photograph 20 minutes after standing at room temperature of 23 ° C., and (c) is 23 It is the photograph after 40-minute progress after leaving still at room temperature of ° C.

[Examples 5 to 7]
As polymer base materials, (1) Mitsubishi Plastics Santo Clear (sequential biaxial stretching), (2) Showa Pax Esclea (unstretched inflation), (3) Oishi Sangyo Styrophane (inflation longitudinal uniaxial stretching) Then, 254 nm light was irradiated at 26 J / cm ² through a photomask having the same shape as in Example 1-2 to form a cell culture substrate.
Thereafter, bovine vascular endothelial cells were seeded at 3 × 10 ⁴ cells / cm ² , and the cells adhered in a pattern. A photomicrograph of the cells on the surface of the cell culture substrate after 3 weeks is shown in FIG. In addition, (a)-(c) in FIG. 19 is a polymer base material, respectively (a) Santo clear (Example 5) by Mitsubishi Plastics, (b) Esclare by Showa Pax (Example 6) (C) Oishi Industry Styrophane (Example 7) is used.
From Examples 5-7, it turned out that the patterning to the polymer base material manufactured with various manufacturing methods by this method is possible.

DESCRIPTION OF SYMBOLS 1 ... Polymer base material 2a ... Cell adhesion area | region 2b ... Cell adhesion inhibition area | region 3 ... Protective layer 4 ... Stimulation responsive layer 10 ... Cell culture substrate 30 ... Cell culture container for pattern formation

Claims (8)

A cell that forms a cell adhesion region having cell adhesion by irradiating energy on the surface of a polymer substrate having a cell adhesion-inhibiting surface and modifying at least part of the surface of the polymer substrate a bonding region formation step possess,
After the cell adhesion region forming step, having a stimulus responsive layer forming step of forming a stimulus responsive layer on the polymer substrate surface,
The stimuli-responsive layer forming step is to form the stimulus-responsive layer so as to cover both the cell adhesion-inhibiting region having cell adhesion-inhibiting property of the polymer substrate and the cell adhesion region. A method for producing a cell culture substrate. The method for producing a cell culture substrate according to claim 1 , further comprising a step of forming a fine uneven shape on the surface of the polymer substrate. The method for producing a cell culture substrate according to claim 1 or 2, wherein the polymer substrate is elongated and energy irradiation is performed while the polymer substrate is conveyed .   The stimuli-responsive material contained in the stimulus-responsive layer is a photo-responsive material, a potential-responsive material, or a magnetic-responsive material, according to any one of claims 1 to 3. A method for producing a cell culture substrate.   The method of forming the stimulus responsive layer in the stimulus responsive layer forming step includes a method of attaching the stimulus responsive layer to the surface of the polymer substrate or a stimulus responsive material on the surface of the polymer substrate. The method for producing a cell culture substrate according to any one of claims 1 to 4, wherein the method is a method of applying a stimulus-responsive layer coating solution, followed by drying and curing.   The method for producing a cell culture substrate according to any one of claims 1 to 5, wherein the polymer material constituting the polymer substrate is polystyrene. A cell adhesion inhibitory region having cell adhesion inhibitory property and a polymer substrate having a cell adhesion region having cell adhesion property formed on a continuous surface;
A cell culture substrate comprising a stimulus-responsive layer formed to cover both the cell adhesion-inhibiting region and the cell adhesion region of the polymer substrate. A cell that forms a cell adhesion region having cell adhesion by irradiating energy on the surface of a polymer substrate having a cell adhesion-inhibiting surface and modifying at least part of the surface of the polymer substrate method for producing a cell culture substrate having a bonding region forming step to produce a cell culture substrate using a cell culture step of culturing to adhere the cells to the cell adhesive region of the cell culture substrate,
An extended cell adhesion region forming step of forming an extended cell adhesion region by irradiating energy to at least a part of the cell adhesion inhibition region adjacent to the cell adhesion region and modifying the surface of the polymer substrate When,
A method for producing a patterned cell sheet, comprising: