JP4553038B2 - Cultured cell handling body, method for producing the same, and use thereof - Google Patents

Description

  The present invention relates to a handling body that is a member for handling cultured cells, a manufacturing method thereof, and use thereof.

  In recent years, it has been practiced to construct cell structures such as sheets by culturing cells collected from animals or the like in vitro. Attempts have been made to use such cell structures as devices for realizing the functions of cells and tissues in vivo and in vitro, including in the medical field such as regenerative medicine.

  For example, a cell structure such as a sheet formed by culturing cells is obtained by culturing cells on the surface of a plastic dish or the like to form a sheet, and then peeling the cultured cells from the adhesive surface. When peeling, treatment with a proteolytic enzyme such as trypsin or a chemical is usually performed. However, in such a method, even if the cells form a certain structure such as a sheet on the dish surface, it is extremely difficult to maintain the structure, and individual cells are separated, and floating single cell It will be collected in the state. For this reason, the interaction between cells and the function as a cell population associated therewith will be lost. Such a collapse of the structure is considered to be due to the fact that cell adhesion factors such as extracellular matrix are decomposed by enzyme treatment or the like, and damage is also caused to cells.

  For this purpose, a temperature-responsive polymer such as poly-N-isopropylacrylamide is graft-polymerized on the surface of a culture substrate such as a plastic dish to form a nanometer-level temperature-responsive polymer layer. A technique for culturing cells is known (Patent Document 1). This type of temperature-responsive polymer does not dissolve in water at a certain temperature range, but completely dissolves in water at a certain temperature (the temperature at which it is completely dissolved is referred to as a lower limit or upper critical solution temperature). Have. Therefore, the hydrophilicity and hydrophobicity of the culture substrate surface to which the temperature-responsive polymer is applied can be controlled by the environmental temperature. And after seed | inoculating a cell on the graft layer of a temperature-responsive polymer at 37 degreeC which does not reach | attain the said critical solution temperature (for example, 32 degreeC as an upper limit critical solution temperature), cell culture is carried out, and it is set as 32 degrees C or less. By doing so, the cultured cells can be detached without any enzyme treatment.

  Also, a temperature-responsive polymer and collagen are mixed, dried on a culture substrate to form an adhesion film, cell culture is performed on this film, and the cultured cells are detached by reaching the above critical dissolution temperature. Technology is also known (Patent Document 2). It is described that by mixing a temperature-responsive polymer and collagen, the cells can be separated from the substrate without impairing the bonding between the cells.

  When expressing a predetermined function as a cell structure, the orientation of the cells in the cell structure may be important. For example, it is considered that muscle cells cannot exhibit their original functions as muscle fibers unless they are arranged with a certain orientation. Then, orientation is provided by forming unevenness on the surface of the cell culture substrate by MEMS technology and arranging the cells along the unevenness (Non-patent Document 1).

JP-A-2-21865 JP-A-3-292882 Charest et al., Biomaterials, 2007, 28, 2202-2210)

  According to the methods described in Patent Documents 1 and 2, the temperature-responsive polymer is fixed to the culture substrate, and the temperature-responsive polymer-containing layer is a layer for detaching cells from the culture substrate. It is used as. According to these methods, a cell sheet can be obtained by obtaining a monolayered cultured cell layer on a culture substrate and dissolving the temperature-responsive polymer. However, the obtained single-layer cell sheet is very weak mechanically and extremely difficult to handle, and the cells are adhered to a separately prepared hydrophobic membrane prior to detachment, and after supply to a predetermined application site, hydrophobicity is obtained. It was necessary to peel off the conductive film.

  In addition, as described in Non-Patent Document 1, cells can be cultured with the orientation controlled on the surface of the culture substrate. However, since the cell arrangement state is formed in accordance with the surface shape of the culture substrate, there is a high possibility that the orientation is lost when the cell structure is peeled from the culture substrate. In fact, at present, there is no report on a technique for detaching cells cultured in a controlled orientation state from the culture substrate while maintaining the orientation state, and a cell structure with controlled orientation has not been obtained.

  As described above, peeling the cultured cell layer from the culture substrate used for cell culture still promotes a decrease in cell orientation as well as handling and stability of the cell structure obtained by cell culture. Was the cause. Moreover, it was a cause of not being able to obtain a functionally superior cell structure.

  An object of the present invention is to provide a cultured cell handling body capable of avoiding a peeling operation of a cultured cell layer obtained by culturing from a culture substrate, a production method thereof, and a use thereof.

  Rather than using a temperature-responsive polymer to separate the cell structure from the culture substrate, the present inventors function as a scaffold during cell culture and support the cell structure until needed after cell culture. It was found that it can be used as a constituent material of a cell support that facilitates handling. Moreover, the knowledge that it can utilize as a template which can provide orientation to a cultured cell by forming predetermined unevenness | corrugation in the cell support surface containing a temperature-responsive polymer was acquired. According to the present invention, the following means are provided based on these findings.

  According to the present invention, there is provided a cultured cell handling body comprising: a molded body comprising a matrix containing a temperature-responsive polymer; and a cell adhesion region to which cells can adhere on at least a part of the surface of the molded body. Is done. In this handling body, the cell adhesion region may contain an extracellular matrix component, and the molded body has two or more kinds of layer structures of different compositions, and the outermost layer is the surface layer. It may include a cell adhesion region. In addition, the cell adhesion region may have a concavo-convex shape in which cells can be oriented and cultured, and may include a linear recess having a width of 5 μm or more and 50 μm or less. The cell adhesion region may include a region where Rz is 2 μm or more and an uneven shape is formed. The cell adhesion region may have an uneven shape obtained by reversing the surface shape obtained by grinding the metal surface in one direction.

  The handling body of the present invention is preferably used for layered modeling of a cell structure in which two or more cultured cell layers adhered to the cell adhesion region are stacked to form a cell structure.

  According to the present invention, the method includes a step of supplying a polymer composition containing a temperature-responsive polymer and an extracellular matrix component to a non-affinity surface from which the cured product can be peeled and curing to produce a molded body. There is provided a method for producing a cultured cell handling body, comprising: a molded body comprising a matrix containing the temperature-responsive polymer; and a cell adhesion region to which cells can adhere on at least a part of the surface of the molded body. The

  In this production method, in the step of producing the molded body, the first polymer composition containing the first temperature-responsive polymer and the extracellular matrix component is supplied to a non-affinity surface from which the cured product can be peeled off. Curing to obtain a first cured layer, and then curing the first polymer composition containing the second temperature-responsive polymer and not containing the extracellular matrix component on the first cured product. It may be a step of obtaining the second cured layer and producing the molded body. Moreover, the manufacturing process of the said molded object may include providing the uneven | corrugated shape which can orient and culture | cultivate a cell to the contact side with the said non-affinity surface of the said molded object. Furthermore, the non-affinity surface may have a surface shape for forming the uneven shape. Furthermore, the uneven shape is a shape obtained by reversing a ground surface shape obtained by grinding a metal surface in one direction, and the non-affinity surface having a surface shape that matches the ground surface shape is prepared. May be.

  According to the present invention, it has a support layer composed of a matrix containing a temperature-responsive polymer, and a cultured cell layer in which cultured cells are supported by being bonded to each other on at least a part of the surface of the support layer. A cultured cell-support layer complex comprising one or more units is provided. In this complex, in at least one of the units, the cultured cells are preferably oriented in a certain direction in at least a part of the cultured cell layer. Moreover, it may have a multilayer structure in which two cultured cell layers are stacked via the support layer. Further, two or more of the units may have a multilayer structure in which the support layer and the cultured cell layer are alternately stacked, and the culture layered via the support layer It is preferable that the cultured cells are oriented in substantially the same direction in at least a part of each cell layer. The cultured cells can be one or more selected from the group consisting of myoblasts, myotubes, smooth muscle cells and cardiomyocytes.

  According to the present invention, there is provided a method for producing a cultured cell-support layer complex according to any one of the above, wherein the cells are cultured in the cell adhesion region of the cultured cell handling body according to any one of the above. There is provided a manufacturing method comprising the step of producing the one unit. This manufacturing method can further comprise a step of laminating the unit produced in the unit production step so that the support layer of the unit is in contact with a separately prepared cultured cell layer, which is prepared separately. The cultured cell layer may be the cultured cell layer of another unit prepared separately.

  According to the present invention, it has a support layer composed of a matrix containing a temperature-responsive polymer, and a cultured cell layer in which cultured cells are supported by being bonded to each other on at least a part of the surface of the support layer. Providing a cultured cell-support layer complex comprising one or more units, and applying a temperature condition based on a critical dissolution temperature of the temperature-responsive polymer to the support layer of the complex. And a step of disrupting the cell structure. In this production method, the disintegration step may be a step of simultaneously removing two or more support layers provided in the composite.

