JP2011223944A - Transfer substrate for micro-patterned vascular endothelial cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a transfer substrate for micro-patterned vascular endothelial cells that allows micro-patterned vascular endothelial cells to be easily and stably transferred to an adherend by bringing the transfer substrate into contact with the adherend.SOLUTION: The transfer substrate for micro-patterned vascular endothelial cells includes: a base material; a stimulus-responsive layer that is arranged on the base material and contains a stimulus-responsive material having cellular adhesiveness and exhibiting cellular non-adhesiveness by stimulation; and a cell adhesion inhibiting layer that is arranged on the base material in a pattern form and contains a cell adhesion inhibiting material having cell non-adhesiveness. The surface of the cell adhesion inhibiting layer is formed to be higher than the surface of the stimulus-responsive layer. The lower-limit height of the cell adhesion inhibiting layer is a height that prevents vascular endothelial cells from going thereover. The upper-limit height of the cell adhesion inhibiting layer is a height that allows collection of micro-patterned vascular endothelial cells formed on the stimulus-responsive layer.

Description

  The present invention relates to a micropatterned vascular endothelial cell transfer substrate capable of transferring micropatterned vascular endothelial cells to an adherend easily and stably by contact.

In recent years, cell sheets and cell micropatterns are often used in regenerative medicine and the like.
Here, the cell sheet refers to a sheet-like cell aggregate in which cells are connected to each other in at least a single layer by intercellular bonding, and a cell pattern is a cell sheet formed as a specific shape desired. It is. A micro pattern refers to a pattern having a width of a micron (μm) scale, and a cell micro pattern is a cell sheet of a micro pattern.
These cell sheets can be obtained by culturing cells on a support such as a petri dish, but the cell sheets formed on the support are firmly bonded to the support surface via adhesion molecules, etc. When used in applications such as regenerative medicine, it is necessary to peel from the support.

  Various methods for detaching the cell sheet from the cell culture support have been studied so far. Conventionally, a method of weakening the bond between the support and the cell using an enzyme reaction, a support having a weak cell adhesion, or a cell adhesion A method using a support having a variety of diameters is used.

  More specifically, the method using an enzyme reaction includes a method of degrading a protein constituting an intercellular adhesion molecule using a protease (proteolytic enzyme) or the like. However, this method weakens not only cell-support surface binding but also cell-cell binding. For this reason, this method has a problem that the cell sheet is damaged to some extent.

In addition, as a method using a support that changes cell adhesion, for example, Patent Documents 1 and 2 disclose a support in which a cell growth surface is coated with a temperature-responsive polymer.
Further, Patent Document 3 discloses a method for recovering cells by adhering cells to a release layer that can be released by irradiation with an ion beam. Patent Document 4 discloses the formation of a cell pattern and a method for recovering the cell pattern. Patent Document 5 discloses culturing desired cells along a desired pattern.
However, such a substrate has a problem that it is difficult to stably transfer the formed cell micropattern to the adherend while maintaining the pattern.

Japanese Patent Publication No. 6-104061 JP-A-5-192130 Japanese Patent Laid-Open No. 2003-082119 JP-A-2005-342112 JP 2006-8975 A

  The present invention has been made in view of the above problems, and provides a micropatterned vascular endothelial cell transfer substrate capable of easily and stably transferring a micropatterned vascular endothelial cell to an adherend by contact. The main purpose.

  To achieve the above object, the present invention provides a stimulus comprising a substrate and a stimulus-responsive material disposed on the substrate, having cell adhesion and capable of expressing cell non-adhesion upon stimulation. A responsive layer, and a cell adhesion inhibiting layer that is arranged in a pattern on the substrate and includes a cell adhesion inhibiting material having cell non-adhesiveness, and the surface of the cell adhesion inhibiting layer has the stimulation response. The lower limit of the height of the cell adhesion-inhibiting layer is a height that cannot be overcome by the vascular endothelial cell, and the upper limit is formed on the stimulus-responsive layer. Provided is a micropatterned vascular endothelial cell transfer substrate characterized in that the height is such that the micropatterned vascular endothelial cells can be collected.

According to the present invention, since the lower limit of the height of the cell adhesion-inhibiting layer is the above-described height, the micropatterned vascular endothelial cells having the exposed stimulus-responsive layer pattern can be accurately formed. it can.
In addition, since the upper limit of the height of the cell adhesion inhibiting layer is the above-mentioned height, the cell recovery layer and the micropatterned vascular endothelial cell can be stably contacted, and the micropatterned vascular endothelium The cells can be easily transferred from the stimulus-responsive layer to the cell collection layer as a transfer destination without damaging the pattern shape due to the contraction of the micropatterned vascular endothelial cells.
Furthermore, the micropatterned vascular endothelial cells are transferred to the cell collection layer or the like under a minimally invasive condition by changing the cell adhesiveness state to the cell nonadhesive state. Can do.

  In the present invention, the stimulus-responsive material is preferably a temperature-responsive material that exhibits cell non-adhesiveness due to a temperature change. This is because it is easy to apply a stimulus.

  In the present invention, the temperature-responsive material is preferably poly-N-isopropylacrylamide (PIPAAm). Since the micropatterned vascular endothelial cells can be cultured under suitable culture conditions and express cell non-adhesiveness under temperature conditions with little influence such as reducing the activity of the vascular endothelial cells, the micropatterned blood vessels This is because endothelial cells can be stably formed and transcribed.

  In the present invention, the exposed surface width of the stimulus-responsive layer is preferably in the range of 15 μm to 500 μm. This is because the micropatterned vascular endothelial cells can be formed with high accuracy.

  In the present invention, the cell adhesion inhibiting material is preferably a polyethylene glycol material. This is because the excellent adhesion inhibition with the vascular endothelial cells and the cytotoxicity are almost absent, so that the micropatterned vascular endothelial cells are formed with high accuracy and have little influence on the cultured cells.

  The present invention relates to a method for producing an artificial tissue containing at least micropatterned vascular endothelial cells, wherein the micropatterned vascular endothelial cell transfer substrate is prepared, and the stimulation-responsive layer expresses cell adhesion. A micropatterned vascular endothelial cell forming step for forming micropatterned vascular endothelial cells by seeding and culturing the vascular endothelial cells on the stimulus-responsive layer under conditions, and after the micropatterned vascular endothelial cell forming step By contacting the micropatterned vascular endothelial cell with a cell recovery layer containing a cell adhesion layer having cell adhesion, under the condition that the stimulus-responsive layer of the cell exhibits non-adhesiveness. And a vascular endothelial cell transfer step of transferring the vascularized endothelial cells to the cell collection layer. To.

According to the present invention, by using the micropatterned vascular endothelial cell transfer substrate, the micropatterned vascular endothelial cells can be formed with high accuracy, and the micropatterned vascular endothelial cells are converted into the cell recovery layer. Can be stably transferred.
For this reason, the artificial tissue containing the micropatterned vascular endothelial cells can be formed with high accuracy.

In the present invention, the cell recovery layer is composed of the cell adhesion layer, and the vascular endothelial cell transfer step is such that the micropatterned vascular endothelial cell and the cell adhesion layer are in direct contact, or the cell recovery layer is The cell adhesion layer and a cell layer composed of cells adhered and laminated on the cell adhesion layer, wherein the vascular endothelial cell transfer step brings the micropatterned vascular endothelial cell into contact with the cell layer; can do. Moreover, it can have a lamination process which adhere | attaches and laminate | stacks the cell layer which consists of a cell on the said micro pattern vascular endothelial cell transcribe | transferred on the said cell collection layer.
It is because the target artificial tissue can be obtained by performing such a process. Moreover, it is because an artificial tissue having a complicated function can be formed by combining a plurality of types of cells.

