JPWO2017077985A1 - Method for producing laminated cell sheet composition, laminated cell sheet composition produced thereby and apparatus for producing the same - Google Patents

Abstract

  The present invention includes a step of bringing a cultured cell migration jig into contact with an upper surface of a sheet-like cell group, and attaching a first cell sheet to the cultured cell migration jig, an upper surface of a second sheet-like cell group, and / or the above-mentioned A step of applying a cell-adhesive substance to the lower surface of the first cell sheet attached to the cultured cell transfer jig, and the lower surface of the first cell sheet attached to the cultured cell transfer jig to the second sheet-like cell A method of producing a laminated cell sheet composition comprising the steps of contacting a top surface of a group and adhering a second cell sheet to the first cell sheet. The present invention also provides a laminated cell sheet composition obtained by the method. The present invention also provides an apparatus for producing a laminated cell sheet composition.

Description

  The present invention relates to a method and an apparatus for producing a laminated cell sheet composition. This application is a priority claim application based on the Japanese application (Japanese Patent Application No. 2015-217784) filed on November 5, 2015.

  In recent years, techniques for producing three-dimensional tissues and organs using cells have been developed for the purpose of use in regenerative medicine and drug-responsive tissue models. Conventionally, many adherent cells could only be cultured two-dimensionally (planar) in vitro. However, many tissues in the living body are constructed by three-dimensional accumulation of cells, and in order to get closer to the state in the living body, a technique for constructing the cells in three dimensions has been required. .

  Examples of attempts to construct cells three-dimensionally include, for example, a method in which cells are seeded on a scaffold called scaffold to construct a three-dimensional tissue and transplant, or a decellularized organ / tissue and a remaining matrix A method has been developed in which cells are seeded to form a three-dimensional structure using the scaffold as a scaffold, a method for constructing a tissue by three-dimensionally laminating cell sheets separated in a sheet shape, and the like. These are all technologies that are expected to be applied in the fields of regenerative medicine and drug discovery, and are expected to be put to practical use at an early stage.

  In order to produce the above-mentioned cell sheet, a cell culture dish (temperature-responsive culture dish) was developed in which the surface of the culture substrate was coated with a polymer having an upper or lower critical solution temperature in water of 0 to 80 ° C. (patent) Reference 1). Using the cell culture dish, cell culture is performed at a temperature below the upper critical solution temperature of the polymer coated on the surface of the culture dish or above the lower critical solution temperature, and after culturing until the cells become confluent, When the temperature is lower than the lower critical solution temperature, the cells can be collected in a sheet-like manner and noninvasively.

  Conventionally, in order to recover adherent cells from culture dishes, proteolytic enzymes such as trypsin and dispase must be used. Matrix proteins expressed on the cell surface and gap junctions that connect cells to each other It was not possible to recover the protein unless the protein constituting it was decomposed. Since the proteins necessary for cell adhesion were decomposed, the cells were collected in a disassembled state. As a result, when transplanted, the cells were difficult to engraft in the tissue. On the other hand, the cell sheet obtained by the temperature-responsive culture dish is superior in the past that the protein on the cell surface is hardly damaged and can be quickly engrafted in the affected area due to the presence of the adhesion protein remaining on the surface. It has the characteristics. As a result, the effect of cell transplantation can be maximized, and the cell transplantation technology has been greatly developed.

  With the establishment of cell sheet engineering, cell therapy technology has undergone dramatic changes, but organ transplantation is still effective for severe patients who do not believe that cell transplantation will be therapeutic. It is a therapeutic tool. However, the number of organs to be supplied is overwhelmingly smaller than the number of patients requiring organ transplantation, and there is a demand for development of a technique for constructing and supplying organs or tissues. The technology of cell sheet engineering is applied also to such a subject, and the trial which builds the biological tissue with thickness by laminating | stacking a cell sheet is performed (patent document 2). In addition, a cell moving jig (Patent Document 3) for efficiently laminating cell sheets and a cell culture treatment apparatus (Patent Document 4) for producing a large number of cell sheets have been developed.

Japanese Patent Laid-Open No. 02-21865 International Publication No. 02/008387 JP 2005-176812 A International Publication No. 12/098931

  As described above, methods and apparatuses for efficiently laminating cell sheets have been developed, but it takes at least about 30 minutes to laminate cell sheets once, and has a certain thickness. In order to produce a textured structure, it is necessary to repeat lamination several times to several tens of times, and there is a problem that it takes a lot of time to produce one structure. Accordingly, an object of the present invention is to provide a method for efficiently producing a laminated cell sheet composition by shortening the time required for laminating cell sheets.

  In order to solve the above-mentioned problems, the present inventors have conducted research and development by adding studies from various angles. As a result, surprisingly, when the cell sheet adhered to the cell transfer jig was applied and contacted before contacting the cell sheet to be laminated, the cell sheet was laminated. It has been found that the time required for is greatly reduced. In addition, before the cell sheet adhered to the cell migration jig is brought into contact with the laminated cell sheet, a cell adhesive substance is applied and contacted while applying a load. It has been found that the time required is greatly reduced. That is, the present invention is as follows.

[1] (1) A cultured cell moving jig is brought into contact with the upper surface of the first sheet-like cell group on the stimulus-responsive culture substrate, and the first sheet-like cell group on the stimulus-responsive culture substrate is peeled off. Applying a stimulus to attach the peeled first cell sheet to the cultured cell moving jig,
(2) A step of applying a cell adhesive substance to the upper surface of the second sheet-like cell group on the stimulus-responsive culture substrate and / or the lower surface of the first cell sheet attached to the cultured cell moving jig. ,
(3) The lower surface of the first cell sheet attached to the cultured cell migration jig is brought into contact with the upper surface of the second sheet-like cell group, and the second sheet-like cell group is separated from the stimulus-responsive culture substrate. A method for producing a laminated cell sheet composition, comprising a step of applying a stimulus for peeling and adhering the peeled second cell sheet to the lower surface of the first cell sheet.

  [2] The method according to [1], wherein when applying the stimulus of the step (3), further loading is performed simultaneously.

[3] For the laminated cell sheet composition attached to the cultured cell migration jig obtained in the step (3),
(4) The upper surface of the third sheet-like cell group on the stimulus-responsive culture substrate and / or the lower surface of the laminated cell sheet composition attached to the cultured cell moving jig obtained in the step (3) Applying a cell adhesive substance to
(5) The lower surface of the laminated cell sheet composition attached to the cultured cell transfer jig is brought into contact with the upper surface of the third sheet-like cell group, and the third sheet-like cell group is in contact with the stimulus-responsive culture medium. Providing a stimulus for peeling from the material, and bonding the peeled third cell sheet to the lower surface of the laminated cell sheet composition;
(6) A step of repeating the steps (4) to (5) any number of times,
The method according to [1] or [2], comprising:

  [4] The method according to any one of [1] to [3], wherein the cell adhesive substance is a cell adhesive protein, a cell adhesive peptide, and / or a cell adhesive polysaccharide.

  [5] The cell adhesive substance is selected from the group consisting of polylysine, collagen, vitronectin, fibrin, a mixture of fibrinogen and thrombin, fibronectin, thrombospondin, gelatin, laminin, chitosan adhesive, and alginate adhesive. The method according to any one of [1] to [3], which is one or more substances.

[6] The step (2)
Applying a cell adhesive substance to the upper surface of the second sheet-like cell group on the stimulus-responsive culture substrate and the lower surface of the first cell sheet attached to the cultured cell transfer jig,
The cell adhesive substance applied to the upper surface of the second sheet-like cell group is fibrinogen and the cell adhesive substance applied to the lower surface of the first cell sheet is thrombin, or
Any of [1] to [5], wherein the cell adhesive substance applied to the upper surface of the second sheet-like cell group is thrombin, and the cell adhesive substance applied to the lower surface of the first cell sheet is fibrinogen. 2. The method according to item 1.

[7] The step (4)
Cell adhesion to the upper surface of the third sheet-like cell group on the stimulus-responsive culture substrate and the lower surface of the laminated cell sheet composition attached to the cultured cell transfer jig obtained in the step (3) A process of applying a substance,
The cell adhesive substance applied to the upper surface of the third sheet-like cell group is fibrinogen, and the cell adhesive substance applied to the lower surface of the laminated cell sheet composition is thrombin, or
[3] to [6], wherein the cell adhesive substance applied to the upper surface of the third sheet-like cell group is thrombin, and the cell adhesive substance applied to the lower surface of the laminated cell sheet composition is fibrinogen. The method according to any one of the above.

  [8] The stimuli-responsive culture substrate is a temperature-responsive culture substrate, and the stimulus is a temperature not higher than the lower critical solution temperature or higher than the upper critical solution temperature. [1] to [7] The method according to any one of the above.

  [9] The stimulation responsive culture substrate according to any one of [1] to [8], wherein the culture substrate is a culture substrate in which poly-N-isopropylacrylamide is coated on at least a part of the culture surface. the method of.

  [10] The cultured cell migration jig is a cultured cell migration jig having a cell adhesion part composed of one or more of cell adhesion protein, cell adhesion peptide, or hydrophilic polymer. [9] The method according to any one of [9].

  [11] The laminated cell sheet composition is one or more selected from the group consisting of cardiomyocytes, hepatocytes, fibroblasts, myoblasts, pancreatic cells, kidney cells, vascular endothelial cells, and epithelial cells. The method according to any one of [1] to [10], comprising a cell.