  ADVANTAGE OF THE INVENTION According to this invention, a cell structure provided with the multilayer structure of the cultured cell by which the cultured cell was arranged with the predetermined orientation is provided. This cell structure is preferably obtained by any one of the methods for producing a cell structure described above.

  The present invention relates to a cultured cell handling body and a method for producing the same, a cultured cell-support layer complex holding a cultured cell in the handling body, a method for producing the same, a cell structure, a method for producing the same, and the like. In various embodiments of the present invention, the cultured cell handling body of the present invention, that is, the cell is cultured in the cell adhesion region on the surface of the molded body comprising a matrix containing a temperature-responsive polymer, thereby culturing the molded body. Based on integrating cell layers, maintaining this composite morphology and subsequent handling. Therefore, the cultured cell layer can be separated from the culture system and handled without being detached from the culture substrate. Moreover, by setting it as such a composite_body | complex, a cultured cell layer is supported by a molded object, and collapse of the structure by the interaction between the cells can be suppressed or avoided.

  In addition, since the molded body in the handling body of the present invention is composed of a matrix containing a temperature-responsive polymer, the cultured cell layer is applied to a site to be adhered or overlaid, for example, when overlaid on other cultured cells. In some cases, the temperature-responsive polymer in the matrix can be water-solubilized by applying an appropriate temperature condition based on the critical dissolution temperature condition, and the matrix can be disintegrated. Since the cultured cell layer is not in a free state but is very close to or close to the application site, it can be easily adhered or overlaid on the application site. For this reason, contraction and deformation of the cultured cell layer due to matrix collapse can be suppressed or avoided. Furthermore, when the cultured cells on the support layer are arranged with a certain orientation by disrupting the matrix in such a state, a decrease in the orientation due to the matrix disruption can be suppressed or avoided.

  Hereinafter, various embodiments of the present invention will be described with reference to the drawings as appropriate. FIG. 1 is a diagram showing an example of a process for obtaining a cell structure from a handling body of the present invention via a complex, FIG. 2 is a diagram showing an embodiment of the handling body of the present invention, and FIG. FIG. 4 is a diagram showing an example of the manufacturing process of the handling body of the present invention, FIG. 4 is a diagram showing various aspects of the composite of the present invention, and FIG. 5 shows an example of the manufacturing process of the composite of the present invention. FIG. 6 is a diagram showing an example of the cell structure of the present invention. In addition, these drawings show an example for description of various embodiments, and do not limit the present invention.

(Support for cultured cells)
(cell)
The cultured cell handling body 10 of the present invention is useful for culturing adhesive cells to construct a structure of the cultured cells. The cell to which the handling body 10 of the present invention is applied is not particularly limited as long as it is an adhesive cell, and preferably includes a cell derived from a human or non-human animal. Examples of adhesive cells include fibroblasts, myoblasts, myotubes, corneal cells, vascular endothelial cells, smooth muscle cells, cardiomyocytes, dermal cells, epidermal cells, mucosal epithelial cells, mesenchymal stem cells, Examples include ES cells, iPS cells, osteoblasts, bone cells, chondrocytes, adipocytes, nerve cells, hair root cells, dental pulp stem cells, beta cells, hepatocytes and the like. In addition, in the present invention, a cell means an individual cell, and includes a cell that is collected from a living body and constitutes a tissue.

  Such cells are preferably autologous cells in view of their use in regenerative medicine etc. in humans and the like, but may be cells derived from different species as long as they have acceptable immunocompatibility, It may be an allogeneic cell or an autologous cell.

(Molded body)
As shown in FIGS. 1 and 2, the cultured cell handling body 10 of the present invention includes a molded body 2 made of a matrix (hereinafter, also referred to as a temperature responsive matrix) 4 containing a temperature responsive polymer. . The molded body 2 has sufficient rigidity and strength to ensure the handleability in the handling body 10. Specifically, the molded body 2 is a self-supporting molded body. In the present specification, the self-supporting molded body means an independent molded body that can be handled without a separate support. By using such a self-supporting molded body 2, the temperature-responsive matrix 4 can be supported while supporting the cultured cell layer rather than being used as a separation layer between the culture apparatus such as a dish or plate and the cultured cell layer. Can be used for proper handling.

  The molded body 2 is not particularly limited, and can have a desired size and a desired three-dimensional shape. The molded body 2 can be, for example, various three-dimensional shapes in addition to sheet shapes, columnar shapes, tubular shapes, spherical shapes having various planar forms. The shape of the molded body 2 can be set based on the cell structure 100 to be constructed using the handling body 10 of the present invention.

  It may be advantageous that the molded body 2 has a sheet shape. For example, as shown in FIG. 1, when the molded body 2 is a flexible sheet-like body, the cultured cell-support complex 50 obtained from the handling body 10 can be appropriately deformed. Become. For this reason, it is possible to obtain the composite 50 having a three-dimensional shape that is tubular or appropriately folded. Depending on the selection of the temperature-responsive polymer and the composition of the matrix 4, flexibility may be imparted to the sheet-like body. Good.

  In addition, in order to make the composite 50 described later into a multilayer structure in which two cultured cell layers 60 are layered via a support layer made of the molded body 2, the molded body 2 is suitable from the viewpoint of being multilayered. Is preferably a sheet-like body. The planar form of the sheet-like molded body 2 is not particularly limited, and does not necessarily match the cross section of the cell structure 100 to be obtained, and may be a larger form or the cross section 100 or the cross section thereof. Some planar forms can also be used.

  The molded body 2 preferably has a thickness of 1 μm or more and 1 mm or less in view of its own self-supporting property, handling property and multi-layer property. When the thickness is less than 1 μm, it is difficult to maintain the self-supporting property, and the handling property is too low. When the thickness exceeds 1 mm, the followability to the application site, the ease of arrangement, the ease of overlaying, and the like are too low. More preferably, they are 10 micrometers or more and 150 micrometers or less.

  The molded body 2 is composed of a matrix 4 containing a temperature-responsive polymer. The temperature-responsive polymer that can be used in the present invention preferably has an upper critical lysis temperature or a lower critical lysis temperature of 0 ° C. or higher and 80 ° C. or lower, more preferably 20 ° C. or higher, in consideration of stress on cells. It is 50 ° C. or lower, more preferably 25 ° C. or higher and 35 ° C. or lower. Preferably, it is a polymer having a lower critical solution temperature that hardly becomes soluble in water when the temperature is raised. The temperature-responsive polymer is not particularly limited, and various temperature-responsive polymers can be used regardless of known homopolymers or copolymers. These may be appropriately cross-linked. Examples of such a temperature-responsive polymer include various polyacrylamide derivatives such as poly-N-isopropylacrylamide (PNIPAAm) and poly-N, N′-diethylacrylamide, as well as Patent Document 1 (Japanese Patent Laid-Open No. 2-211865). No.)).

  The temperature-responsive matrix 4 may be substantially made of a temperature-responsive polymer, or may contain components other than the temperature-responsive polymer. In the case where the composite 50 using the handling body 10 of the present invention is disposed at an application site and adhered, when it is composed only of a temperature-responsive polymer and does not contain other polymer components (excluding an ECM component described later). By giving a temperature condition based on the critical dissolution temperature of the temperature-responsive polymer, it is preferable in that it can suppress or avoid the entire molded body 2 from being collapsed and removed to leave unnecessary materials at the application site. In addition, it does not exclude that it consists only of a temperature-responsive polymer to contain the impurity unavoidable on manufacture.

  As a component other than the temperature-responsive polymer, an extracellular matrix (ECM) component can be contained. By containing the ECM component, the affinity and adhesion to cells can be improved, and the cell growth promoting action of the molded body 2 can be improved during cell culture, thereby enhancing the function as a scaffold. Further, when the temperature-responsive polymer is water-solubilized in the complex 50 and the temperature-responsive matrix 4 is disintegrated, the ECM component is easily held in a state of being bound to the cultured cells. Can be promoted. This also makes it possible to more effectively suppress or avoid a decrease in orientation when the temperature-responsive matrix 4 is collapsed when the cultured cells are given a certain orientation. In this specification, the ECM component includes not only molecules present in the ECM but also cell adhesion peptides.

  Such ECM components are not particularly limited, and various known components can be used. For example, collagen, elastin, proteoglycan, glucosaminoglycan (hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, keratan sulfate, etc.), fibronectin, laminin, hydronectin, gelatin and the like can be mentioned. Moreover, for example, RGD peptide, RGDS peptide, GRGD peptide, and GRGDS peptide can be mentioned.