  In the present invention, the artificial tissue may have a recovery step of peeling from the cell recovery layer. This is because, by combining the artificial tissue with other artificial tissues, it is possible to easily construct and transplant a more complicated artificial tissue.

  INDUSTRIAL APPLICABILITY The present invention has an effect that a micropatterned vascular endothelial cell transfer substrate capable of easily and stably transferring micropatterned vascular endothelial cells to an adherend can be provided.

It is a schematic sectional drawing which shows an example of the micropatterned vascular endothelial cell transfer substrate of this invention. It is process drawing which shows an example of the manufacturing method of the artificial tissue of this invention. It is a schematic sectional drawing which shows the other example of the micro patterning vascular endothelial cell transcription | transfer board | substrate of this invention. It is explanatory drawing explaining the cell collection | recovery layer used for this invention. It is explanatory drawing explaining the collection | recovery process in the manufacturing method of the artificial tissue of this invention.

The present invention relates to a micropatterned vascular endothelial cell transfer substrate and a method for producing an artificial tissue using the same.
Hereinafter, the micropatterned vascular endothelial cell transfer substrate and the artificial tissue manufacturing method of the present invention will be described in detail.

A. Micropatterned Vascular Endothelial Cell Transfer Substrate First, the micropatterned vascular endothelial cell transfer substrate of the present invention will be described.
The micropatterned vascular endothelial cell transfer substrate of the present invention includes a base material and a stimulus-responsive material that is disposed on the base material and has cell adhesiveness and can express cell non-adhesiveness by stimulation. A stimulation-responsive layer, and a cell adhesion-inhibiting layer that is arranged in a pattern on the substrate and includes a cell adhesion-inhibiting material having cell non-adhesiveness, and the surface of the cell adhesion-inhibiting layer is the stimulation It is formed so as to be higher than the surface of the responsive layer, and the lower limit of the height of the cell adhesion-inhibiting layer is a height at which vascular endothelial cells cannot get over, and the upper limit is on the stimulus-responsive layer. The micropatterned vascular endothelial cells formed are of a height that can be collected.

  Such a micropatterned vascular endothelial cell transfer substrate of the present invention will be described with reference to the drawings. FIG. 1 is a schematic cross-sectional view showing an example of a micropatterned vascular endothelial cell transfer substrate of the present invention. As illustrated in FIG. 1, a micropatterned vascular endothelial cell transfer substrate 10 of the present invention is formed on a base material 1 and the base material 1, has cell adhesiveness, and does not adhere to a cell by stimulation. A stimulus-responsive layer 2 containing a stimulus-responsive material capable of expressing sex, and a cell adhesion-inhibiting layer 3 containing a cell adhesion-inhibiting material formed in a pattern on the stimulus-responsive layer 2 and having cell non-adhesive properties; The upper limit and the lower limit of the height of the cell adhesion inhibition layer 3, that is, the height t from the surface of the stimulus responsive layer 2 to the surface of the cell adhesion inhibition layer 3 are the above heights. Is.

Here, in order to explain the micropatterned vascular endothelial cell transfer substrate of the present invention, a method for producing an artificial tissue containing vascular endothelial cells of the present invention will be described with reference to the drawings. FIG. 2 is a process diagram showing an example of the method for producing an artificial tissue of the present invention. As illustrated in FIG. 2, the artificial tissue manufacturing method of the present invention provides the micropatterned vascular endothelial cell transfer substrate 10 described above under the condition that the stimulus-responsive layer 2 expresses cell adhesion. Then, by seeding and culturing the vascular endothelial cells 11 on the stimulation-responsive layer 2 (FIG. 2 (a)), micropatterned vascular endothelial cells 12 are formed (FIG. 2 (b)), and the stimulation response The micropatterned vascular endothelial cell 12 and the cell recovery layer 30 including the cell adhesion layer 20 having cell adhesion are brought into contact with each other under the condition that the sex layer 2 expresses cell non-adhesion (FIG. 2 ( c)), the micropatterned vascular endothelial cells 12 are transferred to the cell collection layer 30 to form an artificial tissue 15 including at least the micropatterned vascular endothelial cells 12 (FIG. 2 (d)).
2A to 2B are micropatterned vascular endothelial cell forming steps, and FIGS. 2C to 2D are vascular endothelial cell transcription steps.

Conventionally, a method of degrading a protein or the like constituting an intercellular adhesion molecule using an enzyme such as a protease (proteolytic enzyme) is used for detaching cells adhered to a support. Such a method weakens not only cell-support surface binding but also cell-cell binding. For this reason, when trying to collect the micropatterned vascular endothelial cells using such a conventional method, there is a problem in that the micropatterned vascular endothelial cells are damaged.
However, in the present invention, since a stimulus-responsive layer that can express cell non-adhesiveness by stimulation is used, the use of such an enzyme can be made unnecessary. Therefore, since the cell-cell bond is not weakened, the vascular endothelial cells are separated from the micropatterned vascular endothelial cells when transferred from the stimulus-responsive layer and transferred, or the micropatterns are separated. It can suppress that the pattern of the vascularized endothelial cell collapses.
Thus, according to the present invention, the micropatterned vascular endothelial cells can be stabilized under minimally invasive conditions by changing from a state expressing cell adhesion to a state expressing cell non-adhesion. Thus, it can be peeled off from the stimulus-responsive layer and transferred to the cell recovery layer or the like.

Furthermore, according to the present invention, the lower limit of the height of the cell adhesion-inhibiting layer is the above-mentioned height, so that the vascular endothelial cells adhered to the stimulus-responsive layer grow over the cell adhesion-inhibiting layer. Since this can be effectively suppressed, micropatterned vascular endothelial cells having a pattern shape of the exposed stimulus-responsive layer can be accurately formed.
In addition, since the upper limit of the height of the cell adhesion inhibiting layer is the above-mentioned height, the cell recovery layer and the micropatterned vascular endothelial cell can be stably contacted, and the micropatterned vascular endothelium The cells can be easily transferred from the stimulus-responsive layer to the cell collection layer without damaging the pattern shape due to the contraction of the cell sheet.
As described above, according to the present invention, the micropatterned vascular endothelial cells can be formed with high accuracy, and the obtained micropatterned vascular endothelial cells can be stably transferred to the cell collection layer. It can be done.

The micropatterned vascular endothelial cell transfer substrate of the present invention has at least the substrate, the stimulus-responsive layer, and the cell adhesion-inhibiting layer.
Hereinafter, each configuration of the micropatterned vascular endothelial cell transfer substrate of the present invention will be described in detail.

1. Stimulus responsive layer The stimulus responsive layer used in the present invention contains a stimulus responsive material. Further, the adhesive force of the stimulus-responsive layer expressing cell non-adhesiveness with the vascular endothelial cell is set lower than the adhesive force of the cell recovery layer with the vascular endothelial cell.

(1) Stimulus responsive material The stimulus responsive material used in the present invention is not particularly limited as long as it has cell adhesiveness and can express cell non-adhesiveness by stimulation. For example, a temperature responsive material, a photoresponsive material, a pH responsive material, a potential responsive material, a magnetic force responsive material, etc. whose degree of cell adhesion changes depending on temperature, light, pH, potential and magnetic force, respectively. it can.
In the present invention, among them, a temperature responsive material is preferable. This is because it is easy to apply a stimulus.
In the present invention, only one kind of each of the above stimulus-responsive materials or a combination of two or more kinds may be used.

