JP2015029468A - Producing method of cell tubular tissue - Google Patents

Abstract

An object of the present invention is to provide a method and a means for facilitating handling in organizing a cell sheet into a tubular shape.
The present invention comprises a film having a cell culture layer on a first surface and an adhesive layer on an end of a second surface, which is the back surface of the first surface, The present invention relates to the cell culture substrate configured to form a tubular structure by adhering an adhesive layer to an end portion of a first surface, and a method of forming a cell tubular tissue using the cell culture substrate.
[Selection] Figure 8

Description

  The present invention relates to a method for efficiently producing a cellular tubular tissue and an apparatus therefor.

  Traditionally, cell culture is essential in the field of cell biology. In recent years, with the proliferation of stem cell research and regenerative medicine research, the importance of cell culture technology development is increasing. A technique called “cell sheet engineering” in which cells are cultured in a sheet form and the cells are recovered in a sheet form by simply lowering the temperature without using an enzyme such as trypsin has attracted attention in the field of regenerative medicine. In this case, a temperature-responsive cell culture substrate to which a temperature-responsive polymer is bound is used.

  Cell sheets obtained by cell sheet engineering have already been confirmed to have a certain therapeutic effect in regenerative medicine such as the cornea and periodontal tissue, and clinical studies and clinical trials are already underway in Europe. In addition, it is possible to create a three-dimensional tissue model by stacking multiple types of cell sheets and to create a mature tissue with vascular tissue in vitro. Treatment is expected.

  Patent Document 1 describes a sheet-like three-dimensional structure for application to a heart disease composed of myoblasts. However, this structure is in the form of a sheet, and there is no description about organizing cells into a tubular shape.

  In Non-Patent Document 1, a cardiomyocyte sheet is prepared by organizing cells into a tube shape, for example, by organizing cardiomyocytes into a tube shape, and wraps the cardiomyocyte around a living artery to perform a heart pump function. Methods for assisting and improving cardiac function are disclosed. However, since the method described in Non-Patent Document 1 is a method in which the obtained cell sheet is wound around a support alone, there is a problem that handling is difficult due to the vulnerability of the cell sheet.

JP 2012-139541 A

Circulation, 2006 Jul 4; 114 (1 Suppl): I87-93

  The present invention has been developed in view of the above problems, and an object of the present invention is to provide a method and a means for facilitating handling in organizing a cell sheet into a tubular shape.

  The present inventors have prepared a film having a cell culture layer on one side and an adhesive layer on the end of the other side, and culturing cells on the cell culture layer of the film to thereby obtain a cell sheet The present invention was completed by finding that the cell sheet can be efficiently organized into a tubular shape by forming the cell sheet together with the film.

That is, the present invention includes the following inventions.
(1) It consists of a film having a cell culture layer on the first surface and an adhesive layer at the end of the second surface opposite to the first surface, and having the adhesive layer on the second surface The cell culture substrate configured to be capable of forming a tubular structure by adhering to an end of the first surface opposite to a distal end with respect to an end on which the adhesive layer is present on the second surface.
(2) The cell culture substrate according to (1), wherein the film has a cell adhesion-inhibiting region at an end of the first surface to which the adhesive layer on the second surface is adhered.
(3) The cell culture substrate according to (1) or (2), wherein the cell culture layer is a stimulus-responsive layer in which cell adhesion changes upon stimulation.
(4) A cell culture container comprising a container and the cell culture substrate according to any one of (1) to (3) disposed on the bottom of the container,
The cell culture container, wherein the cell culture substrate is disposed at the bottom of the container so that the cell culture layer is exposed.
(5) A method for producing a cellular tubular tissue,
a) a step of culturing cells on the cell culture layer of the cell culture substrate according to any one of (1) to (3) to produce a cell sheet; and b) tube formation of the cell sheet together with the cell culture substrate. And forming the cellular tubular tissue.
(6) The method according to (5), wherein in step b), the cell sheet is wound around a support together with a cell culture substrate to form a tube.
(7) The cell culture layer is a stimulus-responsive layer in which cell adhesion changes upon stimulation, and further comprises a step of peeling the cell tubular tissue from the cell culture substrate by stimulation. Method.