  The temperature-responsive matrix 4 may have two or more layer structures having different compositions. That is, you may have the multilayer structure of the matrix of a different composition. For example, as shown in FIG. 2, when the matrix 4 has a multilayer structure of the first layer 6a and the second layer 6b as shown in FIG. The concentration of the two components (including the presence or absence of the component) may be different. For example, different concentrations for the same temperature-responsive polymer, different types of temperature-responsive polymers can be used, and different concentrations or compositions for the ECM components can be used.

  The temperature-responsive matrix 4 tends to improve hydrophilicity and cell adhesion as the ECM component concentration increases, and as the temperature-responsive polymer concentration increases, the rigidity improves and collapses under critical dissolution temperature conditions. Tend to improve. For example, in FIG. 2, when the ECM component concentration is further increased in the layer 6a between the layer 6a and the layer 6b, the cell adhesiveness is improved in the layer 6a. As a result, the molded body 2 is cell-cultured by the layer 6a. Sometimes it functions as a good scaffold. At the same time, in the layer 6b of the molded body 2, the temperature-responsive polymer concentration is relatively high, and better handling properties, shape maintenance properties of the handling bodies 10 themselves, and morphology maintenance properties of the cultured cell layer can be obtained. Such a multilayer structure is advantageous in the construction of the complex 50 for obtaining the cell structure 100 of a complex three-dimensionally shaped tissue.

  When the temperature-responsive matrix 4 has a multilayer structure with different compositions, different thicknesses may be imparted to each layer. If the layer thickness is thick, the rigidity is improved and the collapse property is lowered, and if the layer thickness is thin, the followability and the deformability are improved and the collapse property is improved. For example, in the multilayer structure shown in FIG. 2, the thickness of the layer 6a having a higher ECM component concentration and a lower temperature-responsive polymer concentration is reduced, and the temperature-responsive polymer concentration is lower (or not included). By increasing the thickness of the higher layer 6b, it is possible to form a good cell adhesion region 8 at a low cost with a small amount of ECM component, and the layer 6b can sufficiently ensure good handling properties and rigidity. In addition, since the polymer aqueous solution containing a temperature-responsive polymer and an ECM component takes a long time to dry, there is also a process advantage that the process can be speeded up by reducing the thickness of the layer 6a.

  Further, by reducing the thickness of the layer 6a having a higher ECM component concentration and a lower temperature-responsive polymer concentration, the composite 10 obtained by culturing cells in the handling body 10 is overlaid or engrafted in a desired site. When this is done, it is possible to suppress or avoid biological bonding between the cultured cell layers at the stratified site due to the ECM component and delay of engraftment at the application site. That is, if the temperature-responsive matrix containing a sufficient amount of ECM components for cell adhesion has a thickness sufficient to ensure the rigidity of the handling body 10, a large amount of ECM can be obtained when the temperature-responsive matrix 4 is collapsed. This is because the components remain in the stratification site or the engraftment site, and there are cases where the biological binding of other cultured cell layers and the engraftment to the application site may be prevented.

  As shown in FIG. 2, the layer 6a containing the ECM component at a higher concentration and containing the temperature-responsive polymer at a lower concentration and the temperature-responsive polymer containing (or not containing) the ECM component at a lower concentration. Is provided at a higher concentration, the layer 6a is preferably 0.01 μm or more and 50 μm or less. Within this range, a good cell adhesion region 8 can be obtained within an appropriate amount of ECM component concentration range. This is because if it is less than 0.01 μm, the ECM component present in the cell adhesion region 8 becomes too small and the cell adhesion is lowered. When the concentration of the ECM component is increased, the ECM component is deposited and cannot be formed into a film, so that it is difficult to increase the concentration. On the other hand, if it exceeds 50 μm, the amount of ECM components that are exposed to the cell adhesion region 8 and contribute to cell adhesion is small for the amount of ECM components to be used. It is because it is too much. More preferably, they are 0.05 micrometer or more and 5 micrometers or less. In addition, the layer 6b is preferably 1 μm or more and 1 mm or less in consideration of the self-supporting property of the molded body 2 and the handling property and multi-layer property of the composite 10. When the thickness is less than 1 μm, it is difficult to maintain the self-supporting property, and the handling property is too low. When the thickness exceeds 1 mm, the followability to the application site, the ease of arrangement, the ease of overlaying, and the like are too low. More preferably, they are 10 micrometers or more and 100 micrometers or less.

(Cell adhesion area)
The handling body 10 of the present invention includes a cell adhesion region 8 on at least a part of the surface of the molded body 2. The cell adhesion region 8 may be the surface of the temperature-responsive matrix 4 containing the ECM component already described, or may be a region where the surface of the molded body 2 is coated with the ECM component. The region 12 may be a surface that has been subjected to physical and / or chemical treatment that improves cell adhesion. Examples of such treatment include coating with an ECM component and appropriate surface roughening treatment. Only one type of various methods for imparting cell adhesion may be used, or two or more types may be used in combination. For example, in the example shown in FIG. 2, the surface of the layer 6a can be used as a good cell adhesion region 8 by including an ECM component in the matrix of the layer 6a.

  When the molded body 2 is in the form of a sheet, the entire surface facing the thickness direction may be the cell adhesion region 8 or only a part may be the cell adhesion region 8. Moreover, the cell adhesion region 8 may be patterned according to the planar form of the cultured cell layer to be formed. Such a specific pattern of cell adhesion region 8 can be obtained by site-selective coating of an ECM component, formation of a matrix containing the ECM component, and chemical and / or physical treatment.

  The cell adhesion region 8 can be provided with a concavo-convex shape in which cells can be oriented and cultured. By providing such an uneven shape, the cells of the cultured cell layer can be functionally oriented. Such a concavo-convex shape can be appropriately set according to the orientation or arrangement desired to be imparted to the cultured cells. In the present specification, “orienting cells” means giving a cell a desired orientation, but it may also be used as a meaning including those that give a desired cell arrangement state. .

  For example, when it is desired to give a desired orientation to the cultured cells, a concave portion (the length may be short) along the orientation can be provided. Further, when it is desired to provide a certain arrangement state, for example, when it is desired to arrange the entire cell adhesion region 8 in a desired orientation, a linear concave portion or the like that traverses the entire cell adhesion region 8 is provided. You may have.

  Such a concavo-convex shape can be imparted directly to the cell adhesion region 8 by various etching techniques or MEMS in a semiconductor, or is imparted to the cell adhesion region 8 by a molded body using a mold formed by such a technique. You can also The uneven shape obtained by such a method is preferable in terms of high regularity. A preferable form of the concave portion obtained by such a method is a long groove having a width in the short direction of 5 μm or more and 100 μm or less. If the width is less than 5 μm, the cells are not oriented or arranged. Further, if the thickness exceeds 100 μm, the cells are difficult to orient in the major axis direction of the recess. More preferably, they are 5 micrometers or more and 50 micrometers or less.

  As another uneven | corrugated shape, you may utilize the surface shape of the abrasion form obtained by grinding a metal surface in one direction. Such a concavo-convex shape is less regular than the MEMS technology itself, but exhibits a good cell orientation. Examples of the usage form of the metal grinding surface shape as the concavo-convex portion include a form in which the surface shape that matches the metal grinding surface shape is used as the concavo-convex shape. Moreover, the form which utilizes the surface shape which reversed the metal grinding | polishing surface shape as an uneven | corrugated shape is mentioned. The concavo-convex shape obtained by inverting the metal grinding surface shape tends to effectively orient the cells more than the same concavo-convex shape as the metal grinding surface shape. A method for forming such an uneven shape will be described in detail later.

  The surface roughness (Rz) of the concavo-convex shape forming region using such a metal ground surface is preferably 2 μm or more on average. If it is less than 2 μm, it becomes difficult to orient the cells. Moreover, it is preferable that an upper limit is 20 micrometers or less, More preferably, it is 15 micrometers or less. The surface roughness (Rz) is a parameter defined as a ten-point average height. The reference length is appropriately set according to the surface roughness. Preferably, the average value of two or more points is obtained.

  For cells whose cultured cells are cardiomyocytes, myoblasts, myotubes, smooth muscle cells, etc. whose cell orientation and arrangement state are involved in cell differentiation and function expression, a cell adhesion region having such uneven shape 8 is preferable.