  The change in the degree of cell adhesion of the stimulus-responsive material used in the present invention is particularly limited as long as it can change from a state having cell adhesion to a state having cell non-adhesion. Instead, it may be reversibly changed or irreversibly changed.

  The content of the stimulus-responsive material used in the present invention in the stimulus-responsive layer is not particularly limited as long as the degree of cell adhesion changes by a desired amount due to the stimulus-responsive layer. However, it differs depending on the type of the material.

The temperature-responsive material used in the present invention is not particularly limited as long as the degree of cell adhesion changes by a desired amount due to temperature change.
The temperature at which the cell-responsiveness of the temperature-responsive material in the present invention is exhibited is preferably in the range of 10 ° C to 45 ° C, and particularly preferably in the range of 33 ° C to 40 ° C. This is because the cells can be stably cultured when the temperature region is within the above range.

  The temperature at which cell non-adhesiveness of the temperature-responsive material in the present invention is expressed is preferably within a range of 1 ° C to 36 ° C, and more preferably within a range of 4 ° C to 32 ° C. This is because when the temperature range is within the above range, damage to cells can be reduced.

Specific examples of the temperature-responsive material used in the present invention include poly-N-isopropylacrylamide (PIPAAm), poly-Nn-propylacrylamide, poly-Nn-propylmethacrylamide, poly -N-ethoxyethyl acrylamide, poly-N-tetrahydrofurfuryl acrylamide, poly-N-tetrahydrofurfuryl methacrylamide, poly-N, N-diethyl acrylamide, etc. can be mentioned, among them PIPAAm, poly-N -N-propylmethacrylamide and poly-N, N-diethylacrylamide can be preferably used, and PIPAAm can be particularly preferably used. Since the vascular endothelial cells can be cultured under suitable culture conditions and express cell non-adhesiveness under temperature conditions that have little influence such as reducing the activity of the vascular endothelial cells, the micropatterned vascular endothelial cells are This is because it can be stably formed, peeled off, collected, and transferred.
In the present invention, 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 the present invention is not particularly limited as long as the degree of cell adhesion changes depending on the presence or absence of light irradiation. For example, it is disclosed in JP-A-2005-210936. In addition, photocatalysts and those containing photoresponsive components such as azobenzene, diarylethene, spiropyran, spirooxazine, fulgide and leuco dye can be used.

  The potential responsive material used in the present invention is not particularly limited as long as the degree of cell adhesion is changed by application of a potential. For example, it is disclosed in Japanese Patent Application Laid-Open No. 2008-295382. Further, there may be mentioned those having an electrode and a spacer substance such as alkanethiol, cysteine, alkane disulfide, etc., which have a cell adhesive portion such as a peptide containing an RGD sequence and bind to the electrode surface via thiolate. .

  The magnetic force responsive material used in the present invention is not particularly limited as long as the degree of cell adhesion is changed by the application / removal of magnetic force. For example, it is disclosed in JP-A-2005-312386. Examples thereof include positively charged liposomes encapsulating magnetic particles in which magnetic particles such as ferrite are encapsulated in positively charged liposomes.

(2) Stimulus responsive layer In the stimulus responsive layer used in the present invention, the degree of cell adhesion changes from a state expressing cell adhesion by stimulation to a state expressing cell non-adhesion. To get.

Here, the stimulus-responsive layer expressing cell adhesion means that the cells are easily adhered and spread on the stimulus-responsive layer and have a high cell adhesion extension rate. is there. In the present invention, 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 the present invention, it is preferably 80% or more. This is because cells can be efficiently cultured to form micropatterned vascular endothelial cells.

In the present invention, the cell adhesion spreading rate is such that bovine vascular endothelial cells are seeded in a seeding density range of 4000 cells / cm ² or more and less than 30000 cells / cm ² and stored in a 37 ° C. incubator (CO ₂ concentration 5%). It represents the ratio of the cells that have been spread by adhesion at the time of culturing for 14.5 hours ({(number of cells adhered) / (number of cells seeded)} × 100 (%)).
The cells are seeded by suspending them in a DMEM medium containing 10% FBS (serum) and seeding them on a culture substrate, and then the culture medium on which the cells are seeded so that the cells are distributed as uniformly as possible. This is done by shaking the material 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.

In addition, when the stimulus-responsive layer expresses cell non-adhesiveness, it means that cells are not easily adhered and spread on the stimulus-responsive layer, and the cell adhesion extension rate is low. is there. In the present invention, 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 the present invention, the content is preferably 2% or less.
Therefore, when cells are seeded in the stimulus-responsive layer, the cells adhere to the stimulus-responsive layer when expressing cell adhesion, but the cells are not attached when expressing cell non-adhesiveness. Since adhesion to the stimulus responsive layer is inhibited, the cells adhered to the stimulus responsive layer can be easily peeled off.

In addition, the width of the exposed surface of the stimulus-responsive layer, that is, the width of the opening of the cell adhesion-inhibiting layer, and the width of the micropatterned vascular endothelial cell is the width of the vascular endothelial cell. -It can be cultured and is not particularly limited as long as it has a micropattern, that is, a micron ([mu] m) scale. Specifically, it is preferably within a range of 15 [mu] m to 500 [mu] m, However, it is preferably in the range of 40 μm to 80 μm. This is because, when the width is within the above-described range, it is easy to form vascular endothelial cells into a lumen.
In addition, about the width | variety, height, etc. of each structure in this application, it is a thing at the time of drying unless there is particular notice.

  As the shape of the stimulus-responsive layer used in the present invention, one that covers the entire surface of the substrate is usually used, but it may be formed in a pattern on the substrate.

  The film thickness of the stimulus responsive layer used in the present invention 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 within a range of 1 nm to 100 nm.

  The stimulus-responsive layer used in the present invention contains the above-mentioned stimulus-responsive material, but within a range that does not inhibit the stimulus-responsiveness, a leveling agent, a plasticizer, a surfactant, an antifoaming agent, Additives such as sensitizers, polyvinyl alcohol, unsaturated polyester, acrylic resin, polyethylene, diallyl phthalate, ethylene propylene diene monomer, epoxy resin, phenol resin, polyurethane, melamine resin, polycarbonate, polyvinyl chloride, polyamide, polyimide, Styrene butadiene rubber, chloroprene rubber, polypropylene, polybutylene, polystyrene, polyvinyl acetate, nylon, polyester, polybutadiene, polybenzimidazole, polyacrylonitrile, epichlorohydrin, polysulfide, polyisoprene , It may include polyethylene glycol, MPC polymeric binder resin such as zwitterionic polymer (trade name).

In addition, the stimulus-responsive layer in the present invention may be subjected to surface treatment as necessary. This is because various functions can be imparted depending on the type of surface treatment.
Examples of the surface treatment in the present invention include silane treatment, that is, the surface of the stimulus-responsive layer treated with silane. By applying the silane treatment, for example, when the cell adhesion inhibition layer is formed on the stimulus-responsive layer, the binding with the cell adhesion inhibition layer can be ensured, and the cell adhesion inhibition is achieved. This is because the layer pattern, that is, the pattern shape of the exposed surface of the stimulus-responsive layer can be formed with high accuracy. Therefore, a micropatterned vascular endothelial cell having a desired pattern can be obtained more reliably.

  As such a silane treatment, for example, a silane coupling agent can be applied.

  In the present invention, examples of the silane coupling agent used for the silane treatment include methacryloxy silane, vinyl silane, amino silane, and epoxy silane. Among them, methacryloxy silane can be preferably used.