[12] A laminated cell sheet composition obtained by the method according to any one of [1] to [11].
[13] The laminated cell sheet composition according to [12], wherein the average thickness of the laminated cell sheet composition is 80 μm or more.

  [14] An apparatus for producing a laminated cell sheet composition for carrying out the method according to any one of [1] to [11].

[15] A mounting table provided with stimulation means for stimulating the stimulus-responsive culture substrate and peeling the cell sheet from the stimulus-responsive culture substrate;
A cultured cell transfer jig for attaching the cell sheet;
An arm for attaching the cultured cell moving jig and applying a load to the cultured cell moving jig;
An apparatus for producing a laminated cell sheet composition, comprising: a cell adhesive substance applying means for applying a cell adhesive substance to the cell sheet on the stimulus-responsive culture substrate.

[16] The cultured cell moving jig is brought into contact with the upper surface of the first sheet-like cell group on the stimulus-responsive culture substrate placed on the mounting table, and the stimulus-responsive culture substrate is stimulated by the stimulation means. Giving a stimulus that exfoliates the first sheet-like cell group on the top, and adheres the obtained first cell sheet to the cultured cell migration jig,
Applying the cell adhesive substance supplied by the cell adhesive substance applying means to the upper surface of the second sheet-like cell group mounted on the mounting table and / or the lower surface of the first cell sheet;
The lower surface of the first cell sheet attached to the cultured cell transfer jig is brought into contact with the upper surface of the second sheet-like cell group, and the second sheet-like cell on the stimulus-responsive culture substrate is stimulated by the stimulating means. [15], characterized in that a stimulus is applied to the group, and at the same time a load is applied to the cultured cell moving jig, and the obtained second cell sheet is quickly laminated on the first cell sheet. The manufacturing apparatus of the laminated cell sheet composition of description.

  According to the present invention, the time for laminating the cell sheets is greatly reduced, and the damage to the cells received when laminating the cell sheets is reduced. Moreover, even when a cell type that has been difficult to be laminated is used, the cell sheet can be laminated, and a laminated cell sheet composition having a thickness can be easily and reproducibly obtained. Furthermore, according to the present invention, it is possible to provide a laminated cell sheet composition capable of transplanting a large amount of cells at once.

FIG. 1 is a schematic diagram showing the stamp lamination technique of the present invention. FIG. 2 is a schematic view showing the cell sheet laminating method of the present invention. FIG. 3 is a view showing a laminated cell sheet produced by the cell sheet laminating method of the present invention. (A) Appearance photograph of laminated cell sheets (10 sheets). (B) It is a schematic diagram which shows the cross section of a laminated cell sheet. (C) It is the figure which HE dye | stained the laminated cell sheet (10 sheets). (D) It is the figure which immunostained the laminated cell sheet (10 sheets). (E) HE-stained layered cell sheet (15 sheets). (F) It is the figure which immunostained the laminated cell sheet (15 sheets). (CF) Blue: nucleus (DAPI), green: cardiomyocytes (cTnT). FIG. 4 shows the cell viability when the cell sheets are laminated by the cell sheet lamination method (left) and the conventional method (right) of the present invention (n = 3). FIG. 5 is a view showing the pulsation of the laminated myocardial sheet produced by the cell sheet lamination method of the present invention. 1 to 5 indicate time-series (approximately 0.1 second interval) observation images, and the green portion indicates a portion where the cardiac muscle cells are beating. FIG. 6 is a diagram showing the pulsatile potential of the cardiomyocyte sheet obtained by the present invention. (A) The cardiomyocyte sheet of the present invention and potential measurement points A and B are shown. (B) It is a graph which shows the pulsatile potential of the electric potential measurement point A (upper stage) and the electric potential measurement point B (lower stage). FIG. 7 is a view showing a vascular network form in a laminated cell sheet produced by the cell sheet laminating method of the present invention. (A) It is the schematic diagram which showed the state of the photograph of BD below. (B) It is a figure which shows the vascular network in the cell sheet on a culture dish. (C) It is a figure which shows the vascular network in the cell sheet laminated | stacked two sheets using the method of this invention. Green: CD31 positive cells, blue: nucleus (DAPI). (D) It is a figure which shows the blood-vessel network observed in the state which peeled the cell sheet from the cell culture dish, without using the method of this invention. The vascular network is represented in green (GFP). FIG. 8 is a view showing a state in which a cardiomyocyte sheet produced by the method of the present invention or the conventional method is implanted subcutaneously on the back of a nude rat. (A) It is a figure which shows the mode two weeks after transplanting a myocardial cell sheet subcutaneously on the back of a nude rat. * B on the left of the dotted line indicates a cardiomyocyte sheet prepared by the method of the present invention, and * C on the right of the dotted line indicates a transplanted part of the cardiomyocyte sheet prepared by the conventional method. (B) It is an enlarged view of * B part of (A). (C) It is an enlarged part of * C part of (A). FIG. 9 is a view in which it is confirmed whether the cardiomyocyte sheet produced by the method of the present invention is fixed at the transplant site. (A) It is a figure of the Azan staining of the site | part which transplanted the myocardial cell sheet produced by the conventional method. (B) It is a figure which shows the position of the cell nucleus (blue, Hoechst) and cardiac muscle troponin T (green, cTnT) expression of the site | part which transplanted the myocardial cell sheet produced by the conventional method. (C) Azan staining of a site transplanted with a cardiomyocyte sheet produced by the method of the present invention. (D) It is a figure which shows the position of the cell nucleus (blue, Hoechst) and cardiac muscle troponin T (green, cTnT) expression of the site | part which transplanted the cardiac muscle cell sheet produced by the method of this invention. (E) It is the graph which quantified the fixed area of the transplanted tissue. FIG. 10 is a view showing a state in which a GFP-expressing cardiomyocyte sheet produced by the method of the present invention or a conventional method is transplanted subcutaneously on the back of a nude rat. (A) The state of GFP expression (green) in the part transplanted with the cell sheet produced by the method of the present invention. (B) GFP expression in a part transplanted with a cell sheet prepared by a conventional method (green). (C) It is a graph which shows the fixed area (mm < ² >) after transplant which quantified the GFP expression area of (A) and (B). FIG. 11 is a view showing a laminated hepatocyte sheet produced by the cell sheet laminating method of the present invention. (A, B) The HE-stained figure of HepG2 laminated cell sheet (10 layers) is shown (A: low magnification, B: high magnification). (C, D) HE-stained diagrams of HepG2 laminated cell sheet (15 layers) are shown (C: low magnification, D: high magnification). FIG. 12 is a view showing an example of an apparatus for producing a laminated cell sheet composition capable of performing the production method of the present invention. FIG. 13 is a view showing a tissue image two weeks after transplantation of a laminated cell sheet (10 layers) produced by the cell sheet laminating method of the present invention into a rat. It is a figure which shows the position of the cell nucleus (blue, Hoechst), cardiac muscle troponin T (green, cTnT), and CD31 (red) expression of the site | part which transplanted the myocardial cell sheet produced by the method of this invention. CD31 positive cells represent vascular endothelial cells.

  The present invention relates to a method for producing a laminated cell sheet composition. The “cell sheet” in the present invention refers to a cell group of one layer or plural layers obtained by culturing on a cell culture substrate and peeling off from the cell culture substrate. In this specification, a cell group adhered to a cell culture substrate is referred to as a “sheet cell group”, and a cell group obtained by detaching the sheet cell group from the cell culture substrate is distinguished from a “cell sheet”. To do. The laminated cell sheet composition includes a laminate of a plurality of the cell sheets.

The present invention provides a method for producing a laminated cell sheet composition, which in one embodiment includes the following steps:
(1) A stimulus for bringing the cultured cell moving jig into contact with the upper surface of the first sheet-like cell group on the stimulus-responsive culture substrate and peeling the first sheet-like cell group on the stimulus-responsive culture substrate. Giving and attaching the peeled first cell sheet to the cultured cell moving jig,
(2) A step of applying a cell adhesive substance to the upper surface of the second sheet-like cell group on the stimulus-responsive culture substrate and / or the lower surface of the first cell sheet attached to the cultured cell moving jig. ,
(3) The lower surface of the first cell sheet attached to the cultured cell migration jig is brought into contact with the upper surface of the second sheet-like cell group, and the second sheet-like cell group is separated from the stimulus-responsive culture substrate. The process of giving the irritation | stimulation to peel and adhere | attaching the peeled 2nd cell sheet on the lower surface of this 1st cell sheet.

  According to the method of the present invention, the time for producing the laminated cell sheet composition is greatly reduced, and the damage to the cells received when producing the laminated cell sheet is reduced. Moreover, even when a cell type that has been difficult to be laminated so far is used, a laminated cell sheet composition can be produced easily and reproducibly.

  In addition, the method of the present invention further includes a step of simultaneously loading the stimulus in the above step (3). Thereby, the laminated cell sheet composition which can transplant a lot of cells at once can be manufactured.

In addition, the method of the present invention further provides a layered cell sheet composition attached to the cultured cell transfer jig obtained in the above step (3).
(4) The upper surface of the third sheet-like cell group on the stimulus-responsive culture substrate and / or the lower surface of the laminated cell sheet composition attached to the cultured cell moving jig obtained in the step (3) Applying a cell adhesive substance to
(5) The lower surface of the laminated cell sheet composition attached to the cultured cell transfer jig is brought into contact with the upper surface of the third sheet-like cell group, and the third sheet-like cell group is in contact with the stimulus-responsive culture medium. Providing a stimulus for peeling from the material, and bonding the peeled third cell sheet to the lower surface of the laminated cell sheet composition;
(6) A step of repeating the steps (4) to (5) any number of times,
Is included.