  According to the cultured cell handling body 10 of the present invention described above, cells are seeded in the presence of an appropriate medium by seeding cells in the cell adhesion region 8 or supplying a tissue piece or the like. It can be cultured. Then, from the cell culture system such as a cell culture device that stores liquid medium or the like, the cultured cell layer 60 is taken out together with the handling body 10 as a complex 50 to be described later, and the cell culture layer 60 is taken out from the culture device without being peeled off. It becomes possible to do. For this reason, while being able to suppress or avoid the deformation | transformation of a cultured cell layer, shrinkage | contraction, and the fall of orientation accompanying the peeling of a cultured cell layer, the troublesome operation | work accompanying peeling can be avoided. Further, when the cultured cell region 12 has an uneven shape capable of imparting orientation and alignment to the cultured cells, the cultured cells can be cultured while being oriented and / or aligned, It can be stably maintained until necessary through operations and situations such as conveyance, storage and processing. Furthermore, by obtaining the complex 50, the cell structure 100 having a desired form can be easily constructed at a desired site. That is, the handling body 10 of the present invention can be used for modeling a cell structure (particularly for layered modeling).

(Manufacturing method of cultured cell handling body)
The method for producing a cultured cell handling body of the present invention can include a step of preparing a polymer composition containing a temperature-responsive polymer and a step of curing the polymer composition to produce a molded body. . The various embodiments already described for the molded body in the cultured cell support 10 of the present invention described above are applied as they are to the molded body in the handling body produced in the present invention. According to the present invention, a handling body obtained by such a method for manufacturing a handling body is also provided. Hereinafter, description will be made with reference to FIG. 3 as an example of a manufacturing process suitable for manufacturing the handling body 10 illustrated in FIG. 2 as appropriate.

(Preparation process of polymer composition)
In the manufacturing method of the handling body of the present invention, a polymer composition containing a temperature-responsive polymer is prepared. The polymer composition may be appropriately prepared or may be appropriately obtained. In preparation of the polymer composition for producing the support 10, one or two or more kinds of known temperature-responsive polymers can be appropriately used in combination. For the temperature-responsive polymer, the various embodiments already described for the support 10 are applied as they are. The polymer composition can contain a temperature-responsive polymer, an ECM component, and the like. For the ECM component, the various embodiments already described for the support 10 are applied as they are.

  The polymer composition can have a composition corresponding to the composition of the temperature-responsive matrix 4 of the molded body 2, the shape of the molded body 2, and the molding method. For example, the concentration range of the temperature-responsive polymer in the polymer composition can be 0.01 (w / v)% or more and 20 (w / v)% or less. Within such a range, the temperature-responsive polymer is contained in a high concentration by drying, so that a self-supporting molded body such as a desired sheet can be obtained relatively easily. When the concentration of the temperature-responsive polymer in the polymer composition exceeds 20 (w / v)%, the viscosity of the polymer composition becomes too high and the operability is deteriorated. On the other hand, when the concentration is less than 0.01 (w / v)%, it takes a very long time to cure the polymer composition by drying, and the operability is too low. More preferably, it is 0.5 (w / v)% or more and 10 (w / v)% or less. Within this range, a sheet-like body having a preferable thickness range as the self-supporting molded body 2 can be easily molded. More preferably, they are 0.5 (w / v)% or more and 5 (w / v)% or less.

  Such polymer compositions can be prepared using a suitable solvent. Typically, water, an organic solvent such as an alcohol compatible with water, a mixed solution of water and such an organic solvent, and the like can be given. In general, the polymer composition is prepared in a temperature range where the temperature-responsive polymer is dissolved in the dissolution medium.

  The ECM component concentration in the polymer composition is not particularly limited. The total amount of one or more ECM components can be 0.1 μg / ml or more and 1 mg / ml or less. Within this range, good cell adhesion can be obtained, and the surface of the molded article of the polymer composition becomes a good cell adhesion region. Further, it is preferably 1 μg / ml or more and 100 μg / ml or less. Within this range, cells associated with cell movement such as myoblasts, myotubes, smooth muscle cells and the like are particularly easily adhered.

  In producing the molded body 2, when the different layers 6a and 6b are combined, two or more kinds of polymer compositions corresponding to different matrix compositions are prepared. For example, a first polymer composition containing a temperature-responsive polymer and an extracellular matrix component and a second polymer composition containing a temperature-responsive polymer and no extracellular matrix component may be prepared.

(Curing and molding process of polymer composition)
The molding method for molding the polymer composition to form the molded body 2 is not particularly limited, and is appropriately selected from known resin molding methods in consideration of the three-dimensional shape and size to be obtained for the molded body 2. be able to. For example, in order to form a sheet-like body, a method of casting a polymer composition on an appropriate mold or flat plate can be mentioned.

  In order to easily obtain the molded body 2, as shown in FIG. 3, the polymer composition is supplied to the non-affinity surface 20 from which the cured product of the polymer composition can be peeled to cure the polymer composition. preferable. By doing so, even the thin film-like molded body 2 can be easily obtained. Such a non-affinity surface 20 may be a molding surface of a mold having a cavity, or may be a surface on a flat plate as illustrated in FIG. Moreover, the material which comprises the non-affinity surface 20 can use fluorinated polymers, such as siloxane type polymers, such as polydimethylsiloxane, polytetrafluoroethylene, etc. suitably. In addition, when obtaining the molded object 2 of a multilayer structure, methods, such as casting by superimposing the polymer composition of the layer which overlaps with the layer hardened | cured initially, are employable.

  In order to cure the polymer composition to form the molded body 2, various known methods applied to the curing of the temperature-responsive polymer can be used. For example, under conditions where the temperature-responsive polymer is dissolved in the dissolution medium. It may be by drying and evaporating the dissolution medium.

  In order to form the cell adhesion region 8 in the molded body 2, as shown in FIG. 3A, the polymer composition may contain a necessary amount of an ECM component. An ECM component coating layer may be formed on the surface, or other chemical treatments may be applied. The cell adhesion region 8 is preferably matched with a region where cells are cultured to form a cultured cell layer. Therefore, a necessary form may be patterned in consideration of the form of the cell structure 100 to be finally obtained. Such patterning can also be realized by casting a different polymer composition, but it is easy to use a coating layer or chemical treatment on the surface of the molded body 2.

  Next, a method for forming a concavo-convex shape for aligning and culturing cells in the cell adhesion region 8 of the molded body 2 will be described. The various embodiments already described are applied as they are to the uneven shape, and various methods can be used as described above. Especially, it is preferable to supply and harden a polymer composition to the non-affinity surface which has the surface shape for shape | molding the said uneven | corrugated shape. More preferably, as illustrated in FIG. 3, the metal ground surface shape already described is used. By utilizing the metal ground surface shape, it is possible to easily provide the uneven shape without requiring a large-scale apparatus for etching technology or MEMS, and it is possible to easily provide the uneven shape to a large area. In addition, by preparing the non-affinity surface 20 having a concavo-convex shape that matches or determined the metal grinding surface shape, it is possible to efficiently impart a concavo-convex shape effective for cell orientation to the molded body 2.

  In order to form a concavo-convex shape using a metal ground surface shape, the following method can be typically employed. For example, as shown in FIG. 3, in the case of producing a molded body 2 having a concavo-convex shape obtained by inverting the metal grinding surface shape, as shown in FIG. After grinding with sandpaper in a certain direction and grinding, as shown in FIG. 3 (m), a mold or plate having a non-affinity surface capable of molding the ground surface is produced using the ground surface as a mold. . Furthermore, as shown in FIG. 3 (n), a polymer composition that forms a non-affinity surface is cast using the molding die or the like as a mold, and a non-affinity surface 20 having a concavo-convex shape that matches the ground surface shape is obtained. A molding die provided is obtained (FIG. 3 (o)). And as shown to Fig.3 (a), it has the uneven | corrugated shape which reversed the metal grinding | polishing surface shape by casting etc. the polymer composition containing an ECM component and a temperature-responsive polymer to the non-affinity surface 20. The molded object 2 provided with the cell adhesion area | region 8 can be obtained.

  On the other hand, in order to form a concavo-convex shape that matches the metal grinding surface shape, a non-affinity surface 20 having a concavo-convex shape obtained by reversing the metal grinding surface shape is prepared, and an ECM component and a temperature-responsive polymer are contained therein. What is necessary is just to cast the polymer composition to perform.

  The metal used for preparing the non-affinity surface 20 is not particularly limited, and a metal that is softer than an abrasive used for grinding may be used. For example, iron or aluminum can be used. A grinding material to be ground can be appropriately selected from known grinding materials, and a preferred count can be appropriately selected from a metal file or a so-called sandpaper. The grinding method is not particularly limited, but it is preferable that the grinding direction and the load during grinding can be adjusted.

  In addition, this uneven | corrugated shape is formed according to the orientation to give to the cell in the cultured cell layer 60 or the cell structure 100 to be obtained. In order to be oriented substantially in one direction and linearly arranged, it may be ground in one direction, or may be ground in accordance with the cell structure 100 or the like to be constructed.