  As a coating method of the silane coupling agent in the present invention, it is dissolved using an organic solvent capable of dissolving the silane coupling agent, and this is dissolved by a conventional coating method such as spinner method, die coating method, dipping method, gravure method. The method of apply | coating the said stimulus responsive layer by printing method, CVD (chemical vapor deposition method), etc. can be mentioned. For example, when the spinner method is used, the conditions can be 700 rpm to 2000 rpm and about 3 seconds to 20 seconds.

  In addition, in the stimulus-responsive layer whose surface is treated with silane, the silane-treated layer is not integrated with the stimulus-responsive material or indistinguishable, and is not considered to form separate layers. It is done. For this reason, in the present application, the term “stimulus responsive layer” may include a silane-treated layer. Note that these are theories or assumptions and do not limit the present invention.

  Such polymer coating amount or silane-treated coating amount can be determined by, for example, analysis by coating or non-coating dyeing or fluorescent substance dyeing, surface analysis by contact angle measurement, X-ray photoelectron spectroscopy (XPS) or flight. The time secondary ion mass spectrometer (TOF-SIMS) can be determined alone or in combination.

The method for forming the stimulus-responsive layer used in the present invention is not particularly limited as long as it can be formed with a desired film thickness.
Specifically, a method of applying the stimulus responsive material or the stimulus responsive material composition containing a monomer component capable of forming the stimulus responsive material by a known application method such as a spinner method or a die coating method is exemplified. Can do.
In addition, when the stimulus-responsive layer is formed in a pattern on the substrate, a method of patterning by a photolithography method after forming a coating film of the stimulus-responsive material composition, or the stimulus response A method of applying the conductive material composition in a pattern using a pattern printing method such as gravure printing flexographic printing, screen printing, or an inkjet method can be used.
In addition, when the stimulus-responsive layer is made of only an inorganic substance such as a photocatalyst as the stimulus-responsive material, a method of using a vacuum film-forming method such as a sputtering method, a CVD method, or a vacuum deposition method is exemplified. Can do. This is because a layer having a uniform film thickness can be obtained.

2. Cell adhesion inhibition layer The cell adhesion inhibition layer used in the present invention contains the cell adhesion inhibition material, the lower limit of the height is a height that the vascular endothelial cells cannot overcome, and the upper limit is the stimulation-responsive layer. The height of the micropatterned vascular endothelial cells formed above is recoverable.

(1) Cell adhesion inhibiting material The cell adhesion inhibiting material used in the present invention has cell non-adhesiveness.

As such a cell adhesion inhibiting material, any material having adhesion inhibiting properties with the above vascular endothelial cells may be used. Specifically, diethylene glycol, triethylene glycol, tetraethylene glycol, polyethylene glycol, polyethylene glycol diacrylate, polyethylene Ethylene glycol materials using glycol methacrylate, etc., long-chain alkyl materials such as decylmethoxysilane, fluorine materials such as fluoroalkylsilane, hydrophilic materials such as polyvinyl alcohol (PVA) and polyacrylamide, and phosphorus such as MPC polymer Examples thereof include lipid materials and BSA proteins.
In the present invention, among them, a polyethylene glycol material which is a material containing polyethylene glycol such as polyethylene glycol, polyethylene glycol diacrylate, and polyethylene glycol methacrylate can be preferably used. This is because the micropatterned vascular endothelial cells can be formed with high accuracy by exhibiting little adhesion inhibition and cytotoxicity with the vascular endothelial cells.

  The content of the cell adhesion inhibiting material used in the present invention in the cell adhesion inhibiting layer is not particularly limited as long as the cell adhesion inhibiting layer has a desired cell non-adhesive property. However, it differs depending on the type of the material.

(2) Cell adhesion inhibition layer The cell adhesion inhibition layer used in the present invention is formed such that the surface of the cell adhesion inhibition layer is higher than the surface of the stimulus-responsive layer, and has cell non-adhesiveness. Is.

As such a cell adhesion inhibition layer, specifically, as shown in FIG. 1 already described, it may be formed on a stimulus-responsive layer. As illustrated in FIG. It may be formed directly on the substrate and formed so as to be thicker than the stimulus-responsive layer.
Note that the reference numerals in FIG. 3 indicate the same members as those in FIG.

Micropatterned vascular endothelial cells formed on the stimulation-responsive layer are collected as the upper limit of the height of the cell adhesion-inhibiting layer, that is, the distance from the surface of the cell adhesion-inhibiting layer to the surface of the stimulation-responsive layer. The height is not particularly limited as long as it is possible, but in the present invention, it is preferably 5 μm or less, and particularly preferably 4.6 μm or less. When the height is in the above-described range, the vascular endothelial cells on the stimulus-responsive layer and the cell recovery layer can stably contact, and the micropatterned vascular endothelial cells are in contact with the cell recovery layer. This is because the film can be easily transferred while maintaining the pattern shape without contracting.
Further, the lower limit of the height is not particularly limited as long as the vascular endothelial cell can not overcome, and varies depending on the type of cell adhesion inhibiting material to be used. Specifically, it is 2.5 μm or more. Preferably there is. In the present invention, the thickness is preferably 3.0 μm or more, and particularly preferably 3.5 μm or more. When the lower limit of the height is in the above range, the vascular endothelial cells grow on the cell adhesion-inhibiting layer, or the vascular endothelial cells on the stimulation-responsive layer adjacent to each other through the cell adhesion-inhibiting layer Can be prevented from contacting on the cell adhesion-inhibiting layer. This is because the micropatterned vascular endothelial cells can be formed with high accuracy.

  The degree of cell adhesion of the cell adhesion-inhibiting layer used in the present invention is not particularly limited as long as it exhibits a desired cell non-adhesive property, and the stimulus-responsive layer exhibits cell non-adhesive properties. It can be the same as the degree of cell adhesion.

  The pattern shape of the cell adhesion inhibition layer used in the present invention is not particularly limited as long as it has an opening in the pattern shape of a desired micropatterned vascular endothelial cell. Can do.

  As the width of the cell adhesion inhibiting layer used in the present invention, the vascular endothelial cells adhered to the stimulation-responsive layer do not adhere to each other on the cell adhesion inhibiting layer, and a micropatterned vascular endothelial cell having a desired pattern is used. It is not particularly limited as long as it can be formed. For example, it is preferably 5 μm or more, and more preferably 10 μm or more. It is because it can suppress that a cell proliferates on the said cell adhesion inhibition layer because the said width | variety is the above-mentioned range.

  The additive and binder resin that can be contained in the cell adhesion-inhibiting layer used in the present invention, and the formation method can be the same as those described in the section “1. Stimulus-responsive layer”.

3. Base material The base material used in the present invention is not particularly limited as long as it can support the stimulation-responsive layer and the cell adhesion-inhibiting layer.
Such base materials include polyethylene terephthalate (PET), polystyrene (PS), polycarbonate (PC), TAC (triacetyl cellulose), polyimide (PI), nylon (Ny), low density polyethylene (LDPE), medium density. Acrylic materials such as polyethylene (MDPE), vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyether sulfone, polyethylene naphthalate, polypropylene, urethane acrylate, cellulose, glass, and the like can be given. It may be a biodegradable polymer such as polylactic acid, polyglycolic acid, polycaprolactan, or a copolymer thereof. Further, the substrate may be porous.
In the present invention, among these, polyethylene terephthalate, polystyrene, and polycarbonate can be preferably used, and polyethylene terephthalate can be particularly preferably used. This is because it excels in properties such as transparency, dimensional stability, mechanical properties, electrical properties, and chemical resistance.
Moreover, you may use the said stimulus responsive layer as a base material.