  According to the present invention, the production efficiency of the tubular cell tissue structure can be improved, and the transplantation efficiency can be improved.

It is the schematic which shows the vertical cut surface of one Embodiment of the cell culture substratum of this invention. It is the schematic which shows the vertical cut surface of one Embodiment of the cell culture substratum of this invention. It is the schematic which shows the upper side figure of one Embodiment of the cell culture substratum of this invention. It is the schematic which shows the vertical cut surface of one Embodiment of the cell culture substratum of this invention. It is the schematic which shows the vertical cut surface of one Embodiment of the cell culture substratum of this invention. It is the schematic which shows the upper side figure of one Embodiment of the cell culture substratum of this invention. It is the schematic which shows one Embodiment of the cell culture substratum and cell culture container of this invention. It is the schematic which shows one Embodiment of the production method of the cellular tubular tissue of this invention. It is the schematic which shows one Embodiment of the production method of the cellular tubular tissue of this invention. It is the schematic which shows one Embodiment of the production method of the cellular tubular tissue of this invention.

  For example, as shown in FIG. 1, the cell culture substrate A of the present invention has a cell culture layer B on the first surface 1, and the second surface 2 that is the surface opposite to the first surface 1. It consists of a film 4 having an adhesive layer C at the end 3. For example, as shown in FIG. 2, a tubular structure can be formed by adhering the adhesive layer C of the second surface 2 to the end portion 5 of the first surface 1. The cell culture layer B may be formed on the end portion 5 of the first surface 1 to which the adhesive layer C is bonded. However, from the viewpoint of more easily bonding, the cell culture layer does not exist and the film is It is preferable to be exposed.

  A cut surface perpendicular to the film plane of one embodiment of the cell culture substrate of the present invention is shown in FIG. 1, and a top view of a second surface of the embodiment is shown in FIG. As shown in FIG. 1, in the film 4, the first surface 1 on which the cell culture layer B exists corresponds to a surface (back surface) opposite to the second surface 2 on which the adhesive layer C exists. The shape of the film is not particularly limited as long as it can form a tubular structure, but it is generally a quadrangle, preferably a substantially rectangle. The size of the film is appropriately determined based on the cellular tubular tissue to be formed. Preferably, one side (for example, the side length X and Y in FIG. 3) is the same or different, and is 10 to 200 mm. More preferably, it is a rectangle of 20 to 90 mm, preferably a square. If it is the said size, it will become easy to arrange | position on the bottom face of the cell culture container generally used.

  The thickness of the film is not particularly limited as long as it has flexibility, and is appropriately selected depending on the material of the film, but is usually 10 to 200 μm, preferably 30 to 100 μm. If it is the said thickness, it will become possible to deform | transform into a tubular shape easily via tweezers.

  The material of the film is not particularly limited as long as it has flexibility, but is preferably a plastic film. The plastic is not particularly limited, but polyethylene terephthalate (PET), polystyrene (PS), polycarbonate (PC), TAC (triacetyl cellulose), polyimide (PI), nylon (Ny), low density polyethylene (LDPE), medium Examples include density polyethylene (MDPE), vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyether sulfone, polyethylene naphthalate, polypropylene, and acrylic.

  The shape of the tubular structure formed by the cell culture substrate is substantially the same as the shape of the cellular tubular tissue to be formed, and is elongated, hollow inside, and open at both ends. There is no particular limitation. The length of the tubular structure is usually equal to the length of one side X of the film. It is also possible to cut the film at an arbitrary width in parallel with the side Y (for example, cut along the dotted line in FIG. 3), and in this case, the cut width (X ′) is equal to the length of the tubular structure. Become.

  The cross-sectional shape of the tubular structure is not particularly limited, but is preferably substantially circular as shown in FIG. The substantially circular shape includes a circular shape having a distorted or uneven portion, an elliptical shape, and the like, and preferably a circular shape. The dimension of the hollow portion of the cross section depends on the length Y of one side of the film and depends on the width at which the first surface and the second surface overlap when formed into a tube. It is a 30 mm circle. If it is the said range, in the above-mentioned preferable film size, the deformation | transformation to a tubular shape will be attained satisfactorily.