  Thereby, it becomes possible to obtain a laminated cell sheet composition in which any number of sheets is laminated easily and with good reproducibility.

  The origin of the animal species of the cells used in the present invention is not particularly limited, but for example, human, rat, mouse, guinea pig, marmoset, rabbit, dog, cat, sheep, pig, goat, monkey, chimpanzee or Examples thereof include mammal animals such as immunodeficient animals, birds, reptiles, amphibians, amphibians, fish, and insects. When the composition of the present invention is used for human treatment, it is desirable to use human, swine for pig treatment, monkey for monkey treatment, and chimpanzee-derived cells for chimpanzee treatment. In addition, when the treatment is performed by a human, cells collected from the patient himself (autologous cells), cells collected from other people's cells (transgenic cells), or commercially available cell lines may be used. It may be.

  What is necessary is just to select or adjust suitably according to a use about the cell type used for this invention, the number of cells, the ratio of the cell contained. For example, in the case of a method for evaluating myocardial tissue regeneration or myocardial function, the cells used are cardiomyocytes, myocardial blasts, myoblasts, mesenchymal stem cells, vascular endothelial cells, vascular endothelial progenitor cells, Examples thereof include any one of fibroblasts, bone marrow-derived cells and fat-derived cells, or a mixture of two or more cells.

  For the purpose of regeneration of liver tissue, production of an artificial liver simulating liver tissue, or a method for evaluating the function of liver tissue, for example, the cells used include liver parenchymal cells (also called liver cells), Sinus endothelial cells, Kupffer cells, stellate cells, pit cells, bile duct epithelial cells, vascular endothelial cells, vascular endothelial progenitor cells, fibroblasts, bone marrow derived cells, adipose derived cells, mesenchymal stem cells, or 2 The thing etc. which the cell of the seed | species or more mixed are mentioned.

  For the purpose of regeneration of kidney tissue, production of an artificial kidney simulating kidney tissue, or a method for evaluating kidney function, for example, kidney cells, granule cells, collecting duct epithelial cells, wall side epithelium are used. Cells, podocytes, mesangial cells, smooth muscle cells, tubule cells, interstitial cells, glomerular cells, vascular endothelial cells, vascular endothelial precursor cells, fibroblasts, bone marrow-derived cells, adipose-derived cells, mesenchymal stem cells Any one or a mixture of two or more types of cells may be used.

  For the purpose of regeneration of adrenal tissue, production of an artificial adrenal that simulates the adrenal gland, or a method for evaluating adrenal function, for example, the cells used include adrenal medullary cells, adrenal cortical cells, spherical layer cells, bundled layers Examples include cells, reticuloendothelial cells, vascular endothelial cells, vascular endothelial progenitor cells, fibroblasts, bone marrow-derived cells, adipose-derived cells, mesenchymal stem cells, or a mixture of two or more cells. It is done.

  For the purpose of evaluating skin regeneration or skin function, for example, cells used include epidermal keratinocytes, melanocytes, napped muscle cells, hair follicle cells, vascular endothelial cells, vascular endothelial progenitor cells, fibers Examples include blast cells, bone marrow-derived cells, fat-derived cells, mesenchymal stem cells, or a mixture of two or more cells.

  In the case of aiming at the regeneration of mucosal tissue or the method of evaluating the function of mucosal tissue, for example, as the cells to be used, cells collected from the tissue constituting the mucous membrane may be used. Examples of the mucosa include buccal mucosa, gastric mucosa, intestinal mucosa, olfactory epithelium, oral mucosa and uterine mucosa. Among the cells collected from the mucosal tissue, any one type or a mixture of two or more types of cells can be mentioned, and the type is not limited at all.

  For the purpose of regeneration of the pancreas, production of an artificial pancreas simulating the pancreas, treatment of diabetes or evaluation of pancreatic function, the cells used include, for example, α cells, β cells, δ constituting the pancreas Cells, PP cells, and pancreatic acinar cells (collectively referred to as pancreatic cells) may be used.

  These cells may be cells derived from ES cells, iPS cells, Muse cells, mesenchymal stem cells, and the like.

  As the cells used in the present invention, cells obtained by mincing a living tissue can also be used. In this case, many types of cells are mixed in cells derived from living tissue. For example, in an embodiment of the present invention described later, a rat heart tissue is minced and a cell sheet is prepared using cardiomyocytes contained therein, and the cell sheet includes fibers derived from heart tissue in addition to cardiomyocytes. It includes blast cells, mural cells, vascular endothelial cells and the like. Therefore, a cell sorter or an antibody can be used to remove unnecessary cells depending on the purpose, or conversely, necessary cells can be added. The “cardiomyocyte sheet” in the examples of the present specification refers to a sheet containing fibroblasts, wall cells, vascular endothelial cells and the like in addition to the above-described cardiomyocytes.

  As a method for obtaining a cell sheet, for example, cells are cultured on a stimulus-responsive culture substrate coated with a polymer whose molecular structure is changed by stimulation of temperature, pH, light, etc., and stimulation of temperature, pH, light, etc. By changing the surface conditions of the stimulus-responsive culture substrate and changing the surface conditions, the cells can be detached from the stimulus-responsive culture substrate while maintaining the adhesion state between the cells, or any culture substrate And the like, and a method of physically detaching the cells from the end of the cell culture substrate with tweezers or the like. A preferred form is a method in which a temperature-responsive culture substrate having a surface coated with a polymer whose hydration power changes in a temperature range of 0 to 80 ° C. is used as the stimulus-responsive culture substrate. Culturing the cells on a temperature-responsive culture substrate in a temperature range where the polymer hydration power is weak, and then culturing the cells by changing the culture solution to a temperature at which the polymer hydration power is strong, In this method, cells are detached and collected in a sheet form. At that time, the cells are cultured in a temperature range in which the hydration power of the polymer is weak on a cell culture substrate having a surface coated with a polymer whose hydration power changes in a temperature range of 0 to 80 ° C. The temperature range is usually preferably a temperature for culturing cells, for example, 33 ° C to 40 ° C. The temperature-responsive polymer used in the present invention may be either a homopolymer or a copolymer. Examples of such a polymer include polymers described in JP-A-2-21865.

  Depending on the type of cell, there are cells that are difficult to adhere on the cell culture substrate. In such a case, for example, a cell adhesion protein such as collagen, laminin, laminin 5, fibronectin, or matrigel may be used alone or in combination of two or more. Cells can be cultured on cell culture substrates that have been pre-coated with the mixture. These cell adhesion protein coating methods may be in accordance with conventional methods, for example, a method in which an aqueous solution containing the cell adhesion protein is applied to the surface of the cell culture substrate, and then the aqueous solution is removed and rinsed. .

In the present invention, the number of cells seeded to produce a cell sheet varies depending on the animal species and cell types, but may be, for example, 0.3 × 10 ⁴ to 10 × 10 ⁶ cells / cm ² . 5 × 10 ⁴ ~8 × 10 may be a ^six / cm ^2, may be 0.7 × 10 ⁴ ~5 × 10 ⁶ cells / cm ^2. In the present invention, in order to peel and recover the cell sheet from the temperature-responsive culture substrate, the temperature of the culture substrate to which the cells have adhered and become confluent or sub-confluent is determined by the upper critical solution of the coating polymer. It can be obtained by peeling the film by setting it to a temperature higher than or lower than the lower critical solution temperature. At that time, the production of the cell sheet can be performed in a culture solution or in another isotonic solution, and can be selected according to the purpose. In order to detach and collect the cell sheet more quickly and efficiently, a method of tapping or shaking the culture substrate, a method of stirring the medium using a pipette, a method of using tweezers, etc. alone or in combination It may be used. Culture conditions other than temperature may follow conventional methods. For example, the medium to be used may be a medium to which serum such as known fetal bovine serum (FBS) is added, or a serum-free medium may be used. In the present specification, the “lower surface” of the cell sheet means the surface on the side that is in contact with the stimulus-responsive culture substrate before peeling as a cell sheet. In the present specification, the “upper surface” of the cell sheet means the surface that is not in contact with the stimulus-responsive culture substrate before peeling as the cell sheet, that is, the surface opposite to the lower surface of the cell sheet. .

  A case where poly-N-isopropylacrylamide (PIPAAm) is used as a stimulus-responsive polymer, particularly a temperature-responsive polymer will be described as an example. Poly-N-isopropylacrylamide is known as a polymer having a lower critical solution temperature at 31 ° C. In the free state, dehydration occurs in water at a temperature of 31 ° C. or higher, and polymer chains aggregate and become cloudy. Conversely, at a temperature of 31 ° C. or lower, the polymer chain is hydrated and dissolved in water. In the present invention, this polymer is coated and fixed on the surface of a substrate such as a petri dish. Therefore, when the temperature is 31 ° C. or higher, the polymer on the surface of the culture substrate is similarly dehydrated. However, since the polymer chain is fixed on the surface of the culture substrate, the surface of the culture substrate is hydrophobic. become. Conversely, at a temperature of 31 ° C. or lower, the polymer on the surface of the culture substrate is hydrated, but since the polymer chain is coated on the surface of the culture substrate, the surface of the culture substrate becomes hydrophilic. At this time, the hydrophobic surface is an appropriate surface on which cells can adhere and grow, and the hydrophilic surface is a surface on which cells cannot adhere. Therefore, when the substrate is cooled to 31 ° C. or lower, the cells are detached from the substrate surface. If the cells are cultured until they are confluent on the entire culture surface, the cell sheet can be recovered by cooling the substrate to 31 ° C. or lower.