  According to the manufacturing method of the handling body of the present invention described above, the support body 10 of the present invention can be easily manufactured. In particular, by supplying a polymer composition to a predetermined non-affinity surface and curing the polymer composition to obtain a molded body, the handling body 10 having the multilayered molded body 2 or cells are oriented and cultured. The handling body 10 having a possible uneven shape can be efficiently manufactured.

(Complex of cultured cell layer and support layer)
A cultured cell-support layer complex (hereinafter simply referred to as a complex) 50 of the present invention 50 is a support layer 10 which is a handling body 10 of the present invention comprising a temperature-responsive matrix 4 as shown in FIG. (The same reference numerals are used because they are the same member), and a cultured cell layer 60 in which cultured cells are coupled to and supported by at least a part of the support layer 10. Can be provided. The complex 50 of the present invention can be handled integrally with the support layer 10 by supporting the cell culture layer 60 with the support layer 10. For this reason, the structure and orientation of the cell culture layer 60 can be stably maintained after completion of the culture through various processes until necessary. Moreover, it becomes easy to convey and arrange | position the cultured cell layer 60 to application parts, such as a predetermined | prescribed arrangement | positioning site | part, an overlaying site | part, and an engraftment site | part. Further, in the case where biological bonds are generated between cells layered at the application site, the temperature-responsive matrix 4 of the support layer 10 is given a temperature condition based on the critical dissolution temperature of the temperature-responsive polymer. By disintegrating the matrix 4, it is possible to adhere to the intended location reliably while maintaining the orientation of the cells. The complex 50 of the present invention can be used for modeling the cell structure 100 (particularly for layered modeling). Hereinafter, the various aspects of the composite 50 of the present invention shown in FIG. 4 will be described as appropriate.

  Moreover, the composite 50 of the present invention can provide a unit for constructing the composite 50 having a thicker, larger, or more complicated three-dimensional shape. In particular, when the composite 50 is a sheet-like body because the support layer 10 is in a sheet form or the like, the sheet-like composite 50 is laminated to obtain a thicker, larger or more complicated three-dimensional structure. The shaped composite 50 can be easily shaped and used to provide such a cell structure 100.

  Since the support layer 10 in the composite 50 of the present invention is the handling body 10 of the present invention, the various embodiments already described in the handling 10 of the present invention can be applied as they are.

  As shown in FIG. 4A, the complex 50 of the present invention does not need to include the cultured cell layer 60 on the entire surface of the support layer 10 constituting the unit 70. The cultured cell layer 60 may be provided in an arbitrary pattern in at least a part of the surface of the support layer 10, for example, a region defined by the cell adhesion region 8 or the like. A region other than the cultured cell layer 60 on the support layer 10 can be used as a region for ensuring handling properties in addition to securing a gripping portion function with tweezers or the like or an adhesion function with another cultured cell layer 62.

  As shown in FIG. 4A, the complex 50 according to the present invention may be composed of only one unit 70 including one support layer 10 and one cultured cell layer 60. As shown in FIG. 4 (b) and FIG. 4 (c), it may have a multilayer structure in which two cultured cell layers 60 are stacked with one support layer 10 interposed therebetween. According to such a multilayer structure, cells that are stacked through the support layer 10 are biologically bound by collapsing the temperature-responsive matrix 4 of the support layer 10. More specifically, such a multilayer structure includes, for example, two or more units 70 such that the support layer 10 and the cultured cell layer 60 are alternated as shown in FIG. A multi-layer structure may be used. Moreover, as shown in FIG.4 (c), it is good also as a multilayer structure which laminated | stacked the 1 or 2 or more unit 70 with respect to another cell culture layer 62 which does not comprise the unit 70. FIG.

  The complex 50 of the present invention may have a multilayer structure in which the cultured cell layers 60 are directly stacked. When it is necessary to quickly engraft the cell culture layers 60, such a multi-layer form is preferable. For example, a multilayer structure as shown in FIG. 4D or a multilayer structure in which the cultured cell layer 60 is directly overlaid on another cultured cell layer 62 as shown in FIG. 4E can be adopted. .

  In addition, when the composite 50 has a multilayer structure, the layered form such as the cell culture layer 60 overlaps between the cultured cell layers 60 to be layered or between the cultured cell layer 60 and another cultured cell layer 62. However, it is not necessary to limit the entire layer of one cultured cell layer 60 to overlap with the other cultured cell layers 60 and 62.

  In the complex 50 of the present invention, it is preferable that the cultured cells are oriented in a certain direction in at least a part of the cultured cell layer 60. This is because the orientation of cultured cells may be necessary for differentiation and functional expression. The oriented cultured cells may exist only in the cultured cell layer 60 of one unit 70, but may exist in a plurality of units 70. Preferably, a plurality of cultured cells oriented in substantially the same direction are provided across the plurality of cell culture layers 60. For example, as shown in FIG. 4 (f), it is preferable that the cultured cell layers 60 of two or more units 70 that are successively stacked include cultured cells oriented in substantially the same direction.

  The complex 50 of the present invention may have a single-layer or multi-layer structure of a unit 70 having a cultured cell layer 60 made of the same type of cultured cells, and the cultured cell layers 60 to be stacked are different cultured cells. The multilayer structure which consists of may be sufficient. In the composite 50 of the present invention, even if the cultured cell layer 60 to be overlaid is composed of heterogeneous cells, it is easy to make a composite because it has a multilayer structure through the support layer 10. Further, two or more types of cells may be cultured in the same cultured cell layer 60. The complex 50 of the present invention may have a planar form in which the cultured cell layers 60 in the two or more units 70 to be stacked are the same or different from each other.

  In the case where the composite 50 includes the sheet-like support layer 10 and has a sheet shape as a whole, the composite 50 is appropriately modified regardless of whether the composite 50 has a single-layer structure or a multi-layer structure. There may be. For example, the sheet-like composite 50 can be rounded into a cylindrical body such as a cylinder. When the complex 50 is obtained by laminating the unit 70 having the cultured cell layer 60 having a pattern corresponding to the cross-sectional shape obtained by slicing the cell structure 100 to be obtained, the support layer 10 Since the cell structure 100 is collapsed and removed under an appropriate temperature condition, the cell structure 100 can be constructed as a cell structure 100 having a complicated three-dimensional shape having a desired external shape and internal shape. Accordingly, such a complex 50 is advantageous as a precursor for in vitro and in vivo devices that utilize or substitute for cellular functions. Further, when the orientation is controlled in these cultured cell layers 60 and the orientation is almost the same in the cultured cell layers 60 and the like that are overlaid, cardiomyocytes, myoblasts, myotubes, smooth muscle cells It is even more advantageous as a precursor for in vitro and in vivo devices that utilize or substitute functions such as.

(Production method of composite)
The manufacturing method of the composite 50 of the present invention can include a step of culturing cells in the cell adhesion region 8 of the handling body 10 of the present invention to produce one unit 70. According to the production method of the present invention, a precursor that can be constructed and handled can be easily produced. In addition, by using the unit 70 as a laminated unit, it is possible to accurately and stably form a laminated state as the complex 50 or the cultured cell layer 60. According to the present invention, a composite obtained by such a method for producing a composite is also provided.

  That is, when the cultured cell layer 60 supported by the support layer 10 is laminated, the form of the cultured cell layer 60 and the orientation of the cells are maintained in the support layer 10 and stress on the cultured cell layer 60 is applied. Therefore, the cultured cell layer 60 can be laminated in a state suitable for biological bonding while maintaining a good state. In addition, since the cultured cell layer 60 is supported on the sheet-like support layer 10 in a state in which the orientation is controlled, when the cultured cell layer 60 with the controlled orientation is stacked, the support layer 10 By paying attention to the orientation, the cultured cell layer 60 can be easily laminated while controlling the orientation of the cells.

  In producing the complex 50 of the present invention, cells are cultured in the cell adhesion region 8 of the handling body 10 of the present invention. Typically, the handling body 10 of the present invention is placed in a culture apparatus capable of storing a liquid medium or the like, and in the presence of the medium, cells or tissue pieces are supplied to the cell adhesion region 8 and cultured. Culture conditions can be appropriately set by those skilled in the art according to the type of cells used. Based on the upper or lower critical temperature of the temperature-responsive polymer used in the temperature-responsive matrix 4 of the handling body 10 of the present invention, the temperature at which the temperature-responsive polymer constituting the matrix 4 does not dissolve is set as the culture condition.

  In obtaining the multilayer structure 50, as shown in FIGS. 4B and 4D, the complex 50 of the present invention has a multilayer structure in which two or more units 70 are stacked. In some cases, as shown in the left of FIG. 5, by preparing or obtaining a plurality of units 70 and stacking them so that the support layer 10 is in contact with the cultured cell layer 60 of another unit 70. Obtainable. Further, as shown in FIGS. 4 (c) and 4 (e), when it has a multilayer structure laminated on another cultured cell layer 62 that does not constitute the unit 70, as shown on the right side of FIG. Then, it is laminated on another cultured cell layer 62. The other cultured cell layer 62 may be a cultured cell layer 60 that has already been left after the support layer 10 has been disintegrated and removed from the unit 70 as described later.