  The adhesive layer is a layer containing an adhesive and is present at least at the end of the second surface 2. By providing an adhesive layer on at least the end 3 of one surface of the film, the adhesive layer C of the second surface 2 can be adhered to the end of the first surface 1 to form a tubular structure. The adhesive layer C exists at the end 3 of the second surface 2 for forming a tubular structure, and the end refers to a so-called marginal portion. The end portion may be substantially an end portion, and is not necessarily the endmost portion. If the film is approximately square, it refers to a strip area along either side. The width of the band-like region, for example, the width Z in FIG. 3, is preferably about 20 to 50%, more preferably about 30 to 45% of the length Y of one side of the quadrangle. More specifically, the width of the band-like region, for example, the width Z in FIG. 3, is preferably 10 to 40 mm, more preferably 15 to 30 mm. If it is the said range, it is thought that it is enough to adhere | attach the 1st surface and 2nd surface of a film. Moreover, the effective area of a cell is fully securable by setting it as 40 mm or less. As long as a tubular structure can be formed, the adhesive layer C may be formed on the entire surface of the band-shaped region, or may be partially formed, for example, may be formed in a stripe shape or a dot shape. .

  By providing an adhesive layer, it is possible to fix the overlapping portion of the film when the film is rolled to form a tube, and to maintain the tubular structure firmly, which is advantageous in the formation of cellular tubular tissue. In addition, since the adhesive layer exists on the surface opposite to the cell culture layer in the step of culturing cells on the cell culture layer of the film before forming the film to form a cell sheet, the adhesive component The effect of cell elution on cell culture is small.

  The adhesive constituting the adhesive layer is not particularly limited as long as it does not inhibit cell culture. For example, a silicone-based or acrylic-based adhesive is preferable. Even when cell culture is carried out using a commercially available silicone-based (deoxime type) adhesive, no cytotoxicity is observed.

  The thickness of the adhesive layer is appropriately determined according to the adhesive to be used, and is not particularly limited as long as the first surface and the second surface of the film can be bonded, but is usually 5 to 50 μm, preferably 10 to 30 μm. It is. By setting the thickness to 50 μm or less, it is possible to suppress a risk that the cell sheet breaks due to the step difference after being deformed into a tubular shape. By setting it as 5 micrometers or more, the function as an adhesive agent is insufficient and the risk that a tubular structure cannot be maintained can be suppressed.

  The cell culture layer is a layer for culturing cells on the surface thereof, and is a layer in which the surface expresses cell adhesiveness at least during cell culture. As the cell culture layer, for example, a cell adhesion layer that always expresses cell adhesion, and a stimulus responsive layer in which cell adhesion changes upon stimulation can be used. The cell adhesion layer that always expresses cell adhesion is a layer containing a cell adhesion material. Examples of the cell adhesive material include cell adhesive proteins such as collagen, fibronectin, laminin and elastin. The stimulus responsive layer will be described later.

  The expression of cell adhesion means that the cells are easy to adhere and spread, and the cell adhesion spread rate is high, specifically, the cell adhesion spread rate is 60% or more, preferably 80% or more. Say. In the case where cell non-adhesiveness is expressed, the cells are difficult to adhere and spread, and the cell adhesion spreading rate is low. Specifically, the cell adhesion spreading rate is 5% or less, preferably 2% or less. The state which is.

The cell adhesion spreading rate is, for example, inoculated with bovine vascular endothelial cells 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%) for 3 hours. It represents the ratio of the cells that have spread and adhered at the time of culture ({(number of cells that have adhered) / (number of cells that have been seeded)} × 100 (%)).

  When cells are seeded on a film having a cell culture layer, the cells grow by adhering to the cell culture layer, so that a cell sheet is formed in a region where the cell culture layer is present on the film. Therefore, on the first surface of the film, the cell culture layer is appropriately formed with a planar shape similar to the planar shape of the cell sheet to be formed. The cell culture layer may be formed on the entire first surface of the film or may be formed in a pattern. The shape of the pattern is not particularly limited as long as the resulting cell sheet can be tubed with a cell culture substrate to form a cell tubular tissue. For example, a polygon including a quadrangle, a circle including an ellipse, a lattice shape, and a mesh shape Is mentioned. In addition, the cell adhesion layer may be multifaceted according to any cutout position.