  The thickness of the substrate in the present invention is not particularly limited as long as it can stably support the stimulus-responsive layer and the like. For example, it is within the range of 10 μm to 1000 μm, preferably , In the range of 50 μm to 200 μm.

4). Cell Recovery Layer The cell recovery layer in the present invention includes at least a cell adhesion layer having cell adhesiveness, and is brought into contact with the micropatterned vascular endothelial cells formed on the stimulation responsive layer, whereby Patterned vascular endothelial cells can be transferred and recovered from the stimulus-responsive layer.

(1) Cell adhesion layer As the cell adhesion layer used in the present invention, the micropattern on the stimulus-responsive layer having adhesion to cells containing vascular endothelial cells and expressing cell non-adhesion. It is not particularly limited as long as the cells can be recovered after contacting with the vascularized endothelial cells for a predetermined time, and it is preferable to use those containing cell adhesion materials used for general cell culture substrates and the like. it can.

Examples of the cell adhesive material used in the present invention include a material having cell adhesiveness due to physicochemical properties and a material having biochemical cell adhesiveness.
In the present invention, examples of the material having cell adhesion due to the above physicochemical properties include basic polymers such as hydrophilic polystyrene and polylysine, aminopropyltriethoxysilane, N- (2-aminoethyl) -3- Examples include basic compounds such as aminopropyltrimethoxysilane and condensates containing them.
Examples of biochemical cell adhesive materials include fibronectin, laminin, tenascin, vitronectin, RGD (arginine-glycine-aspartic acid) sequence-containing peptide, YIGSR (tyrosine-isoleucine-glycine-serine-arginine). Examples include sequence-containing peptides, collagen, atelocollagen, gelatin, and mixtures thereof such as Matrigel, PuraMatrix, fibrin and the like. Further examples include various glasses, plasma-treated polystyrene, polypropylene, nonwoven fabric, and paper.
In the present invention, among them, gelatin can be preferably used, and in particular, gelatin from porcine skin can be preferably used. This is because the micropatterned vascular endothelial cells can be stably transferred by having an excellent adhesive force with the vascular endothelial cells and low cytotoxicity. Moreover, it is because the artificial tissue containing the micropatterned vascular endothelial cells can be easily detached from the cell recovery layer and recovered by heating the cell recovery layer. As a result, it is easy to use the obtained artificial tissue.

  The content of the cell adhesion material used in the present invention in the cell adhesion layer is not particularly limited as long as it can stably transfer the vascular endothelial cells, and varies depending on the kind of the material. is there.

  The additive and binder resin that can be contained in the cell adhesion layer used in the present invention, the film thickness, and the formation method can be the same as those described in the section “1. Stimulus-responsive layer”.

(2) Cell recovery layer The cell recovery layer used in the present invention contains at least the cell adhesion layer, and if necessary, has a cell layer composed of cells on the cell adhesion layer. Can do. Since the cell collection layer has a cell layer, such a cell layer can be combined with the micropatterned vascular endothelial cells to form a complex artificial tissue containing a plurality of types of cells. is there. For example, as illustrated in FIG. 4A, the micropatterned vascular endothelial cell 12 includes the cell adhesion layer 20 and a cell recovery layer 20 including cells formed on the cell adhesion layer 20. 30, that is, by adhering to the cell layer 14 included in the cell recovery layer 30 and transferring from the stimulus-responsive layer 2 (FIG. 4B), the cell layer 14 and the micropatterned vascular endothelium An artificial tissue 15 including cells 12 can be formed (FIG. 4C).
In addition, about the code | symbol in FIG. 4, since it shows the member same as FIG. 1 and FIG. 2, description here is abbreviate | omitted.

The cells used in the present invention are not particularly limited as long as they are used together with the above-mentioned vascular endothelial cells, and various cells, for example, epithelial cells and endothelial cells constituting each tissue and organ in a living body, contraction Sexual skeletal muscle cells, smooth muscle cells, cardiomyocytes, neurons that make up the nervous system, glial cells, fibroblasts, liver parenchymal cells, non-hepatic parenchymal cells and fat cells involved in biological metabolism, have differentiation potential As cells, stem cells existing in various tissues, bone marrow cells, ES cells, iPS cells, stem cells, various types of cells induced to differentiate from ES cells or iPS cells, and the like can be used.
Moreover, the said cell may be only 1 type and may use 2 or more types.

  As a method for forming the cell layer used in the present invention, any method can be used as long as the cell layer can be laminated with high accuracy. For example, a method of performing the lamination step described in the section of “B. Can be mentioned.

As the degree of cell adhesion of the cell recovery layer used in the present invention, the adhesion with the vascular endothelial cells is greater than the stimulation-responsive layer exhibiting desired cell adhesion and expressing cell non-adhesion, It is particularly limited as long as the micropatterned vascular endothelial cells can be collected on the cell collection layer by contacting with the micropatterned vascular endothelial cells on the stimulus-responsive layer expressing cell non-adhesiveness. For example, it may be the same as the degree of cell adhesion when the stimulus-responsive layer expresses cell adhesion.
The degree of cell adhesion of the cell collection layer includes the cell layer when the cell collection layer has cells, that is, when the cell collection layer has a cell layer on the cell adhesion layer. It is the measured value of the surface of a cell collection layer.

5). Micropatterned vascular endothelial cell transfer substrate The micropatterned vascular endothelial cell transfer substrate of the present invention comprises at least a base material, a stimulus-responsive layer, and a cell adhesion-inhibiting layer.
In the present invention, the adhesive layer is formed between the base material and the stimulus responsive layer, and has an adhesive layer containing an adhesive that can make the base material and the stimulus responsive layer have excellent adhesion. There may be.
Moreover, you may have the adhesion layer for fixing to the bottom face of a polystyrene dish and a petri dish in the said base material layer.

  Examples of the adhesive used in the present invention include polyester, acrylic ester, polyurethane, polyethyleneimine, silane coupling agent, perfluorooctane sulfonic acid (PFOS) and the like. Among them, polyester, acrylic ester, Polyurethane and the like are preferable. This is because the adhesion between the base material and the stimulus-responsive layer can be further improved.

  The method for forming the adhesive layer in the present invention is not particularly limited as long as an adhesive layer having a desired film thickness can be formed. For example, the adhesion-imparting paint can be applied on the substrate by an in-line coating method or an offline coating method. Examples of the adhesion-imparting paint include a combination of the above adhesive and a melamine resin as a crosslinking agent component. The in-line coating method is a method of applying in the film forming process of the base material, and the off-line coating method is a method of applying the coating material to the coater and applying and drying the paint. . Examples of paint application methods include roll coating, gravure coating, micro gravure coating, reverse coating, reverse gravure coating, bar coating, roll brushing, air knife coating, curtain coating, and die coating. Arbitrary application methods can be applied singly or in combination as appropriate.

  The pressure-sensitive adhesive layer used in the present invention can be obtained by applying and forming a known pressure-sensitive adhesive. Moreover, the separator may be formed in the said adhesion layer surface as needed. It is because it can prevent that a dust etc. adhere to the above-mentioned adhesion layer by peeling a separator just before pasting on a dish etc.