  In the case of the complex 50 in which two or more cultured cell layers 60 are provided with cells oriented in substantially the same direction, preferably, the support 10 provided with the cell adhesion region 8 having a concavo-convex shape capable of controlling the orientation of the cultured cells. A plurality of units 70 obtained by culturing cells are prepared, and these units 70 are stacked so that their orientations are substantially the same. Since the unit 70 is supported by the support layer 10, the two or more cultured cell layers 60 can be reliably stacked so that the cultured cells are oriented in the same direction.

  The complex 50 can be produced in vitro or in vivo as shown in FIG. It can select suitably according to the use of the cell structure 100 which is finally going to obtain from the composite 50. For example, when the complex 50 is manufactured at a different site from the site to be used and moved from the site to be manufactured, the non-affinity surface 20 made of PDMS or the like already described may be used. By doing so, the complex 50 is easily peeled off from the production site, and the cultured cell layer 60 is not easily stressed. For example, as an in vitro example, the non-affinity surface 20 on which the handling body 10 of the present invention is manufactured may include a molded body (including a single-layer molded body 2 containing an ECM component) 2 at the time of molding. The cultured cell layer 60 may be formed by culturing cells on the surface of the molded body 2 while being maintained and placed. Alternatively, the complex 50 may be formed by the non-affinity surface 20 prepared separately for producing the complex 50.

  As an in vivo example, the handling body 10 is arranged at a site where the cell structure 100 is to be engrafted, such as a living tissue of a human or non-human animal, and the cells are cultured on the spot, and the handling body 10 is stacked and the cells are cultured. Or the composite 50 produced in another place may be laminated | stacked. Alternatively, another composite 50 prepared separately at the engraftment site may be arranged and another composite 50 may be laminated, or only the handling body 10 may be laminated on the composite 70 arranged at the engraftment site. The cells may be cultured on the handling body 10. That is, the complex 50 may be generated in situ at the engraftment site.

(Method for producing cell structure)
The method for producing a cell structure of the present invention includes a step of preparing the complex 50 of the present invention, a step of causing the support layer 10 of the complex 50 to collapse under a temperature condition based on the critical dissolution temperature of the temperature-responsive polymer, Can be provided. According to the method for producing a cell structure of the present invention, the cultured cell layer is biologically bound to an engraftment site or another cultured cell layer by collapsing the support layer in the complex of the present invention. A cell structure maintaining the structure and orientation of the cultured cell layer can be obtained. In particular, in the case where the complex 50 has a multilayer structure, the support layer interposed in the complex 50 is disrupted to reliably maintain the shape of the two or more cultured cell layers 60 and the orientation of the cells. Can be integrated. Therefore, a cell structure excellent in structural stability and orientation can be obtained as compared with the case where the cultured cell layer itself is directly laminated. Furthermore, according to the manufacturing method of the present invention, an in vitro device or an in vivo device that uses or substitutes for the function of a cell or tissue can be easily manufactured. According to the present invention, a cell structure obtained by such a method for producing a cell structure is also provided.

  Regarding the composite 50 prepared in the production method of the present invention, its support layer 10 and the temperature-responsive polymer, various embodiments of the composite 50, the support layer 10 and the temperature-responsive polymer of the present invention already described are used. Applied.

  The complex 50 includes one or more cell culture layers 60 and one or more support layers 10 (FIG. 4). In order to collapse the support layer 10 of the composite 50, a temperature condition based on the critical dissolution temperature of the temperature-responsive polymer constituting the temperature-responsive matrix 4 is applied to the composite 50. Such temperature conditions can be higher or lower than the upper or lower critical solution temperature inherent to the temperature-responsive polymer used. In order to promote the collapse, the temperature may be higher or lower than the upper or lower critical solution temperature. Such temperature conditions take into consideration the critical dissolution temperature of the temperature-responsive polymer, the type and amount of other ECM components that constitute the temperature-responsive matrix 4, the site where the cell structure 100 is produced, and the cultured cells that constitute it. It can be set appropriately.

  A method for applying the temperature condition to the composite 50 is not particularly limited. In the simplest case, as shown in FIG. 6, the support 10 can be collapsed by adjusting the ambient temperature at which the composite 50 exists. In addition, a gas whose temperature is adjusted can be supplied to the composite 50 to selectively give a desired temperature condition to the composite 50 or a part of the support layer 10.

  Production of the cell structure 100 by disintegrating the support layer 10 can be performed in vitro or in vivo, as in the case of the complex 50, but can be appropriately selected depending on the use of the cell structure 100. In consideration of the stability of the cell structure 100 and the like, it is preferable to manufacture the cell structure 100 by placing the complex 50 at the site where the cell structure 100 is used. That is, it is preferable to dispose the support layer 10 on the spot after placing the complex 50 at the site (in vivo or in vitro) where the cell structure 100 is used. By carrying out like this, handling of the cell structure 100 itself can be avoided, and the structure collapse of the cell structure 100 and the orientation deterioration of a cell can be avoided or suppressed.

  In addition, when the handleability of the cell structure 100 obtained after the collapse of the support layer 10 can be expected, it is not always necessary to collapse the support layer 10 at a site where the cell structure 100 is to be applied. Moreover, you may collapse the support body layer 10 on the suitable support | carrier which can support the cell structure 100 after the support body layer 10 collapses. In the case where the support layer 10 of the complex 50 is collapsed at a site other than the use site, the support 10 of the present invention is prepared so that the cell structure 100 can be easily transported and handled from the site. It is preferably carried out on the non-affinity surface used in the above.

  For example, as an in vitro production example, when the cell structure 100 is used at a site different from the production site after a certain time, the cell structure 100 can be produced on the non-affinity surface 20. In addition, when the cell structure 100 is a multilayer structure, since the whole intensity | strength is improved by multilayering, it is easy to handle. Further, the cell structure 100 may be produced in a container (for storage or transportation) in which the cell structure 100 can be accommodated. By doing so, stress on the cell structure 100 can be suppressed or avoided.

  Further, for example, as already described in vivo, as described above, the composite 50 may be produced at a site where the composite 50 is to be engrafted, and then the support 10 may be collapsed as it is. Moreover, the composite body 50 produced in another place may be arrange | positioned in an engraftment site | part etc., and the support body layer 10 may be collapsed on the spot.

  In the production method of the present invention, when the complex 50 has a multilayer structure including two or more units 70, the cell structure 100 is removed at once by removing the plurality of support layers 10 provided in the complex 50 simultaneously. Can be obtained. Further, by applying a temperature condition to the complex 10 in a site-selective manner, the support layer 10 at the specific site is collapsed, and the cultured cell layer 60 near the specific site is combined, and then the other support layer 10 May be disrupted and other cultured cell layers 60 may be combined.

  When the complex 50 is obtained by laminating the unit 70 having the cultured cell layer 60 having a pattern corresponding to the cross-sectional shape obtained by slicing the cell structure 100 to be obtained, Due to the collapse, it is possible to construct a cell structure 100 having a complicated three-dimensional shape such as having a desired outer shape and size, and having a penetrating portion and a hollow portion. When the cultured cells are oriented in the cultured cell layer 60, the cell structure 100 further includes the oriented cells.

  By further laminating the cell structure 100 obtained in this manner so that the support layer 10 of the complex 50 of the present invention is in contact with the cell structure 100, a more complicated three-dimensional cell structure 100 can be constructed.

  As described above, according to the method for manufacturing the cell structure 100 of the present invention, unlike the conventional method, the cell structure 100 is obtained by disintegrating the support layer 10 of the complex 50 including the support layer 10. Since the cultured cell layer 60 stably maintained by the support layer 10 can be bonded, the stable cell structure 100 can be manufactured in a desired form.

(Cell structure)
As shown in FIGS. 1 and 6, the cell structure 100 of the present invention can have a structure in which cultured cells in which cultured cells are arranged in a predetermined orientation are stacked. Such a cell structure 100 is useful as an in vitro device or a cell structure for an in vivo device in which cell orientation is an important factor for differentiation and functional expression. In particular, it is useful as an in vitro or in vivo device that utilizes or substitutes for the function of muscle tissue such as smooth muscle tissue in which cell orientation is particularly important.

  The three-dimensional form of the cell structure 100 of the present invention is not particularly limited. It can also be set as a sheet-like body as a whole. In the case of a sheet-like body, there is an advantage that it can be used in various forms and can easily follow the application site. The cell structure 100 of the present invention can have a complicated three-dimensional shape such as a hollow part or a penetrating part. The cell structure 100 of the present invention is useful when such a structure is important or essential in terms of function.