  As the shape of the cell culture substrate for cell sheet preparation used in the present invention, for example, a dish, a multiplate, a flask, or a flat membrane can be used. As the material for the cell culture substrate, compounds such as glass, modified glass, polystyrene, polymethyl methacrylate, polycarbonate, etc., which are usually used for cell culture, and substances that can generally be given form, such as polymers other than those described above, are used. Examples thereof include compounds and ceramics.

  The cell culture substrate for cell sheet preparation used in the present invention may be a cell culture substrate provided with both a region to which cells adhere and a region to which cells do not adhere on the same culture surface. It is possible to produce a plurality of cell sheets at a time by using a cell culture substrate provided with a plurality of circular cell adhesion regions and a region where other cells do not adhere on the same culture surface. In this case, the shape of the cell adhesion region may be a desired shape according to the purpose, such as a circle, a square, a triangle, a rectangle, or the like, and the size can be changed as appropriate. A method for providing a region to which cells do not adhere is not particularly limited. For example, poly-N-acryloylmorpholine, polyacrylamide, polydimethylacrylamide, polyethylene glycol, cellulose and the like, which are non-cell-adhesive polymers with low affinity to cells. And a method of coating a hydrophilic polymer, or a strongly hydrophobic polymer such as a silicone polymer or a fluorine polymer.

  Since the cell sheet in the present invention does not use a proteolytic enzyme such as dispase and trypsin conventionally used for collecting adherent cells, the protein expressed on the cell surface is hardly damaged. For this reason, the lower surface of the cell sheet peeled from the cell culture substrate (the surface on the side in contact with the cell culture substrate) is rich in undamaged adhesive protein, and the cell − The desmosome structure between cells is retained. By having such a structure, the cell sheet is suitable for sticking to a living body affected part or laminating cell sheets. Dispase, a proteolytic enzyme, is known to be able to detach cells in a state where 10-40% of the cell-cell desmosome structure is retained. Proteins are destroyed at the same time. On the other hand, the cell sheet used in the present invention can be peeled and collected in a state where both the desmosome structure and the basement membrane-like protein remain in 60% or more, and can obtain various effects as described above. it can.

  As a method for producing a laminated cell sheet, a cell sheet floating in a culture solution is sucked together with a culture solution by a pipette or the like, discharged onto a cell sheet of another culture dish, and laminated by a flow of a liquid medium, cells The method of laminating | stacking using a moving jig is mentioned. In the method for producing a laminated cell sheet composition of the present invention, the method using a cell migration jig is preferable because it can be laminated without damaging the cell sheet. The cell migration jig only needs to have a function of capturing a cell sheet. Examples of the material include polyvinylidene difluoride (PVDF), silicone resin, polyvinyl alcohol, urethane, cellulose and derivatives thereof, chitin, Chitosan, collagen, gelatin, fibrin gel and the like can be used. Examples of the shape of the cell migration jig include a stamp shape, a membrane shape, a porous membrane shape, a nonwoven fabric shape, and a woven fabric shape. In the embodiment of the present invention, the cell migration jig only needs to have a function of collecting a cell sheet without damaging it and laminating it on another cell sheet, such as a cell adhesive protein, a cell adhesive peptide, or a hydrophilic substance. It is preferable that it is a cultured cell movement jig which has a cell adhesion part which consists of 1 type or 2 types or more of a property polymer. For example, Japanese Patent Application Laid-Open No. 2005-176812 discloses a stamp-type cultured cell moving jig having a cell attachment portion. The stamp-type cultured cell transfer jig can be recovered while preventing the cell sheet from being damaged by the cell attachment portion, and preventing the cell sheet from shrinking when the cell sheet peels from the culture dish. To do. The cell sheet can be stacked without shrinking the cell sheet while easily moving the cell sheet onto another cell sheet. By stacking the cell sheets without shrinking, the cell sheets are stacked without any gap, and a stacked cell sheet having a high-density three-dimensional structure can be obtained.

The cell adhesive substance used in the present invention is not particularly limited as long as it has a function of attaching the cell sheet to the cell sheet. For example, a cell adhesive protein, a cell adhesive peptide, a cell adhesive polysaccharide, Other medical adhesives are included. For example, cell adhesion proteins include collagen, vitronectin, fibrin (including a mixture of fibrinogen and thrombin), fibronectin, thrombospondin, gelatin, laminin and the like. Examples of the cell adhesion peptide include polylysine. Examples of the cell-adhesive polysaccharide include chitosan-based adhesives and alginic acid-based adhesives (including a mixture of an aqueous sodium alginate solution and Mg ²⁺ or Ca ²⁺ ). Other medical adhesives that are commercially available can be used. Among them, fibrin is a fibrous protein involved in blood coagulation in the living body, and because it causes a hemostasis effect by forming a blood clot by polymerizing with platelets due to wounds, etc. It is known to be often used to close wounds. Fibrin is a cell adhesion protein gel having a network structure obtained by converting fibrinogen, which is a precursor thereof, into fibrin by thrombin, a kind of serine protease. In addition, polylysine, collagen, vitronectin, fibronectin, thrombospondin, gelatin or laminin, chitosan-based adhesives, alginic acid-based adhesives, and other substances used as medical adhesives also have a cell-adhesive action. By applying these cell adhesive substances between the cell sheets, the adhesion between them can be temporarily supported until the cell sheets adhere to each other.

  Conventionally, it took 30 minutes or more to laminate a cell sheet on another cell sheet and to prevent the two cell sheets from peeling off. The time required for this is that it takes 30 minutes or more for the extracellular matrix on the lower surface of the upper cell sheet to adhere to the upper surface of the lower cell sheet and adhere to the extracellular matrix on the lower surface of the upper cell sheet. It is thought that it is necessary. For this reason, a time of 30 minutes or more is required every time the cell sheets are laminated, and the more time is required as the number of laminated sheets increases. As a result, it took time until a laminated cell sheet composition was obtained, and stressed the cells. As in the embodiment of the present invention, by applying a cell adhesive substance between the cell sheets, the time required for the cell sheets to adhere to each other can be greatly shortened and given to the cells. The stress has been reduced. The cell adhesive substance is not limited as long as it has an effect of enhancing the adhesion between the cell sheets, and it is particularly preferable to use both thrombin and fibrinogen. Although thrombin and fibrinogen themselves do not have cell adhesion, cell adhesion occurs only when thrombin and fibrinogen react. Therefore, it is preferable from the viewpoint of operability that thrombin and fibrinogen are applied to the laminated surfaces of different cell sheets, and fibrinogen is converted into fibrin gel only when laminated to produce fibrin gel. A solution in which thrombin and fibrinogen are mixed may be applied immediately before application to the cell sheet. As a result, the temporary adhesion between the cell sheets is improved, and the time required for stacking the cell sheets can be greatly shortened. The amount to be applied may be appropriately adjusted depending on the type of cell adhesive substance, the amount or type of cells, etc., but it is preferable to adjust the amount so as not to inhibit the adhesion and functionality between cell sheets. For example, when the cell sheet to be laminated is a cell sheet containing cardiomyocytes, it is preferable to keep the coating amount so that the upper and lower cell sheets do not impair electrical connection. As a method of applying, a solution containing a cell adhesive substance on the cell surface may be applied to the entire laminated surface or sprayed, or a cell sheet is attached to a solution containing a cell adhesive substance in advance. The cell migration jig may be dipped and applied.

  A factor that induces angiogenesis may be further added to the cell adhesive substance applied between the cell sheets. Examples of factors that induce angiogenesis include vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), angiopoietin, platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), Matrix metalloproteases (MMP), VE-cadherin, ephrin, plasminogen activator, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), placental growth factor (PlGF) and the like. By adding these between the cell sheets, angiogenesis in the laminated cell sheet composition is promoted. In addition, angiogenesis at the transplant site is further promoted.

When stacking cell sheets, it is preferable that the stacked cell sheets are brought into close contact with each other by applying a load to the cell sheets. Thereby, the clearance gap between a cell sheet and a cell sheet reduces, and the coupling | bonding between a cell sheet and a cell sheet becomes closer. Moreover, since the time required in order to laminate | stack a cell sheet is shortened by applying a load, it is preferable. The load varies depending on the type of cell used and the type of cell adhesive substance used, but for example, 1 g / cm ² or more, 2 g / cm ² or more, 3 g / cm ² or more, 4 g / cm ² or more, 5 g / Cm ² or more, 6 g / cm ² or more, 7 g / cm ² or more, 8 g / cm ² or more, 9 g / cm ² or more, 10 g / cm ² or more, 11 g / cm ² or more, 12 g / cm ² or more, 13 g / cm ^The load is preferably ² or more, 14 g / cm ² or more, and 15 g / cm ² or more. In addition, the upper limit of the load should be kept to the extent that damage to the cell is minimized or the cell attachment part of the cell movement jig is not damaged, and the upper limit varies depending on the cell type and the like Is, for example, 50 g / cm ² or less, 45 g / cm ² or less, 40 g / cm ² or less, 39 g / cm ² or less, 38 g / cm ² or less, 37 g / cm ² or less, 36 g / cm ² or less, 35 g / cm ² Hereinafter, it is preferably 34 g / cm ² or less, 33 g / cm ² or less, 32 g / cm ² or less, 31 g / cm ² or less, or 30 g / cm ² or less. When the load exceeds 50 g / cm ² , the cell is excessively loaded and the cell is damaged, which is not preferable. The method of applying a load may be a method of applying a load by the weight of the stamp type device, or a method of applying a load by a mechanically applying mechanism.