B. Next, a method for manufacturing an artificial tissue according to the present invention will be described.
The method for producing an artificial tissue according to the present invention is a method for producing an artificial tissue containing at least micropatterned vascular endothelial cells, wherein the micropatterned vascular endothelial cell transfer substrate is prepared, and the stimulus-responsive layer is attached to a cell. A micropatterned vascular endothelial cell forming step for forming the micropatterned vascular endothelial cell by seeding and culturing the vascular endothelial cell on the stimulus-responsive layer under a condition expressing sex, and the micropattern Contact the micropatterned vascular endothelial cell with the cell recovery layer including the cell adhesion layer having cell adhesion property under the condition that the stimulation-responsive layer after the step of forming the vascular endothelial cell expresses cell non-adhesiveness And a vascular endothelial cell transfer step for transferring the micropatterned vascular endothelial cells to the cell recovery layer. It is intended.

  Such a method for producing an artificial tissue of the present invention can be exemplified by the already described FIG.

According to the present invention, by using the micropatterned vascular endothelial cell transfer substrate, the micropatterned vascular endothelial cells can be formed with high accuracy, and the micropatterned vascular endothelial cells are converted into the cell recovery layer. Can be stably transferred.
For this reason, the artificial tissue containing the micropatterned vascular endothelial cells can be formed with high accuracy.

The method for producing an artificial tissue of the present invention includes at least the micropatterned vascular endothelial cell formation step and the vascular endothelial cell transcription step.
Hereinafter, each process of the manufacturing method of the artificial tissue of this invention is demonstrated in detail.

1. Micropatterned vascular endothelial cell forming step In the micropatterned vascular endothelial cell forming step in the present invention, the micropatterned vascular endothelial cell transfer substrate is prepared, and the stimulus-responsive layer expresses cell adhesiveness. In this step, micropatterned vascular endothelial cells are formed by seeding and culturing the vascular endothelial cells on the stimulus-responsive layer.

  The vascular endothelial cell used in this step is not particularly limited as long as it is a cell that forms a blood vessel by culture, and those obtained from each organism, particularly an animal, can be used.

In this step, the method for making the stimulus-responsive layer have cell adhesiveness is different depending on the type of the stimulus-responsive material constituting the stimulus-responsive layer, and exhibits the above-mentioned cell adhesiveness. The method is not particularly limited as long as it can be a condition that can be performed.
Specifically, when the stimulus-responsive material is PIPAAm, a method of allowing the stimulus-responsive layer to stand in an incubator set to a temperature condition higher than 32 ° C. can be used.

  The method for seeding the vascular endothelial cells in this step is not particularly limited as long as it is a method for uniformly seeding vascular endothelial cells on the stimulation-responsive layer, and a general seeding method may be used. it can. For example, a method of seeding the vascular endothelial cells in a state where the micropatterned vascular endothelial cell transfer substrate is immersed in a medium can be mentioned.

  The method for culturing the vascular endothelial cells in this step is not particularly limited as long as it is a method capable of forming micropatterned vascular endothelial cells adhered to the stimulus-responsive layer. The method can be used.

  The micropatterned vascular endothelial cells formed in this step are those in which vascular endothelial cells are bound to each other and can maintain a state in which the cells are bound to each other even when detached from the stimulus-responsive layer. Is. Further, it has a micro pattern, that is, a pattern of the stimulus-responsive layer having a micron (μm) scale width, that is, a pattern shape of an opening of the cell adhesion inhibition layer formed with a micron scale width.

  Note that the micropatterned vascular endothelial cell transfer substrate used in this step is the same as that described in the section “A. Micropatterned vascular endothelial cell transfer substrate”, and the description thereof is omitted here. .

2. Vascular endothelial cell transfer step The vascular endothelial cell transfer step in the present invention is performed under the condition that the stimulus-responsive layer after the micropatterned vascular endothelial cell formation step expresses cell non-adhesiveness. An artificial tissue containing at least the micropatterned vascular endothelial cells by transferring the micropatterned vascular endothelial cells to the cell recovery layer by contacting the cells with a cell recovery layer including a cell adhesion layer having cell adhesion. Is a step of forming.

In this step, the method for setting the stimulus-responsive layer as a condition in which cell non-adhesiveness is expressed varies depending on the type of the stimulus-responsive material contained in the stimulus-responsive layer.
Specifically, when the stimulus-responsive material is PIPAAm, the micropatterned vascular endothelial cell transfer substrate to which the micropatterned vascular endothelial cells are attached is placed in an incubator set at a temperature of 32 ° C. or lower. A method of standing still can be used.

In this step, as a method for transferring the micropatterned vascular endothelial cells to the cell recovery layer by bringing the micropatterned vascular endothelial cells into contact with the cell recovery layer, the micropatterned vascular endothelial cell formation is performed. It is not particularly limited as long as the method is a contact method under the condition that the stimulus-responsive layer after the step expresses cell non-adhesiveness, and the micropatterned vascular endothelial cell and the cell recovery layer are brought into contact with each other. After the treatment, the stimulation-responsive layer may be transferred as a condition that expresses cell non-adhesiveness, and after the condition that the stimulus-responsive layer expresses cell non-adhesiveness, The micropatterned vascular endothelial cell may be brought into contact with the cell collection layer.
In this step, in particular, after the micropatterned vascular endothelial cells and the cell recovery layer are brought into contact, the transcription is performed under the condition that the stimulus-responsive layer expresses cell non-adhesiveness. Is preferred. This is because the micropatterned vascular endothelial cells can be transferred while maintaining the micropattern more stably.

  The place where the micropatterned vascular endothelial cells and the cell collection layer are brought into contact with each other in this step is preferably in the medium.

  Moreover, in this process, after making the said stimulus responsive layer express cell non-adhesiveness, the process which adds external stress, such as pipetting, to the said stimulus responsive layer may be performed. This is because the micropatterned vascular endothelial cells can be more stably transferred.

The cell recovery layer in this step is composed of a cell adhesion layer, and this step may directly contact the micropatterned vascular endothelial cells and the cell adhesion layer. And a cell layer composed of cells adhered and stacked on the cell adhesion layer, and this step may contact the micropatterned vascular endothelial cell and the cell layer. .
It is because the target artificial tissue can be obtained by performing such a process. Moreover, it is because an artificial tissue having a complicated function can be formed by combining a plurality of types of cells.
Such a cell recovery layer is the same as the contents described in the above-mentioned section “A. Micropatterned Vascular Endothelial Cell Transfer Substrate”, and the description thereof is omitted here.

3. Method for Producing Artificial Tissue The method for producing an artificial tissue of the present invention includes at least the micropatterned vascular endothelial cell formation step and the vascular endothelial cell transcription step, but may have other steps as necessary. good.
Examples of such other processes include a lamination process in which a cell layer made of cells is adhered and laminated on the micropatterned vascular endothelial cells transferred onto the cell collection layer. This is because an artificial tissue having a more complicated function can be formed by combining a plurality of types of cells with the micropatterned vascular endothelial cells.
Moreover, the collection | recovery process etc. which peel the said artificial tissue from the said cell collection layer can be mentioned. This is because a more complex artificial tissue can be constructed and transplanted easily by combining the artificial tissue with another artificial tissue.

(1) Lamination process The lamination process in this invention is a process which adhere | attaches and laminate | stacks the cell layer which consists of a cell on the said micropatterned vascular endothelial cell transcribe | transferred on the said cell collection layer.
In this step, as the method of laminating the cell layer, for example, a method of seeding and culturing cells directly on the micropatterned vascular endothelial cells, or a cell layer obtained by culturing cells can be obtained. And a method of using a cell transfer substrate capable of transferring a cell.
In this embodiment, the method using the cell transfer substrate is particularly preferable. It is because a cell layer can be laminated | stacked stably.