  The cell structure 100 of the present invention can be used for regenerative medicine for the purpose of substituting various cells, tissues, organs and organs in humans or non-human animals. In particular, a part of the myocardium where cell orientation is important can be used as a regeneration material. In addition, the cell structure 100 of the present invention can be used for an actuator or the like that uses functions such as skeletal muscle in vitro.

  Hereinafter, specific examples of the present invention will be described with reference to examples. The following examples do not limit the present invention.

(Manufacture of handling bodies and preparation of composites)
In this example, a handling body for handling cultured cells and a complex of a cultured cell-handling body using the handling body were prepared. First, a 0.25 (w / v)% aqueous solution of poly-N-isopropylacrylamide PNIPAAm (lower critical solution temperature: 32 ° C.) was prepared. The final concentration of collagen IV was 14 μg / ml, and the final concentration of laminin was 10 μg / ml. The ECM component-containing polymer composition A was prepared so as to be ml. This polymer composition A was cast on a surface of a plate made of polydimethylsiloxane (PDMS) in a rectangular shape so as to be 100 μg / cm ² and dried at 4 ° C. for 18 hours.

Next, a 5 (w / v)% ethanol solution of PNIPAAm was prepared as a polymer composition B, and cast and overlaid on the surface of the dried and cured polymer composition A at 50 μl / cm ² at room temperature. Dried. The obtained cured product was peeled from PDMS to obtain a rectangular handling body. The handling body could be easily peeled from the PDMS surface and had rigidity and strength suitable for handling.

Next, in the presence of DMEM (Dulbecco's modified Eagle Medium) medium containing 10% FBS (Fatal Bovine Serum) and 1% penicillin / streptomycin, a mouse myoblast cell line is formed on the cured surface of the polymer composition A of the handling body. C2C12 cells were supplied (2 × 10 ³ cells / cm ² ) and cultured at 37 ° C. When the cells were observed after culturing for 24 hours, it was confirmed that the cells were engrafted on the cured layer of the polymer composition A and proliferated. The cultured cells engrafted on the surface of the cured layer of the handling body have high mechanical strength when they are integrated with the cured layer, and these can be handled sufficiently with tweezers or the like as an integral composite. Moreover, the obtained composite_body | complex had moderate flexibility and was able to be rounded and made into the tubular body.

(Manufacture of a handling body that can be handled in a state in which the orientation of cultured cells is controlled and the orientation control of cultured cells)
In this example, the surface of the cured layer of PNIPAAm was provided with an uneven shape, and the cells were cultured.

  First, an uneven shape was imparted to the PDMS surface on which the handling body was cast using the unevenness produced by the optical lithography technique as a mold. The concavo-convex shape has a length of 200 μm to 1000 μm, a height of 10 to 100 μm, a width of 5 μm to 200 μm (5 μm, 10 μm, 20 μm, 30 μm, 50 μm, and 100 μm), and variously different at intervals of twice these line widths. The convex part which can form a recessed part in a handling body was formed. The same polymer composition A as prepared in Example 1 was cast on the PDMS surface having such convex portions in the same manner and cured under the same conditions. Furthermore, the polymer composition B was cast in the same manner as in Example 1. Thus, a handling body having a multilayer structure was produced.

The mouse myoblast cell line C2C12 cells were supplied to the surface of the cured layer having a large number of recesses of the polymer composition A of the handling body in the same manner as in Example 1 and cultured at 37 ° C., and the cell culture process was observed. . For the observation, the cultured cells were stained in DMEM for 30 minutes with Cell Tracker manufactured by Lonza, washed with DMEM, and then used with a fluorescence microscope (BZ-8000 manufactured by Keyence). The results are shown in FIGS.

  C2C12 cells did not engraft when the width of the recess was less than 5 μm and were not oriented when the width was more than 100 μm, but were aligned along the major axis direction of the recess when the width of the recess was 5 μm or more and 100 μm or less. Further, as shown in FIG. 7, it was found that C2C12 cells divide while extending the protrusion along the recess and moving toward the long axis of the recess. Further, as shown in FIG. 8, this orientation continued until the cell density became dense.

  From the above, it was found that an uneven shape capable of controlling the orientation of cultured cells can be set on PNIPAAm.

(Manufacturing of a handling body having a concavo-convex shape based on a metal ground surface and manufacturing of a composite)
In the present Example, the uneven | corrugated shape which was excellent in the mobility of the cultured cell which can accelerate | stimulate the differentiation of a cultured cell on a handling body was examined.

  The surface of the iron block was shaved with sandpaper (No. 150) in one direction at a constant load, and a PDMS plate was produced using the ground surface of the metal block as a mold. The polymer composition of Example 1 was formed on the surface of the PDMS plate. A handling body 1 having a concavo-convex shape corresponding to the ground surface on the surface of the cured layer of the polymer composition A was prepared by supplying A and the polymer composition B in the same manner as in Example 1 and curing them.

  Also, a PDMS plate having a concavo-convex shape matching the ground surface was prepared, and polymer composition A and polymer composition B of Example 1 were supplied on the surface of this PDMS plate in the same manner as in Example 1 and cured. As a result, a handling body 2 having a concavo-convex shape in which the grinding surface was inverted on the surface of the cured layer of the polymer composition A was produced.

  The mouse myoblast cell line C2C12 cells were supplied to the surface of the cured layer having the uneven shape of each of the handling bodies 1 and 2 in the same manner as in Example 1 and cultured at 37 ° C., and the cell culture process was observed. . Mouse myoblast cell line C2C12 cells were supplied to the surface of the cured layer in the same manner as in Example 1 and cultured at 37 ° C., and the cell culture process was observed. The observation was performed in the same manner as in Example 2 while observing with a phase contrast microscope (BZ-8000 manufactured by Keyence Corporation). The results for the handling body 2 are shown in FIG.

  With the handling body 1 having an uneven shape that coincides with the ground surface of the metal block, the cells can be engrafted and cultured, and the cells were oriented along the shape of the recesses. Further, as shown in FIG. 9, even in the handling body 2 having an inverted shape obtained by transferring the grinding surface of the metal block, the cells can be engrafted and cultured, and further, the cells are oriented along the shape of the recesses, The orientation was maintained even when the cell density increased.

Further, a DMEM medium containing 2% CS (Calf Serum) and 1% penicillin / streptomycin is supplied to the C2C12 cells cultured on the surface of the handling body 2, and the C2C12 cells are cultured in a CO ₂ incubator (5% CO ₂ , 37 ° C. ) To differentiate myoblasts. After culture, cells washed with PBS (myoblasts, myotubes) were fixed with 4% p-formaldehyde. Thereafter, the cells were treated with 0.2% Triton X as a surfactant and blocked with PBS containing 1 mg / ml bovine serum albumin (BSA). After blocking, nuclei were stained with Alexa546-Palliioidin containing DAPI and f-actin, and actinin with mouse anti-actinin antibody and secondary antibody Alexa488-anti-mouse IgG antibody. Observation was performed with a fluorescence microscope, and cells stained with actinin were determined to be myotubes. The results are shown in FIG. As shown in the left of FIG. 10, myocytes oriented in the direction of the concavo-convex shape could be obtained. It was found that the obtained myocytes constitute multinucleated cells and have characteristics as muscles. Further, as shown on the right side of FIG. 10, it was found that the actin filaments in the muscle cells were also oriented along the direction of the irregular shape.

  From the above, it was found that the concavo-convex shape capable of controlling the orientation of cultured cells can be produced by a simple method and a molding method without requiring an apparatus such as photolithography and hot embossing. In addition, according to such a technique, there are various types of materials that can give the uneven shape, and there is no restriction on the area where the uneven shape can be given, and it is possible to produce a cultured cell layer with controlled orientation in a large area. all right.

(Examination of metal grinding surface shape as uneven shape for orientation control)
In this example, metal grinding surface shapes suitable for controlling the orientation of cultured cells were examined. Abrasives with different counts on the surface of the iron block (Plate 1: 9 μm diamond suspension (Buhler, USA), Plate 2: Sandpaper No. 400, Plate 3: Sandpaper No. 150, Plate 4: Sandpaper No. 40) The PDMS has fine line-shaped irregularities formed by rubbing in one direction, and has four types of grinding surfaces with different surface roughnesses Rz. Plates (plates 1-4) were formed. The measurement results of the surface roughness Rz on the metal grinding surface are shown in the following table. The surface roughness Rz was measured using three Hommel Tester LV15 (MITUTOYO) at three different locations of the iron block.