  The method of the present invention makes it possible to obtain a laminated cell sheet composition having a desired thickness by repeating the above-described method for laminating cell sheets a desired number of times. In particular, the present invention can reduce the lamination time per one time to about 1/6 as compared with the conventional cell sheet lamination method, and the effect of shortening is more effective if the number of times of lamination increases. Become prominent. The number of times of lamination can be appropriately changed according to the purpose. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 and 20 times. 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, or more. Further, the cell sheets may be laminated one layer at a time, or two or more layers may be laminated at one time, and there is no limitation. By laminating two or more cell sheets at a time, it is possible to further shorten the lamination time.

  The present invention provides a laminated cell sheet composition having a thickness that cannot be obtained by a conventional method by having a cell adhesive substance between the cell sheets. Moreover, this invention provides the laminated cell sheet composition of the form which each cell sheet layer contact | adhered. When a conventional laminated cell sheet is transplanted into a living body, due to its thickness, no nutrients or oxygen can reach the inside of the cell sheet, resulting in necrosis. Therefore, the thickness of the cell sheet that can be transplanted at one time was less than 80 μm. In order to transplant a cell sheet having a thickness of 80 μm or more, a plurality of transplantation operations are required, and an operation is required each time the transplantation is performed, which places a burden on the subject. On the other hand, the laminated cell sheet of the present invention can be transplanted with a cell sheet having a thickness of 80 μm or more by one transplantation, and can greatly reduce the burden on the subject.

  In the present invention, the thickness of the laminated cell sheet composition means an average thickness, and is a value obtained by measuring the thickness of at least two places of the laminated cell sheet. The method for measuring the thickness of the laminated cell sheet composition of the present invention is not particularly limited. For example, a section specimen of the laminated cell sheet composition can be prepared and obtained from a microscopic image thereof.

  The average thickness of the laminated cell sheet composition of the present invention is preferably 80 μm or more, for example, 85 μm or more, 90 μm or more, 95 μm or more, 100 μm or more, 110 μm or more, 120 μm or more, 130 μm or more, 140 μm or more, 150 μm or more, 160 μm or more, 170 μm or more, 180 μm or more, 190 μm or more, 200 μm or more, 210 μm or more, 220 μm or more, 240 μm or more, 250 μm or more, 260 μm or more, 270 μm or more, 280 μm or more, 290 μm or more, 300 μm or more, 400 μm or more, 500 μm or more , 600 μm or more, 700 μm or more, 800 μm or more, 900 μm or more, 1000 μm or more, or more. The upper limit is not particularly limited.

  The average thickness of the laminated cell sheet composition of the present invention is, for example, 80 μm to 1000 μm, 80 μm to 800 μm, 80 μm to 600 μm, 80 μm to 500 μm, 80 μm to 300 μm, 90 μm to 1000 μm, 90 μm to 800 μm, 90 μm to 90 μm It may be 600 μm, 90 μm to 500 μm, 90 μm to 300 μm, 100 μm to 1000 μm, 100 μm to 800 μm, 100 μm to 600 μm, 100 μm to 500 μm, 100 μm to 300 μm, but is not limited thereto.

  The method of the present invention may be embodied by an apparatus for automatically producing a laminated cell sheet composition. When the stimulus-responsive culture substrate is a temperature-responsive culture substrate, the thin sheet-like cells formed on the cell-adhesive surface of the temperature-responsive culture substrate change the temperature response by decreasing (decreasing) the temperature. Peel from the sex culture substrate. Therefore, a cell sheet is obtained by lowering the temperature of the temperature-responsive culture substrate with a cooler (Peltier) or the like, and a laminated cell sheet composition is obtained by superimposing a plurality of sheets with a robot or an actuator. Can do. When the cell sheets are stacked, the cell sheet further includes means for applying a cell adhesive substance between the cell sheets. Thereby, a cell sheet can be laminated | stacked rapidly compared with the conventional cell sheet | seat lamination | stacking apparatus.

As a configuration of such a device, for example,
A mounting table provided with stimulation means for stimulating the stimulus-responsive culture substrate and peeling the cell sheet from the stimulus-responsive culture substrate;
A cultured cell transfer jig for attaching the cell sheet;
An arm for attaching the cultured cell moving jig and applying a load to the cultured cell transplanting jig;
Cell adhesive substance applying means for applying a cell adhesive substance to the cell sheet on the stimulus-responsive culture substrate,
The cultured cell moving jig is brought into contact with the upper surface of the first sheet-like cell group on the stimulus-responsive culture substrate placed on the mounting table, and the stimulus-responsive culture substrate on the stimulus-responsive culture substrate is contacted by the stimulation means. Giving a stimulus to peel off the first sheet-like cell group, and attaching the obtained first cell sheet to the cultured cell moving jig;
Applying the cell adhesive substance supplied by the cell adhesive substance applying means to the upper surface of the second sheet-like cell group mounted on the mounting table and / or the lower surface of the first cell sheet;
The lower surface of the first cell sheet attached to the cultured cell transfer jig is brought into contact with the upper surface of the second sheet-like cell group, and the second sheet-like cell on the stimulus-responsive culture substrate is stimulated by the stimulating means. A laminated cell sheet composition characterized by applying a load to the cell movement jig and rapidly laminating the obtained second cell sheet on the first cell sheet simultaneously with giving a stimulus for detaching the group Apparatus for manufacturing a product.

  The configuration of the above apparatus is an embodiment of the present invention, and is not particularly limited as long as the method of the present invention is embodied and has the same effect.

  The laminated cell sheet composition obtained by the method of the present invention is finally separated from the cell migration jig. The method of separating from the cell transplantation jig is not limited, and examples thereof include a method of physically peeling it from the cell transfer jig and a method of obtaining by dissolving or melting a substance contained in the cell attachment part. For example, when the cell adhesion part is made of a gel containing gelatin, the cell adhesion part is dissolved by keeping the temperature at 37 ° C., and only the laminated cell sheet composition can be recovered.

  Hereinafter, the present invention will be described in more detail based on examples, but these do not limit the present invention in any way. The experiment in this example was approved by the animal experiment screening committees established at both universities in accordance with the detailed regulations for the biological experiment safety management regulations of Waseda University and Tokyo Women's Medical University.

<Used animals, cells, reagents, etc.>
In this example, the following animals, cells, reagents and the like were used unless otherwise indicated.
・ Rat (1 day after birth, Nippon Medical Science Animal Materials Laboratory, Hsd: Sprague Dawley SD (strain name))
・ HepG2 (ATCC, HB-8065)
-Temperature-responsive culture dish (UpCell (registered trademark)) (Cellseed)
-Rat cardiomyocyte culture solution (medium 199 40% + HANKS balanced salt solution 54% + FBS 6% + penicillin-streptomycin solution 1% +166 mmol / L NaCl, 1.0 mmol / L NaH ₂ PO ₄ , 0.8 mmol / L MgSO ₄ 2 mmol / L NaHCO ₃ , 0.9 mmol / L CaCl ₂ , 5 mmol / L glucose)
-HepG2 cell culture solution (DMEM + FBS 10% + penicillin-streptomycin solution 1%)
・ FBS (Japan Bioseam, S1650-500)
・ Hanks balanced salt solution (Nacalai Tesque, 17460-15)
・ DMEM (SIGMA, D6429)
-Penicillin-streptomycin solution (Life Technologies, 15140-122)
・ 4% paraformaldehyde fixative (Muto Chemical Co., 3311-1)
・ 50% sodium hydroxide solution (Wako Pure Chemical Industries, 197-12375)
Anti-rat myocardial troponin T (Thermo Fisher Scientific, MS-295-P1)
CD31 antibody mouse anti rat PECAM-1 [CD31] (Millipore, MAB 1393)
Secondary staining antibody Alexa Fluor 488 goat anti-mouse (Life technologies, A11017)
Secondary staining antibody Alexa Fluor 594 goat anti-rabbit (Life technologies, A11037)
・ Nuclear staining ProLong Antifade reagent with DAPI (Life technologies, P36935)
Anti-fibrotic antibody (abcam, ab2413)
・ Fibrin gel (fibrinogen + thrombin) (Teijin Pharma Limited for bonding Bolheel tissue)
Collagen solution (Kokensha, collagen acidic solution I-AC 5 mg / mL)
Gelatin (SIGMA, G1890-100G)
<Devices used>
・ Stamp device (Reference: Ngo TX, et al. Endothelial cell behavior in myoblast sheets with differential thickness. Biotechnol Lett (2013) 35: 1001-1008)
・ Microscope (Nikon, ECLIPSE, E800)
・ Confocal microscope (Olympus, FV1200)
・ Optical coherence tomography (OCT, Panasonic Healthcare, Reference: Kobayashi M., et al., Real-time, non-innovative optical coherence of the cross-slection of the biotechnology-in-the-life RESEARCH B: APPLIED BIOMATERIALS vol.103 (6), 1267-1273, Aug.2015)

<Method for producing cardiomyocyte sheet>
The myocardial cell sheet was prepared according to the method of Sakaguchi et al. (Sakaguchi K. et al., In Vitro Engineering of Viscosified Tissue Surrogates, Scientific Reports, 3, 1316, 2013.). Specifically, the heart collected from a 1-day-old pup rat was minced to collect cardiomyocytes. The collected cells were washed with a buffer (a mixture of HANKS and FBS at 1: 1) and suspended in a rat myocardial culture medium. Cardiomyocytes were seeded in a temperature-responsive 24-well plate culture dish (UpCell (registered trademark), Cellseed) at a concentration of 900,000 cells / well, and 5% CO _{2 in} 0.5 mL of rat myocardial medium for 4 days. The cells were cultured in a 37 ° C. incubator under saturated steam. Then, it collect | recovered as a cell sheet | seat by heat-retaining at 20 degreeC.