  The end of the first surface to which the adhesive layer C is bonded is, for example, as shown in FIG. 1, facing the distal end with respect to the end 3 where the adhesive layer is present on the second surface 2. An end 5 on the surface 1. It is preferable that a cell adhesion inhibition region is present at the end portion 5 of the first surface to which the adhesive layer C adheres. The cell adhesion-inhibiting region is a non-cell-adhesive region. When cells are seeded on the first surface of a film having a cell culture layer and a cell adhesion-inhibiting region, the cells adhere to the cell culture layer and proliferate. A sheet is formed, but no cell sheet is formed because cells do not adhere to the cell adhesion-inhibiting region.

  In the present invention, after the cell sheet is formed on the cell culture layer, the end of the second surface having the adhesive layer C is adhered to the end of the first surface to form a tubular structure, thereby Form a tubular tissue. Even if cells are present at the end of the first surface to which the adhesive layer C is bonded, the end of the first surface and the end of the second surface are bonded by destroying the cells at the time of bonding. However, the absence of cells is preferable from the viewpoint of fixing the overlapping portions of the films and maintaining the tubular structure. Moreover, it is preferable also from a viewpoint of not damaging a part of cell sheet.

  For example, as shown in FIG. 4, the cell adhesion inhibition region is a region having a cell adhesion inhibition layer D whose surface is non-cell-adhesive. The cell adhesion inhibition layer D may be formed on the cell culture layer B on the first surface 1 as shown in FIG. 4a, or directly formed on the first surface 1 as shown in FIG. 4b. May be. Alternatively, when the film itself is non-cell-adhesive, it may simply be an area where the cell culture layer is not present and the film is exposed. In the cell culture substrate shown in FIG. 4a, the cut surface when the tubular structure is formed by adhering the adhesive layer on the second surface to the cell adhesion inhibition layer D on the end of the first surface is shown in FIG. Show.

  The cell adhesion-inhibiting region is present at least at the end of the first surface 1. Since the adhesive layer C of the second surface 2 is adhered to the cell adhesion-inhibiting region existing at the end 5 of the first surface 1 to form a tubular structure, the end 5 is formed on the second surface. It is a distal end relative to the end portion 3 where the adhesive layer C exists, and refers to a so-called marginal portion existing on the first surface. The end portion may be substantially an end portion, and is not necessarily the endmost portion. A top view of the first surface of the embodiment shown in FIG. 4a is shown in FIG. As shown in FIG. 6, the cell adhesion inhibition region is a belt-like region along the opposite side of the side where the belt-like region of the adhesive layer C is present when the film is substantially square, and exists on the first surface. This is a belt-like region. The width of the belt-like region, for example, the width W in FIG. 6, is preferably about 10 to 30%, more preferably about 15 to 25% of the length Y of one side of the quadrangle. More specifically, the width of the belt-like region, for example, the width W in FIG. 6 is preferably 5 to 20 mm, more preferably 7 to 15 mm. If it is the said range, it will become possible to obtain a favorable tubular structure | tissue, without a cultured cell and an adhesive agent contacting. By setting it to 20 mm or less, a cell adhesion region can be sufficiently secured. Moreover, the lack of the adhesion | attachment area | region after deform | transforming into a tubular shape can be prevented by setting it as 5 mm or more. The cell adhesion inhibition region may be formed on the entire surface of the band-shaped region or may be partially formed as long as a tubular structure can be formed. For example, the cell adhesion inhibition region may be formed in a stripe shape or a dot shape. .