(Table 1)
Type Rz (μm)
Plate 1 0.3
Plate 2 2
Plate 3 6-8
Plate 4 13 or higher

  A laminin PBS solution (10 μg / ml) is supplied and dried on the surface of the PDMS plate having an irregular shape to form a laminin coating layer, and the mouse myoblast cell line C2C12 cells are seeded and cultured in the same manner as in Example 1. did. A laminin coating layer was similarly formed using a PDMS plate having no irregular shape as a control, and C2C12 cells were cultured. The results are shown in FIG. Visualization was performed in the same manner as the myoblast observation in Example 3.

  As shown in the upper part of FIG. 11, myoblasts were aligned along the uneven shape in the plates 2 to 4, but no alignment was observed in the control and the plate 1. In addition, as shown in the lower part of FIG. 11, the myotube was also oriented only in the plates 2 to 4, and the orientation of the myotube was not observed in the control and the plate 1.

  From the above, it was found that in order to orient myoblasts, the uneven shape must have a certain size or more. It was also found that myoblast cell orientation control can be realized at the same time by controlling the orientation of myoblasts using such irregularities.

(Modeling of cellular structure by stacking composites)
In this example, a composite comprising a unit comprising one support layer and one cultured cell layer is laminated, and temperature conditions based on the critical dissolution temperature of the temperature-responsive polymer contained in the matrix constituting the support layer In addition, the support was disintegrated by imparting, and the two cultured cell layers were integrated to form a cell structure.

The handling body prepared in Example 1 was seeded with rat cardiomyocytes and cultured in Medium 199 containing 10% FBS, 1% penicillin / streptomycin in a 37 ° C., 5% CO ₂ environment. A cultured cell layer-handled complex having a cultured cardiomyocyte layer in which cardiomyocytes were oriented along the direction was obtained. The two composites obtained in this manner were laminated on the electrode array so that the cultured cardiomyocyte layers overlapped one above the other to obtain a composite having a multilayer structure. The composite of this multilayer structure was placed at 22 ° C. for 12 hours based on the critical dissolution temperature (32 ° C.) of the temperature-responsive polymer used to collapse the temperature-responsive matrix. After 12 hours, this complex was washed with water at 22 ° C. and observed at room temperature. As a result, the cultured cardiomyocyte layers were integrated at portions overlapping each other, and partially multi-layered cardiomyocytes A structure was formed.

  On the electrode array, the cardiomyocyte structure is located at two locations (one in the cultured myocardium on the lower layer side and the other in the cultured myocardium on the upper layer side) across the overlapping portion of the cultured cardiomyocyte layer. ) To measure the spontaneous action potential of the myocardium. As a result, it was found that the phase of the spontaneous action potential in each place was uniform, and a physiological connection was formed in the overlapping part of the cultured cardiomyocyte layer.

  From the above, it was found that the two cultured cardiomyocyte layers overlaid by the collapse of the temperature-responsive matrix are physiologically bound.

It is a figure which shows an example of the process of obtaining a cell structure from the handling body of this invention through a composite_body | complex. It is a figure which shows the one aspect | mode of the handling body of this invention. It is a figure which shows an example of the manufacturing process of the handling body of this invention. It is a figure which shows the various aspects of the composite_body | complex of this invention. It is a figure which shows an example of the manufacturing process of the composite_body | complex of this invention. It is a figure which shows an example of the cell structure of invention. 4 is a view showing a photograph of a cell culture process in Example 2. FIG. It is a figure which shows the photograph which is maintaining orientation, even if a cell becomes dense. It is a figure which shows the photograph of the state of the myoblast cultured on the surface which has the uneven | corrugated shape which reversed the metal grinding | polishing surface shape. It is a figure which shows the photograph of the state of the myocyte obtained by differentiating the myoblast cultured on the surface which has the uneven | corrugated shape which reversed the metal grinding | polishing surface shape. It is a figure which shows the photograph of the state of the myoblast cultured on the surface which has the uneven | corrugated shape which reversed the metal grinding | polishing surface shape. The upper part shows a state in which myoblasts are oriented, and the lower part shows a state in which myotubes are oriented.

Explanation of symbols

2 molded body, 4 matrix 6a, 6b layer, 8 cell adhesion region, 10 handling body, 12 20 non-affinity surface, 50 cultured cell-support layer complex, 60 cultured cell layer, 62 another cultured cell layer, 70 units, 100 cell structures

Claims (9)

A self-supporting molded article comprising a matrix containing a temperature-responsive polymer and having a cell adhesion region, which can be placed in a cell culture device, and cells are cultured on the cell adhesion region from the cell culture device. The sheet is separable together with the obtained cultured cell layer, and includes a first layer that includes the cell-adhesive region and includes the temperature-responsive polymer and an extracellular matrix component, and is laminated on the first layer. A second layer containing the temperature-responsive polymer at a higher concentration than the first layer and not containing the extracellular matrix component or containing the extracellular matrix component at a lower concentration than the first layer. a support layer and a laminate at least in part on the cultured cells of the surface of the support layer are laminated units 2 or more with a cultured cell layer supported bind to each other There, complex. A self-supporting molded article comprising a matrix containing a temperature-responsive polymer and having a cell adhesion region, which can be placed in a cell culture device, and cells are cultured on the cell adhesion region from the cell culture device. The sheet is separable together with the obtained cultured cell layer, and includes a first layer that includes the cell-adhesive region and includes the temperature-responsive polymer and an extracellular matrix component, and is laminated on the first layer. A second layer containing the temperature-responsive polymer at a higher concentration than the first layer and not containing the extracellular matrix component or containing the extracellular matrix component at a lower concentration than the first layer. a support layer, a laminate of at least a part the cultured cells are layered units 2 or more with a cultured cell layer supported coupled to each other on the surface of said support layer Ri,
In at least one of the units, the cell adhesion region has a concavo-convex shape for orienting and culturing cells, and the cultured cells are oriented in a certain direction in at least a part of the cultured cell layer. . The complex according to claim 1 or 2 , wherein two or more of the units have a multilayer structure in which the support layer and the cultured cell layer are alternately stacked. The complex according to any one of claims 1 to 3 , wherein the cultured cells are oriented in substantially the same direction in at least a part of each of the cultured cell layers stacked via the support layer. The complex according to any one of claims 1 to 4 , wherein the cultured cells are one or more selected from the group consisting of myoblasts, myotubes, smooth muscle cells and cardiomyocytes. A method for producing a complex of cultured cell-support layer according to any one of claims 1 to 5 ,
A self-supporting molded article comprising a matrix containing a temperature-responsive polymer and having a cell adhesion region, which can be placed in a cell culture device, and cells are cultured on the cell adhesion region from the cell culture device. The sheet is separable together with the obtained cultured cell layer, and includes a first layer that includes the cell-adhesive region and includes the temperature-responsive polymer and an extracellular matrix component, and is laminated on the first layer. A second layer containing the temperature-responsive polymer at a higher concentration than the first layer and not containing the extracellular matrix component or containing the extracellular matrix component at a lower concentration than the first layer. Culturing cells in the cell adhesion region of the support for handling cultured cells provided with a support layer to produce one unit;
Laminating the one unit produced in the unit production step and the other unit, and
In at least one of the above units, the cell adhesion region has a concavo-convex shape for orienting and culturing cells, and the cultured cells are oriented in a certain direction in at least a part of the cultured cell layer. .   The said lamination process is a manufacturing method of Claim 6 which is a process of laminating | stacking so that the said support body layer of said one unit may contact the cultured cell layer of said other unit. A self-supporting molded article comprising a matrix containing a temperature-responsive polymer and having a cell adhesion region, which can be placed in a cell culture device, and cells are cultured on the cell adhesion region from the cell culture device. The sheet is separable together with the obtained cultured cell layer, and is laminated on the first layer including the cell-adhesive region, the temperature-responsive polymer and the extracellular matrix component, and the first layer. A second layer containing the temperature-responsive polymer at a higher concentration than one layer and not containing the extracellular matrix component or containing the extracellular matrix component at a lower concentration than the first layer; support layer and a laminate at least in part on the cultured cells of the surface of the support layer are laminated units 2 or more with a cultured cell layer supported coupled to one another with There, cultured cells - a step of preparing a composite of the support layer,
Providing the support layer of the composite with a temperature condition based on a critical dissolution temperature of the temperature-responsive polymer to disintegrate the laminate of the cultured cell layer ,
With
In at least one of the units, the cell adhesion region has a concavo-convex shape for orienting and culturing cells, and the cultured cells are oriented in a certain direction in at least a part of the cultured cell layer. Body manufacturing method. The manufacturing method according to claim 8 , wherein the collapsing step is a step of collapsing two or more support layers included in the composite at the same time .