<Cell sheet stacking method (pipetting method)>
Cell sheet stacking by pipetting was performed according to the method of Haraguchi et al. (Haraguchi Y. et al., Fabrication of functional three-dimensional tissues by stacking cell sheets in vitro, 50, Nature 7 Proto. Specifically, the cell sheets suspended in the culture solution were sucked together with the cell culture solution with a pipette, and the sucked culture solution was discharged onto another cell sheet to stack the cell sheets.

<Cell sheet stacking method (stamp method)>
Stacking of cell sheets by the stamp method is basically performed according to the method of Haraguchi et al. (Haraguchi Y. et al., Fabrication of functional three-dimensional tissues by stacking cell sheets in vitro, Prot. , Partially modified (FIGS. 1 and 2). Specifically, powdery gelatin was dissolved in Hank's balanced salt solution at a concentration of 65 mg / ml, and 50% sodium hydroxide solution was added so that the pH was around 7.4. The gelatin solution adjusted for pH was poured slowly into the stamp device mold so that no bubbles would enter, and was allowed to stand at 4 ° C. for 30 minutes to complete the stamp device with gelatin gel. The completed stamp device was placed on a sheet-like cell group on a 24-well plate culture dish from which the culture solution was removed, and a weight was placed so that a total load of 30 g was applied together with the stamp device from above, and left on ice for 30 minutes. . After the first sheet-like cell group and gelatin are adhered, 2 μL of thrombin is added to the cell sheet side adhered to the gelatin after the second sheet, and 2 μL of fibrinogen is added to the sheet-like cell group side on the 24-well plate culture dish by Pipetman Then, similarly to the first sheet, a total of 30 g of load was applied from the top and laminated. When the lamination to the desired number of sheets was completed, the cell sheet was cut out from the stamp device together with the gelatin gel using Scalpel, and the laminated tissue was collected.

  As the examination method of lamination conditions, load, fibrin gel amount, adhesion time, gelatin concentration are set as examination items, and 10 rat cardiomyocyte sheets can be laminated stably in a short time by changing the conditions The lamination conditions were searched. Regarding the load and gelatin gel concentration, refer to the study by Kikuchi et al. (Kikuchi T. et al., Automatic fabrication of 3-dimensional tissue use cell sheet manipulator technique. Biomaterials. Went. Specific examination values were carried out at a load of 10, 20, 30 g, a gelatin concentration of 60, 65 or 75 mg / mL, an adhesion time of 3 or 5 minutes, and a fibrin gel amount of 4 μL or 6 μL.

<Hematoxylin and eosin (HE) staining, immunostaining>
The collected tissue was fixed in paraffin, sliced at a thickness of 5 μm, and then the morphology of the laminated tissue was evaluated by HE staining or immunostaining.

<Method for producing laminated cell sheet of HepG2 cells>
After diluting the collagen solution 10-fold with hydrochloric acid water adjusted to pH 3 with hydrochloric acid, 300 μL was added to a temperature-responsive 24-well plate culture dish and allowed to stand for 30 minutes. Thereafter, the collagen solution was removed and air-dried for 2 hours to give a collagen coating. Thereafter, HepG2 cells are seeded there at a concentration of 200,000 cells / well and cultured in a 37 ° C. incubator for 3 days. Note that DMEM (containing 1% penicillin-streptomycin solution and 10% FBS) was used as the culture solution. The stamp lamination was performed in the same manner as the lamination of the cardiomyocyte sheets described above.

<Example 1>
(Examination of stamp lamination conditions using fibrin gel)
As a result of examining the conditions of the cell sheet lamination method using a stamp device and fibrin gel, any of the examined conditions (load: 10, 20, 30 g, gelatin concentration: 60, 65 or 75 mg / mL, fibrin gel amount: 4 μL) Or 6 μL, adhesion time: 3 minutes or 5 minutes), it was possible to shorten the lamination time which required 30 minutes or more in the conventional method. Among them, it was possible to laminate the cardiomyocyte sheets with the most reproducible conditions shown in Table 1. According to the present invention, the stacking time of the second and subsequent cell sheets has been required to be 30 minutes or longer, but can be shortened to 5 minutes or shorter.

  The result of producing a tissue section from the laminated cell tissue constructed by this method is shown in FIGS. 3 (C) and 3 (E), it can be seen that a cell tissue having a dense cell thickness can be constructed. The presence of cardiomyocytes could also be confirmed from the results of immunostaining in FIGS. 3 (D) and 3 (E). From the above results, it is possible not only to reduce the stacking time per cell sheet to 1/6 of the conventional method, but also to stack the myocardial cell sheets that cannot be thickly stacked by the conventional stamp stacking method. I can do it now.

<Example 2>
(Evaluation of cell viability)
Cells collected from the hearts of 1-day-old pups were seeded in a temperature-responsive culture dish (24 wells) at a concentration of 900,000 cells / well and cultured in a 37 ° C. CO ₂ incubator for 4 days. Thereafter, the stamp device on which gelatin was formed was placed on the cells from which the culture solution had been removed, and allowed to stand for 30 minutes while applying a load of 30 g, thereby allowing the first cell to adhere to the stamp device. The second lamination was prepared by the conventional lamination method over 30 minutes and the lamination method of the present invention in which the adhesion time was 5 minutes by adding fibrin gel in between. After the two layers were completed by two methods, the gelatin sheet was dissolved and removed by moving the cell sheet of two layers together with gelatin to a 60 mm dish and culturing in a 37 ° C. incubator for 5 minutes. After collecting two layers of cell sheets, trypsin enzyme treatment was performed to separate the cell sheets into individual cells, and dead cells and living cells were counted using trypan blue staining. For control, cells before stacking were prepared, and after the same treatment, dead cells and live cells were counted.
The cell viability was calculated by the following formula (FIG. 4).

  The results are shown in FIG. As is clear from this figure, it was suggested that there is no difference in cell viability between the cell sheet laminating method according to the method of the present invention and the conventional laminating method.

<Example 3>
(Pulse synchronization evaluation of laminated myocardial tissue)
It is known that the cardiomyocyte sheets are pulsating, and it has also been confirmed that the pulsations of the two cardiomyocyte sheets are synchronized by laminating them. Therefore, it was confirmed by observation with an OCT and a bright field microscope whether the solid myocardial tissue constructed by this method is pulsating or whether the pulsation is synchronized (FIG. 5).

  A tissue in which five cardiomyocyte sheets were laminated according to the present invention was adhered to a φ60 mm dish and allowed to stand in a 37 ° C. incubator under saturated steam for 5 minutes in order to dissolve the gelatin gel. Then, after washing once with a rat myocardial medium warmed to 37 ° C., the culture solution was removed and observed by OCT.

  FIG. 5 shows the result of observation of pulsation by OCT (optical coherence tomography apparatus) after recovering the tissue laminated with five stamps by the method of the present invention. It can be seen from FIG. 5 that the pulsation is propagated throughout the tissue from one ignition point. This suggests that even when fibrin gel is sandwiched between the cell sheets, the pulsations of the cardiomyocytes are synchronized and have a function as a myocardial tissue.

<Example 4>
(Synchronous evaluation of pulsatile potential of laminated myocardial tissue)
It was confirmed whether or not the pulsatile potential was synchronized between the cardiomyocyte sheets laminated with fibrin gel. Cells collected from the hearts of 1-day-old pups were seeded in a temperature-responsive culture dish (24 wells) at a concentration of 900,000 cells / well and cultured in a 37 ° C. CO ₂ incubator for 4 days. Thereafter, the stamp device on which gelatin was formed was placed on the cells from which the culture solution had been removed, and allowed to stand for 30 minutes while applying a load of 30 g to adhere the first cell to the stamp device. When two sets of two cardiomyocyte sheets laminated by this method are prepared, the first set is adhered to a special culture dish for potential measurement, and cultured in a 37 ° C. CO ₂ incubator for 5 minutes to dissolve gelatin. Removed. Next, the second set of laminated cell sheets was adhered so as to partially overlap the first set of laminated cell sheets (FIG. 6A). At this time, 4 μL of fibrin gel was sandwiched between the two laminated cell sheets. After culturing in a 37 ° C. CO ₂ incubator for 5 minutes to dissolve and remove gelatin, the pulsatile potential was measured. The potential was measured by the method of Matsuura et al. (Matsuura K. et al., Creation of mouse embryonic stem cell-derived cardiac cell sheets, Biomaterials 32 (2011) 7355-7362.).