  A hydrophilic polymer is mentioned as a cell non-adhesive material which comprises a cell adhesion inhibition layer. Specific examples of the hydrophilic polymer include polyalkylene glycol, polyvinyl pyrrolidone, polypropylene glycol, polyvinyl alcohol, polyethylene imine, polyallylamine, polyvinyl amine, polyvinyl acetate, polyacrylic acid, poly (meth) acrylamide and the like. Examples include copolymers with monomers and graft polymers. Among them, polyalkylene glycols having various molecular weights are commercially available and are excellent in biocompatibility, so that they can be suitably used. Specific examples of the polyalkylene glycol include polyethylene glycol, polypropylene glycol, a copolymer of ethylene glycol and propylene glycol, and polyethylene glycol diacrylate. Polyethylene glycol is preferably used. The thickness of the cell adhesion-inhibiting layer is not particularly limited because the steps required for exhibiting the cell adhesion-inhibiting function differ depending on the material. For example, in the case of polyethylene glycol, the range of 1 to 5 nm is sufficient. In the case of polyacrylamide (gel) or polyethylene glycol gel, the cell adhesion inhibiting function is sufficiently exerted in the range of 1 μm to 20 μm.

  The method for forming a cell culture layer, an adhesive layer, a cell adhesion inhibition layer, or the like on the film is not particularly limited. Specifically, a composition containing a material constituting each layer is applied onto the film using a known application method such as spin coating. When forming a pattern for each layer on the film, use a patterning method by photolithography, or a patterning method using a known pattern coating method such as gravure printing, flexographic printing, screen printing, or inkjet method. it can. You may produce by carrying out an electron beam or an ultraviolet-ray process in a pattern shape through the metal mask etc. which have a through-hole (through-hole).

  The present invention also relates to a cell culture container provided with the cell culture substrate. For example, as shown in FIG. 7, the cell culture container of the present invention comprises a container 6 such as a culture dish and a cell culture substrate A made of a film, and a cell culture layer B of the cell culture substrate is present. The cell culture substrate can be arranged on the bottom surface of the container so that one surface is exposed. Since the cell culture substrate is taken out together with the cell sheet after cell culture, it is arranged so that it can be taken out from the bottom of the container. For example, the cell culture substrate is detachably disposed on the bottom surface of the container. Specifically, culture is performed in a system in which the culture solution does not leak when the culture solution is placed inside with the cell culture surface facing upward and a rectangular side wall made of PDMS (polydimethylsiloxane). I do.

  As a container in which the cell culture substrate is disposed, a petri dish having a bottom surface of a single plane and a diameter of 30 to 60 mm is preferably used. This is preferable because it is equivalent to a petri dish used for conventional cell culture, can be easily produced from a general-purpose petri dish, and is easily adapted to an existing culture apparatus. As a material constituting the container of the cell culture container, metals, glass, ceramics, plastics, elastomers and the like and composite materials thereof can be used, but are not limited thereto. A transparent material is particularly desirable. This is because it facilitates microscopic observation of cells. If the vapor deposition layer of gold or the like is about 50 nm in thickness, it can be suitably used because it has optical transparency. The example of plastic is the same as described above.

The present invention also relates to a method of making a cellular tubular tissue, the method comprising, for example, as shown in FIG.
a) a step of culturing the cell 7 on the cell culture layer B of the cell culture substrate A to produce a cell sheet; including.

  In the method of the present invention, since the cell sheet is tubed together with the cell culture substrate to form the cell tubular tissue, the tube structure can be formed while supporting the fragile cell sheet, and the cell tubular tissue can be efficiently formed. Can be produced. The cell sheet is a sheet in which cells are bound to each other in at least a single layer by intercellular bonding.

  In the step of pipetting the cell sheet together with the cell culture substrate, as shown in FIG. 8b, the cell sheet may be piped so as to be inside the pipe, or as shown in FIG. Although it may be tubed so that it is outside the tube, it is preferably tubed so that the cell sheet is inside the tube. The cell sheet on the inside has advantages such as easy removal of the substrate after making it tubular. It is thought that when the cell sheet is peeled off from the cell adhesion layer, it shrinks, and the outer diameter of the tubular cell sheet is smaller than the inner diameter of the tubular base material, which is considered to be easy to remove.

  Since the cell tubular tissue is formed by pipetting a cell sheet formed on a cell culture substrate together with the cell culture substrate, the shape thereof is substantially the same as the tubular structure of the cell culture substrate described above. It is. That is, it has a shape that is elongated and hollow inside, and both ends are open to the outside, and the length of the cellular tubular tissue is usually approximately equal to the length X of one side of the film. When the film is used after being cut at an arbitrary width parallel to the side Y, the cut width (X ′) is substantially equal to the length of the cellular tubular tissue. The film may be cut before the cell sheet is formed, or the cell sheet and the film may be cut together after the cell sheet is formed.