  The cell transfer substrate used in this step may be any substrate that can form the cell layer and transfer the cell layer onto the micropatterned vascular endothelial cells. Specifically, the cell transfer substrate has cell adhesiveness. And what has a cell culture layer from which the adhesive force with the said cell becomes lower than the adhesive force with respect to the said cell of the said micropatterned vascular endothelial cell can be used.

  Such a cell culture layer may be one that does not change the degree of cell adhesion, and in particular, has a cell responsiveness and a stimulus responsiveness that can express cell non-adhesiveness by stimulation. It is preferable to have it. The layering step is performed by preparing a cell transfer substrate having the cell culture layer, and seeding and culturing the cells on the cell culture layer under the condition that the cell culture layer expresses cell adhesion. The cell layer is contacted with the micropatterned vascular endothelial cell under the condition that the cell layer forming step for forming the cell layer and the cell culture layer after the cell layer forming step express cell non-adhesiveness A cell transfer step of transferring the cell layer onto the micropatterned vascular endothelial cells, and the formation and transfer of the cell layer can be facilitated. is there.

  Moreover, the cell transfer substrate used in this step may have a cell adhesion inhibiting layer in a pattern on the cell culture layer as necessary. This is because the cell layer can be formed in a pattern.

Specific examples of the cell culture layer and cell adhesion-inhibiting layer having stimulus responsiveness used in this step include the stimuli-responsive layer and cells described in the above section “A. Micropatterned vascular endothelial cell transfer substrate”. It can be the same as the adhesion-inhibiting layer.
The cell culture layer in which the degree of cell adhesion does not change can be the same as the cell adhesion layer included in the cell collection layer described in the section “A. Micropatterned vascular endothelial cell transfer substrate” above.
In addition, as a method of seeding and culturing cells on the cell transfer substrate and a method of transferring the cell layer, a general method can be used. For example, the above-mentioned “1. Micropatterned vascular endothelial cell formation step” And “2. Vascular endothelial cell transcription step”.
In addition, when the cell culture layer in the cell transfer substrate has a stimulus responsiveness, a method for providing cell adhesion and a method for making the cell culture layer non-adhesive Can be the same as the method described in “1. Micropatterned vascular endothelial cell formation step” and “2. Vascular endothelial cell transcription step”.

  The cells constituting the cell layer in this step are appropriately selected according to the use of the artificial tissue produced by the method for producing an artificial tissue of the present invention, and “A. Micropatterned vascular endothelial cell” This can be the same as that described in the section “Transfer substrate”.

(2) Recovery step The recovery step in the present invention is a step of peeling the artificial tissue from the cell recovery layer.

In this step, the method for peeling the artificial tissue from the cell recovery layer is not particularly limited as long as the artificial tissue can be peeled stably.
As such a method, for example, a method of peeling due to a difference in adhesive force with an adherend such as a transplant destination or other artificial tissue, more specifically, at the time of peeling from the cell recovery layer, Examples thereof include a method using a cell recovery layer that can have a lower adhesion to an artificial tissue than the adherend.
Specifically, in this step, when the cell adhesion layer contained in the cell collection layer contains gelatin, a method of peeling from the cell collection layer is performed by attaching the artificial tissue and the cell adhesion layer. The method is preferably a method in which the body is kept in contact with the body and then kept at the culture temperature. Gelatin can be dissolved and removed from the artificial tissue by heating in the vicinity of a temperature suitable for culture, specifically within a range of 33 ° C. to 40 ° C. Therefore, as illustrated in FIG. 5, the artificial tissue 15 is brought into contact with the adherend 40 (FIG. 5 (a)), and the cell adhesion layer 20 is kept at the culture temperature (heated) in the contacted state. This is because the artificial tissue can be easily recovered by removing the cell adhesion layer (FIG. 5C) (FIG. 5B).
Moreover, when the cell adhesion layer to be used is a cellulose film, a PVDF film, parchment paper or the like, as a method of peeling from the cell adhesion layer, water is dropped into the cell adhesion layer to absorb moisture, thereby the cell adhesion layer. The method of reducing the adhesive force with the artificial tissue can be used.
In addition, about the code | symbol in FIG. 5, since it shows the same member as FIG. 1 and FIG. 2, description here is abbreviate | omitted.

  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]
(1-1) Preparation of temperature-responsive film N-isopropylacrylamide was dissolved in isopropyl alcohol (IPA) so as to have a final concentration of 20% by weight. A commercially available easy-adhesive polyethylene terephthalate film (obtained from Sanko Sangyo Co., Ltd., transparent 50-F Sepa 1090) (hereinafter sometimes abbreviated as “easily-adhesive PET”) was procured and cut into 10 cm squares. The above solution was spread on the easy-adhesion surface of easy-adhesion PET and coated with a Miya bar. The sample was irradiated with an electron beam using an electron beam irradiation apparatus (manufactured by Iwasaki Electric Co., Ltd.), and the solution was graft polymerized. The electron beam irradiation dose at this time was 300 kGy.

(1-2) Immobilization of Cell Adhesion Inhibiting Material Methacryloxysilane (obtained from Momentive Performance Materials, TSL8370) was prepared and diluted with isopropyl alcohol (IPA) to 0.1 wt%.
The film obtained above was fixed to glass with a tape, coated with methacryloxysilane using a spinner (Mikasa), and subjected to silane treatment.
Next, polyethylene glycol diacrylate (hereinafter sometimes referred to as PEGDA) (molecular weight 300, obtained from Aldrich) is diluted with pure water so that the final concentration is 50% by weight. As a polymerization initiator, 2-hydroxy-4 ′-(2-hydroxyethoxy) -2-methylpropiophenone (obtained from Aldrich) was added to a final concentration of 1% by weight. This solution was stirred with a stirrer for 15 minutes.
1 mL of this solution was developed on a silane-treated film, and spin coating was performed at a final concentration of 50% and 900 rpm. Thereafter, using a photomask, the exposure apparatus was processed with an energy of 150 mJ to form a cell adhesion inhibition layer, and a micropatterned vascular endothelial cell transfer substrate was formed.
Then, it confirmed visually that the poly erylene glycol diacrylate was fix | immobilized on the silane processing base material.

[Example 2]
A micropatterned vascular endothelial cell transfer substrate was formed in the same manner as in Example 1 except that the spin conditions of the solution containing polyethylene glycol diacrylate were changed to a final concentration of 50% and 750 rpm.

[Comparative Example 1]
A micropatterned vascular endothelial cell transfer substrate was formed in the same manner as in Example 1 except that the spin condition of the solution containing polyethylene glycol diacrylate was changed to a final concentration of 40% and 1000 rpm.

[Comparative Example 2]
A micropatterned vascular endothelial cell transfer substrate was formed in the same manner as in Example 1 except that the spin conditions of the solution containing polyethylene glycol diacrylate were changed to a final concentration of 40% and 750 rpm.

[Comparative Example 3]
A micropatterned vascular endothelial cell transfer substrate was formed in the same manner as in Example 1 except that the spin condition of the solution containing polyethylene glycol diacrylate was changed to a final concentration of 60% and 900 rpm.

[Comparative Example 4]
A micropatterned vascular endothelial cell transfer substrate was formed in the same manner as in Example 1 except that the spin condition of the solution containing polyethylene glycol diacrylate was changed to a final concentration of 60% and 825 rpm.

[Comparative Example 5]
A micropatterned vascular endothelial cell transfer substrate was formed in the same manner as in Example 1 except that the spin conditions of the solution containing polyethylene glycol diacrylate were changed to a final concentration of 60% and 750 rpm.

[Evaluation]
(1) Height measurement of cell adhesion inhibition layer The height of the cell adhesion inhibition layer of the micropatterned vascular endothelial cell transfer substrate prepared in Examples and Comparative Examples was measured using a three-dimensional white light interference profiler. The measurement results are shown in Table 1 below.