  As a result, it was confirmed that the pulsatile potentials at two points A and B having different potential measurement locations were synchronized (FIG. 6B). This suggests that electrical synchronization between cardiomyocyte sheets is maintained without problems even in the presence of fibrin gel.

<Example 5>
(Vessel network evaluation in laminated myocardial tissue)
Previous studies have shown that vascular network construction is indispensable for culturing three-dimensional cell tissues in vitro. Therefore, the state of the vascular network in the laminated tissue constructed by this method was confirmed by antibody immunostaining using CD31, which is a vascular endothelial cell-specific antigen. A rat cardiomyocyte sheet laminated with two stamps according to the present invention was fixed with 4% paraformaldehyde for 15 minutes and then blocked with goat serum diluted 10% with PBS for 2 hours. Thereafter, the CD31 antibody diluted 100: 1 with PBS was allowed to react overnight at 4 ° C., and then the secondary staining antibody Alexa Fluor 488 goat anti-mouse (Life technologies, A11017) diluted 100: 1 with PBS was used for 1 hour. And reacted at room temperature. Finally, the cell nuclei were stained with Hoechst 33258 diluted 300: 1 with PBS for 15 minutes and observed with a confocal microscope.

  FIG. 7 shows the result of confirming the vascular network in rat heart-derived cells cultured in a temperature-responsive culture dish for 4 days and the vascular network in the laminated cell sheet laminated by the novel stamp laminating technique by immunoantibody staining with CD31. It is. From the result shown in FIG. 7C, it was confirmed that the vascular network was maintained as it was in the cell sheet obtained by stacking two stamps even though it was just stacked. On the other hand, in the cell sheet prepared by peeling without using a stamp, the network of vascular endothelial cells was not maintained (FIG. 7D). From these results, the rat cardiomyocyte tissue laminated by the new stamp lamination technique has a better vascular network state than the rat cardiomyocyte tissue laminated by the pipetting lamination technique, and is used when cultured or transplanted in vitro. Suggested that a thicker tissue could be constructed.

<Example 6>
(Transplantation evaluation of high-speed stamp laminated tissue)
Cells collected from the hearts of 1-day-old pups were seeded in a temperature-responsive culture dish (UpCell (registered trademark), 24 well) at a concentration of 900,000 cells / well and cultured in a 37 ° C. CO ₂ incubator for 4 days. Thereafter, the stamp device on which gelatin was formed was placed on the cells from which the culture solution had been removed, and allowed to stand for 30 minutes while applying a load of 30 g to adhere the first cell to the stamp device. From the second and subsequent laminations, the laminated cell sheet is obtained by a method of laminating again for 30 minutes, and a method of laminating by adding fibrin gel between the cell sheet and the adhesion time of 5 minutes. Got ready. Up to 5 layers were stacked by two methods. In addition, the start time of lamination was adjusted so that the completion time of the cell sheet laminated | stacked five layers by two methods became simultaneous. When the lamination was completed, the back of a nude rat (strain name: F344 / NJcl-rnu / rnu, Claire Japan, Inc.) immediately subjected to anesthesia (isoflurane) 8 to 12 weeks old was opened, and two laminated cells were subcutaneously applied. The sheet was transplanted with the gelatin gel separated. Thereafter, a silicon sheet (As One, 6-611-01) was placed on the transplanted tissue so that the transplanted site could be identified, and the open back was closed and reared for 2 weeks (FIGS. 8 and 9). Two weeks after transplantation, the back was opened in the same manner, and the transplanted site was collected and immersed in 4% paraformaldehyde for 24 hours to fix the tissue. After the tissue was fixed, it was washed with 1 × PBS, and finally paraffin substitution was performed to prepare a paraffin block. After preparing the paraffin block, it was sliced to a thickness of 5 μm using a microtome to prepare a tissue section. When tissue sections were prepared, morphological evaluation was performed by Azan staining by a conventional method. In addition, the tissue sections were deparaffinized using xylene and ethanol, treated with an activation (Target Retrieval Solution 10 × concentrate (Dako, S1699)) solution, and then Normal Goat Serum (for 1 hour at room temperature). Abaqam, ab7481) Blocking treatment, overnight at 4 ° C. with primary antibody (anti-cardiac Toroponin T) diluted 200-fold with PBS, and 2 hour room temperature reaction with secondary antibody (Alexafluor 488) diluted 200-fold with PBS The antibody immunostained section was prepared by reacting with Hoechst 33258 at room temperature for 15 minutes and encapsulating with an encapsulant, and observed with a confocal microscope.

  In addition, the fixed area of the transplanted tissue was quantified from antibody immunostained images. Four cut sections were prepared from one tissue at intervals of 300 to 400 μm, and after immunostaining, the portion that was positive for cardiac troponin T was imageJ (free software, reference: http: //rsb.info.nih. gov / ij / index.html) (FIG. 9E).

  As a result, it was confirmed that the site where the cardiomyocyte sheet prepared by the method of the present invention was transplanted was more angiogenic than the site where the cardiomyocyte sheet prepared by the conventional technique was transplanted (Fig. 8). This suggests that the cardiomyocyte sheet produced according to the present invention enhances the blood vessel induction effect after transplantation and has a high therapeutic effect. It was also revealed that the cell sheet produced by the method of the present invention is more firmly established after transplantation than the cell sheet produced by the conventional method.

  Using a GFP positive SD rat (SD-Tg (CAG-EGFP) (Sankyo Lab Service)), a laminated cardiomyocyte sheet was prepared by the method of the present invention and the conventional method in the same manner as in FIGS. An experiment for transplantation was performed (FIG. 10). Two weeks after transplantation, the percentage of transplanted cell sheet colonies attached to rats was calculated from the ratio of GFP positive area (the calculation method is the same as the method performed in FIG. 9). Similar to the results confirmed in FIGS. 8 and 9, the laminated cell sheet produced by the present invention has a larger fixed area after transplantation and more blood vessels than the cell sheet produced by the conventional method. Was confirmed to be induced (FIG. 10).

<Example 7>
(Lamination of HepG2 cell sheet by stamp lamination technology)
All studies on the new stamp lamination technique using fibrin gel were performed using rat heart-derived cell sheets. Therefore, in order to investigate whether this method can be applied to cell sheets of other cell types, a stacking experiment was performed in the same manner as the rat cardiomyocyte sheet using a HepG2 cell sheet (human liver cancer-derived cell), and a tissue section was prepared and the morphology was determined. evaluated. A HepG2 cell sheet was prepared by seeding HepG2 cells in a collagen-coated temperature-responsive culture dish and culturing for 3 days, and the results of lamination using a new stamp lamination technique are shown in FIG. From the result of FIG. 7, it was confirmed that a thick HepG2 cell tissue was constructed, and it was suggested that the layering technique of the present invention can be applied to other cell types.

<Example 8>
(Transplantation evaluation of laminated cell sheet composition (10 layers))
Similar to Example 6, a 10-layer laminated cell sheet was prepared using cells collected from the heart of a 1-day-old pup rat by a method using fibrin gel. The obtained laminated cell sheet was transplanted to nude rats in the same manner as in Example 6. Tissue sections were prepared from around the transplant site 2 weeks after transplantation, and myocardial troponin T (green, indicating the position of cardiomyocytes) and CD31 (red, indicating the position of vascular endothelial cells) were stained by immunostaining. Cell nuclei were stained with Hoechst 33258. The obtained tissue section was observed with a confocal microscope (FIG. 13).

  In the laminated cell sheet produced by the conventional method, the thickness of the cell sheet that can be transplanted at one time is less than 80 μm. When transplanting a laminated cell sheet with a thickness exceeding that, multiple transplantation operations are performed. It was necessary to repeat. However, even if the laminated cell sheet of the present invention is a 10-layer laminated cell sheet having a thickness of 200 μm or more, it can be engrafted in vivo without necrosis. Vascular structures were also observed in the transplanted laminated cell sheet. That is, the laminated cell sheet obtained by the method of the present invention exhibited an excellent effect that even a cell sheet having a thickness exceeding 80 μm can be engrafted without necrosis after transplantation.

<Cell sheet laminating device>
The apparatus for manufacturing the laminated | stacked cell sheet composition of this invention is illustrated. FIG. 12 is a perspective view showing the cell sheet laminating apparatus 1 of the present invention. Reference numeral 10 denotes a cultured cell moving jig 10 that holds a cell sheet. The cultured cell moving jig 10 can be attached to and detached from the lower end of the arm 11. The cell attachment that contacts the cell sheet of the cultured cell transfer jig 10 may have any function that can capture the cell sheet. For example, the material may be polyvinylidene difluoride (PVDF), silicone resin, polyvinyl Alcohol, urethane, cellulose and derivatives thereof, chitin, chitosan, collagen, gelatin, fibrin gel and the like can be used. Thereby, a cell sheet can be stuck and lifted. The arm 11 is attached to an upper portion of a base member (in the shape of a quadrangular prism) so as to be swingable within a vertical plane. A cultured cell moving jig 10 is provided on the left side of the figure across the swing rotation shaft 12 of the arm 11, and a pressing force measuring means 13 for measuring the pressing force (weight) is provided on the right side of the figure. A lower portion of the base member 15 is sealed with a bellows seal 14. Below the base member, there are provided drive means (not shown) that allows the base member 15 to move in the X and Y directions in the horizontal plane and lift drive means that moves the base member up and down in the Z direction.