  In step b), for example, as shown in FIG. 9, it is preferable to form a tube by winding the cell sheet around the support 8 together with the cell culture substrate. It is possible to improve the cardiac function by wrapping a cell sheet on a support derived from a living body such as an artery to assist the pump function of the heart and transplanting the cell sheet. From the viewpoint of forming a cell tubular tissue by winding a cell sheet, the support derived from a living body is preferably a rod-like or tubular support such as a blood vessel.

  Using a tissue obtained from a living body as a support derived from a living body, the support can be wound outside the living body, and then transplanted into the living body. Alternatively, a model structure can be constructed and used for an assay by winding a cell sheet around a support that is not derived from a living body and forming a tube. As the support in that case, materials usually used for cell culture, for example, metal, glass, ceramics, plastics, elastomers, etc., and rod-like supports and tubular supports of these composite materials can be used, but are not limited thereto. .

  In the method of the present invention, as a cell culture layer, a stimulus-responsive layer in which cell adhesion is changed by stimulation is used to stimulate cell tubular tissue 9 from cell culture substrate A as shown in FIG. Can be peeled off. The stimulus-responsive layer can attach and detach cells to be cultured by changing the adhesion of the cells by stimulation. The stimulus-responsive layer is preferably capable of changing from a state expressing cell adhesion with high cell adhesion to a state expressing cell non-adhesion with low cell adhesion by a specific stimulus. Is.

  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 exposed to the stimulus-responsive layer when non-adhesive properties are expressed. Since the adhesion to the cell is inhibited, the adhered cells can be detached. Therefore, cells are seeded and adhered to a stimulus-responsive region that expresses cell adhesion, and a cell sheet is formed by culturing the cells, forming cell tubular tissue, and then stimulating with specific stimuli. By changing the responsive region to a state in which cell non-adhesiveness is expressed, the cellular tubular tissue can be detached and obtained.

  By exfoliating the cell tubular tissue from the cell culture substrate, only the cell tubular tissue can be obtained and can be suitably used for transplantation into a living body. If the film, cell culture layer, and adhesive layer that make up the cell culture substrate are biocompatible, it can be transplanted to the living body without removing the cell culture substrate. When assisting the pump function of the heart by moving it, it is preferable to transplant the cell culture substrate after removing the cell culture substrate from the viewpoint of more efficiently expressing the cell function.

  The stimulus responsive layer is a layer containing a stimulus responsive material. The stimulus-responsive material is not particularly limited as long as the adhesion of cells changes depending on the presence or absence of stimulation, and can adhere and peel cells to be cultured. For example, temperature, light, pH Examples thereof include a temperature-responsive material, a photo-responsive material, a pH-responsive material, a potential-responsive material, and a magnetic-responsive material in which cell adhesion changes depending on potential and magnetic force. In the present invention, among them, a temperature responsive material is preferable.

  The temperature-responsive material is not particularly limited as long as the cell adhesiveness changes due to temperature change. The temperature range in which the cell-responsiveness of the temperature-responsive material is exhibited is preferably in the range of 10 ° C to 45 ° C, and more 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-mentioned range. It is preferable that the temperature range which exhibits the cell non-adhesiveness of a temperature-responsive material is in the range of 1 ° C to 36 ° C, and in particular, it is preferably in the range of 4 ° C to 32 ° C. When the temperature region is within the above-described range, damage to cells can be reduced.

  Specific examples of the temperature-responsive material include poly-N-isopropylacrylamide (PIPAAm), poly-Nn-propylacrylamide, poly-Nn-propylmethacrylamide, poly-N-ethoxyethylacrylamide, poly -N-tetrahydrofurfuryl acrylamide, poly-N-tetrahydrofurfuryl methacrylamide, and temperature-responsive polymers such as poly-N, N-diethylacrylamide can be exemplified, among which PIPAAm, poly-Nn- Propylmethacrylamide and poly-N, N-diethylacrylamide can be preferably used, and PIPAAm can be particularly preferably used. This is because the temperature region having cell adhesiveness and the temperature region having cell non-adhesiveness are the above-described temperature regions and can be reduced in damage.