(2) Cell adhesion evaluation Moreover, the degree of cell adhesion about the material (PEGDA, N-isopropylacrylamide) used by the Example and the comparative example was evaluated.
In the evaluation method, bovine vascular endothelial cells (HH cells) were seeded on each of the above materials at 22000 cells / cm ² , the medium was changed after 14.5 hours, and the number of expanded cells (observation field 0.88 mm × 0.67 mm). = 0.59 mm ² ).
As a result, 0 to 2 on the surface of the PEGDA polymer forming the cell adhesion inhibiting layer, 0 on the most area (per visual field), that is, 0 to 340 cells / cm ² , and cell adhesion spreading rate Was less than 1.6%.
When the surface after graft polymerization of N-isopropylacrylamide (surface of the stimulus-responsive layer (temperature-responsive layer)) expresses cell adhesion (37 ° C.), 84 to 133 (one field of view) Around), that is, 14200 to 22500 cells / cm ² , and the cell adhesion extension rate was 60% or more.
On the other hand, when the surface of the stimulus-responsive layer (temperature-responsive layer) expresses cell non-adhesiveness (20 ° C.), 0 to 2 in the most region, 0 (per visual field), That is, it was 0 to 340 cells / cm ² and the cell adhesion extension rate was less than 1.6%.

(3) Cell culture (evaluation of pattern maintenance)
Micropatterned vascular endothelial cell transfer substrates prepared in Examples and Comparative Examples were cut into 20 mmφ, the adhesive layer was exposed, and attached to the bottom of a 35 mmφ polystyrene dish (manufactured by Becton Dickinson), which was 70% ethanol Sterilized. The sterilization time was 1 hour.
HUVEC was adjusted to 4 × 10 ⁵ cells / cm ² and seeded in a micropatterned vascular endothelial cell transfer substrate.
At this time, EGM-2 was used as the medium used, and the following day, the medium was replaced with a high serum medium (FBS 30%, ascorbic acid ester, copper sulfate added). Cultivation was performed in a CO ₂ incubator at 37 ° C. and 5% CO _2. After 4 days of culturing, whether or not the cells maintained the pattern (pattern sustainability) was confirmed with a phase contrast microscope and judged according to the following criteria. . The results are shown in Table 1 below.
○: Cells adhere and grow only on the stimulus-responsive layer.
(Triangle | delta): The cell has adhere | attached and extended by the ratio of 30% or less (1 visual field or less among 5 visual fields) to parts other than a stimulus response layer.
X: Cells adhere to and spread at a ratio of 30% or more (2 or more out of 5 fields of view) other than the stimulus-responsive layer.

(4) Artificial tissue production (evaluation of cell transcription)
Under room temperature conditions, Tsuda et al. Biomaterials, 28 (2007) A cell recovery layer consisting of a gelatin stamp prepared by the method described in 4939-46 was brought into contact with patterned HUVEC, allowed to stand at 20 ° C. for 30 minutes, and then carefully gelatinized. The stamp was lifted and contacted on another human skin fibroblast (NHDF) sheet. Thereafter, it was incubated for 15 minutes in a 37 ° C. incubator, and the stamp was carefully lifted. Thereafter, whether or not the HUVEC pattern was satisfactorily transferred onto the NHDF sheet (cell transferability) was confirmed with a phase contrast microscope, and evaluated according to the following criteria. The results are shown in Table 1 below.
○: All cell patterns were collected in the area contacted with gelatin.
Δ: Poor cell recovery is observed at a ratio of 30% or less (1 field or less out of 5 fields) in the area contacted with gelatin.
X: Poor cell recovery is observed at a rate of 30% or more (2 or more out of 5 fields) in the area contacted with gelatin.

  As shown in Table 1, when using the micropatterned vascular endothelial cell transfer substrate prepared in the examples, a desired pattern can be formed on the micropatterned vascular endothelial cell transfer substrate, and It was confirmed that stable transfer was possible.

DESCRIPTION OF SYMBOLS 1 ... Base material 2 ... Stimulus responsive layer 3 ... Cell adhesion inhibition layer 10 ... Micropatterned vascular endothelial cell transfer substrate 11 ... Vascular endothelial cell 12 ... Micropatterned vascular endothelial cell 14 ... Cell layer 15 ... Artificial tissue 20 ... Cell Adhesive layer 30 ... Cell recovery layer 40 ... Adherent

Claims (10)

A substrate;
A stimulus-responsive layer comprising a stimulus-responsive material disposed on the substrate and having cell adhesion and capable of expressing cell non-adhesion by stimulation;
A cell adhesion-inhibiting layer that is arranged in a pattern on the substrate and contains a cell adhesion-inhibiting material having cell non-adhesiveness;
Have
The surface of the cell adhesion inhibition layer is formed to be higher than the surface of the stimulus responsive layer,
The lower limit of the height of the cell adhesion-inhibiting layer is a height that cannot be overcome by vascular endothelial cells, and the upper limit is a height at which the micropatterned vascular endothelial cells formed on the stimulus-responsive layer can be recovered. A micropatterned vascular endothelial cell transfer substrate characterized in that:   2. The micropatterned vascular endothelial cell transfer substrate according to claim 1, wherein the stimulus-responsive material is a temperature-responsive material that exhibits cell non-adhesiveness due to a temperature change.   The micropatterned vascular endothelial cell transfer substrate according to claim 2, wherein the temperature-responsive material is poly-N-isopropylacrylamide (PIPAAm).   The micropatterned vascular endothelial cell transcription according to any one of claims 1 to 3, wherein the width of the exposed surface of the stimulus-responsive layer is in the range of 15 µm to 500 µm. substrate.   The micropatterned vascular endothelial cell transfer substrate according to any one of claims 1 to 4, wherein the cell adhesion-inhibiting material is a polyethylene glycol-based material. A method for producing an artificial tissue comprising at least micropatterned vascular endothelial cells,
Preparing a micropatterned vascular endothelial cell transfer substrate according to any one of claims 1 to 5,
A micropatterned vascular endothelium that forms the micropatterned vascular endothelial cell by seeding and culturing the vascular endothelial cell on the stimulus responsive layer under a condition in which the stimulus responsive layer expresses cell adhesion A cell formation process;
A cell recovery layer comprising a cell adhesion layer having cell adhesion with the micropatterned vascular endothelial cells under the condition that the stimulus-responsive layer after the micropatterned vascular endothelial cell formation step expresses cell non-adhesiveness A vascular endothelial cell transfer step of transferring the micropatterned vascular endothelial cells to the cell recovery layer by contacting with
A method for producing an artificial tissue, comprising: The cell recovery layer comprises the cell adhesion layer;
The method for producing an artificial tissue according to claim 6, wherein the vascular endothelial cell transfer step directly contacts the micropatterned vascular endothelial cell and the cell adhesion layer. The cell recovery layer comprises the cell adhesion layer, and a cell layer composed of cells adhered and laminated on the cell adhesion layer,
The method for producing an artificial tissue according to claim 6, wherein the vascular endothelial cell transfer step is to bring the micropatterned vascular endothelial cells into contact with the cell layer.   9. The method according to claim 7, further comprising a laminating step of adhering and laminating a cell layer composed of cells on the micropatterned vascular endothelial cells transferred onto the cell recovery layer. Method for manufacturing artificial tissue.   The method for producing an artificial tissue according to any one of claims 6 to 9, further comprising a recovery step of peeling the artificial tissue from the cell recovery layer.

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