  The temperature-responsive material may be composed of only one type, or may include two or more types. Moreover, in order to adjust a temperature range, what was copolymerized with temperature-responsive materials and / or another polymer may be used.

  The photoresponsive material is not particularly limited as long as the adhesiveness of the cells changes depending on the presence or absence of light irradiation. For example, a photocatalyst disclosed in JP-A-2005-210936, Those containing photoresponsive components such as azobenzene, diarylethene, spiropyran, spirooxazine, fulgide, and leuco dye can be used.

  The potential responsive material is not particularly limited as long as the cell adhesiveness is changed by application of a potential. For example, an electrode as disclosed in JP-A-2008-295382, 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 is not particularly limited as long as the cell adhesiveness is changed by applying / removing the magnetic force. For example, as disclosed in JP-A-2005-312386, ferrite, etc. There can be mentioned positively charged liposomes encapsulating magnetic particles in which positively charged magnetic particles are encapsulated.

  The thickness of the layer of the stimulus responsive material is not particularly limited as long as it can exhibit stimulus responsiveness and cell adhesiveness, 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. Moreover, it is preferable that it is 0.3-6.0 microgram / cm <2> as the coating amount of the layer of a stimulus responsive material. The same applies to cell adhesive materials.

  As the cells constituting the cell sheet and the cell tubular tissue, any tissue existing in a living body and cells derived from it can be used, except for non-adherent cells such as blood cells and cells with weak intercellular bonds. Specifically, epithelial cells and endothelial cells that constitute each tissue and organ in the living body, skeletal muscle cells that exhibit contractility, smooth muscle cells, cardiomyocytes, neurons that constitute the nervous system, glial cells, fibroblasts, As hepatocytes, non-hepatocytes and adipocytes related to metabolism in the living body, cells having differentiation ability, stem cells existing in various tissues, bone marrow cells, ES cells and the like can be used. Only one type of cell may be used, or two or more types of cells may be used.

  According to the present invention, the cell sheet can be handled together with the cell culture substrate, so that the transportation and transplantation to the living body becomes much easier, the engraftment is improved, and the transplantation efficiency of the tissue can be greatly improved. become.

A: Cell culture substrate B: Cell culture layer C: Adhesive layer D: Cell adhesion inhibition layer 1: First side of film 2: Second side of film 3: Edge part of second side 4: Film 5: End portion of first surface 6: Container 7: Cell 8: Support 9: Cell tubular tissue

Claims (7)

  It consists of a film having a cell culture layer on the first surface and an adhesive layer at the end of the second surface opposite to the first surface. The cell culture substrate is configured such that a tubular structure can be formed by adhering to the end of the first surface opposite to the distal end with respect to the end on which the adhesive layer is present.   The cell culture substrate according to claim 1, wherein the film has a cell adhesion-inhibiting region at an end of the first surface to which the adhesive layer on the second surface is adhered.   The cell culture substrate according to claim 1 or 2, wherein the cell culture layer is a stimulus-responsive layer in which cell adhesion changes upon stimulation. A cell culture container comprising a container and the cell culture substrate according to any one of claims 1 to 3 disposed at the bottom of the container,
The cell culture container, wherein the cell culture substrate is disposed at the bottom of the container so that the cell culture layer is exposed. A method for producing a cellular tubular tissue comprising:
a) a step of culturing cells on the cell culture layer of the cell culture substrate according to any one of claims 1 to 3 to produce a cell sheet; and b) pipetting the cell sheet together with the cell culture substrate. Said method comprising the step of forming cellular tubular tissue.   6. The method according to claim 5, wherein in step b), the cell sheet is tubed by winding it around a support together with a cell culture substrate.   The method according to claim 5 or 6, further comprising a step of detaching the cell tubular tissue from the cell culture substrate by stimulation, wherein the cell culture layer is a stimulus responsive layer in which cell adhesion changes upon stimulation.

Images