  Reference numeral 17 denotes a cell adhesive substance applying means for applying a cell adhesive substance to a sheet-like cell group cultured on the cell culture substrate 20. A solution containing the cell adhesive substance is supplied from the cell adhesive substance applying means 17 and applied to the upper surface of the cells on the cell culture substrate 20. Two or more cell adhesive substance applying means 17 may be provided. In this case, the cell adhesive substance applying means 17 is a substance that forms a gel by mixing (for example, thrombin, fibrinogen, sodium alginate solution). And calcium ion solution, etc.) may be supplied separately, or may be bundled and mixed immediately before application. The means for applying may be a means for dropping a liquid or a means for applying in a spray form.

  By measuring the pressing force by the pressing force measuring means 13, the base member 15 is lowered by the operation of the elevating means, and the force for pressing the cultured cell moving jig 10 against the cell sheet in the cell culture substrate 20 is adjusted. It becomes possible. At that time, the cultured cell moving jig 10 (power point) attached to the tip of the arm 11 swings about the swinging rotation axis (fulcrum) and presses the pressing force measuring means 13 (working point). The pressing force measuring means 13 is, for example, a load cell.

  Reference numeral 16 denotes a mounting table on which a plurality of cell culture substrates 20 and the like can be mounted. The mounting table 16 includes a rotation shaft 16a. The rotary shaft 16a is sealed with an O-ring or the like, and can be driven to rotate at the lower part of the base 30 by the operation of a rotary drive means. Further, the part of the mounting table 16 on which the cell culture substrate 20 is placed is provided with a stimulus providing means 18 that gives a stimulus to the cell culture substrate 20. For example, when the cell culture substrate 20 is a temperature-responsive culture dish, the stimulus providing unit 18 is a unit that changes the temperature (cooler or the like, for example, Peltier), and the cell culture substrate 20 is light-responsive. In the case of a culture dish, for example, it is a light source (for example, LED) that gives a light stimulus.

  By using this apparatus, it is possible to carry out the method for producing a cell sheet laminate of the present invention and to produce a laminated cell sheet composition efficiently and reproducibly. As a specific procedure, the base member 15 is lowered by the raising / lowering driving means, the arm 11 and the cultured cell moving jig 10 connected to the base member 15 are lowered, and the cell culture substrate 20 from which the culture solution has been sucked in advance. The cultured cell moving jig 10 is brought into contact with the upper surface of the upper sheet-like cell group. In this state, stimulation is applied by a stimulus providing means provided at the lower part of the cell culture substrate 20 of the mounting table 16, and the sheet-like cell group is peeled off as a cell sheet from the cell culture substrate 20, and the cultured cell moving jig 10 Paste to. Thereafter, the lifting drive means is raised, and the mounting table 16 is rotated so that the cell culture substrate 20 on which another sheet-like cell group is cultured is positioned directly below the cultured cell moving jig 10 to which the cell sheet is attached. Rotate around the axis 16a. A solution containing a cell adhesive substance is supplied from the cell adhesive substance applying means 17 and applied to the upper surface of the cells on the cell culture substrate 20. Thereafter, the base member 15 is lowered by the lift driving means to lower the cultured cell moving jig 10 again, and the cultured cell moving jig 10 is brought into contact with the upper surface of the sheet-like cell group on the cell culture substrate 20. In this state, stimulation is applied by a stimulus providing means provided at the lower part of the cell culture substrate 20 of the mounting table 16, and the sheet-like cell group is peeled off as a cell sheet from the cell culture substrate 20, and the cultured cell moving jig 10 Paste to. By repeating this operation, a laminated cell sheet composition can be produced.

  Note that this embodiment is merely an example, and any configuration can be applied as long as the manufacturing method of the present invention can be implemented, and the configuration is not limited at all. Moreover, all the structures of the invention disclosed in International Publication No. 2012/098931 are also included in the scope of the present invention, and constitute a part of this specification.

  If it is the manufacturing method shown by this invention, a thick laminated cell sheet composition can be produced now simply and at high speed. Such a thick cell composition is useful as a living body-like tissue for regenerative medicine of various tissues and organs, and at the same time, as a model for evaluating the effect of a drug intended for treatment. .

DESCRIPTION OF SYMBOLS 1 Cell sheet | seat lamination | stacking apparatus 10 Cultured cell moving jig | tool 11 Arm 12 Oscillating rotating shaft 13 Lateral pressure measuring means 14 Bellows seal 15 Base member 16 Mounting base 16a Rotating shaft 17 Cell adhesive substance application means 18 Stimulation donating means 20 Cell culture Base material 30 Base 31 Carry-in / out entrance

Claims (15)

(1) A stimulus for bringing a cultured cell moving jig into contact with the upper surface of the first sheet-like cell group on the stimulus-responsive culture substrate and peeling the first sheet-like cell group on the stimulus-responsive culture substrate. Giving and attaching the peeled first cell sheet to the cultured cell moving jig,
(2) A step of applying a cell adhesive substance to the upper surface of the second sheet-like cell group on the stimulus-responsive culture substrate and / or the lower surface of the first cell sheet attached to the cultured cell moving jig. ,
(3) The lower surface of the first cell sheet attached to the cultured cell moving jig is brought into contact with the upper surface of the second sheet-like cell group, and the second sheet-like cell group is separated from the stimulus-responsive culture substrate. Providing a stimulus for peeling, and bonding the peeled second cell sheet to the lower surface of the first cell sheet;
A method for producing a laminated cell sheet composition.   The method according to claim 1, wherein when applying the stimulus of the step (3), further loading is performed simultaneously. For the laminated cell sheet composition attached to the cultured cell transfer jig obtained in the step (3),
further,
(4) The upper surface of the third sheet-like cell group on the stimulus-responsive culture substrate and / or the lower surface of the laminated cell sheet composition attached to the cultured cell moving jig obtained in the step (3). Applying a cell adhesive substance to
(5) The lower surface of the laminated cell sheet composition attached to the cultured cell transfer jig is brought into contact with the upper surface of the third sheet-like cell group, and the third sheet-like cell group becomes the stimulus-responsive culture medium. Providing a stimulus for peeling from the material, and bonding the peeled third cell sheet to the lower surface of the laminated cell sheet composition;
(6) A step of repeating the steps (4) to (5) any number of times,
The method according to claim 1, comprising:   The method according to any one of claims 1 to 3, wherein the cell adhesive substance is a cell adhesive protein, a cell adhesive peptide and / or a cell adhesive polysaccharide.   1 or 2 wherein the cell adhesive substance is selected from the group consisting of polylysine, collagen, vitronectin, fibrin, fibrinogen and thrombin, fibronectin, thrombospondin, gelatin, laminin, chitosan adhesive, alginate adhesive The method of any one of Claims 1-3 which is the above substance. The step (2)
Applying the cell adhesive substance to the upper surface of the second sheet-like cell group on the stimulus-responsive culture substrate and the lower surface of the first cell sheet attached to the cultured cell transfer jig,
The cell adhesive substance applied to the upper surface of the second sheet-like cell group is fibrinogen, and the cell adhesive substance applied to the lower surface of the first cell sheet is thrombin, or
The cell adhesive substance applied to the upper surface of the second sheet-like cell group is thrombin, and the cell adhesive substance applied to the lower surface of the first cell sheet is fibrinogen.
The method of any one of claims 1-5. The step (4)
Cell adhesion to the upper surface of the third sheet-like cell group on the stimulus-responsive culture substrate and the lower surface of the laminated cell sheet composition attached to the cultured cell transfer jig obtained in the step (3) A process of applying a substance,
The cell adhesive substance applied to the upper surface of the third sheet-like cell group is fibrinogen, and the cell adhesive substance applied to the lower surface of the laminated cell sheet composition is thrombin, or
The cell adhesive substance applied to the upper surface of the third sheet cell group is thrombin, and the cell adhesive substance applied to the lower surface of the laminated cell sheet composition is fibrinogen.
The method according to any one of claims 3 to 6.   The said stimulus responsive culture base material is a temperature responsive culture base material, The said stimulus is the temperature below the lower critical solution temperature or higher than the upper critical solution temperature. The method described.   The method according to any one of claims 1 to 8, wherein the stimulus-responsive culture substrate is a culture substrate in which poly-N-isopropylacrylamide is coated on at least a part of the culture surface.   The culture cell migration jig according to any one of claims 1 to 9, wherein the culture cell migration jig is a culture cell migration jig having a cell attachment part composed of one or more of cell adhesion protein, cell adhesion peptide, and hydrophilic polymer. The method according to claim 1.   The laminated cell sheet composition includes one or more cells selected from the group consisting of cardiomyocytes, hepatocytes, fibroblasts, myoblasts, pancreatic cells, kidney cells, vascular endothelial cells, and epithelial cells. The method of any one of Claims 1-10.   The laminated cell sheet composition obtained by the method of any one of Claims 1-11.   The laminated cell sheet composition according to claim 12, wherein an average thickness of the laminated cell sheet composition is 80 µm or more. A mounting table provided with stimulation means for stimulating the stimulus-responsive culture substrate and peeling the cell sheet from the stimulus-responsive culture substrate;
A cultured cell transfer jig for attaching the cell sheet;
An arm for attaching the cultured cell moving jig and applying a load to the cultured cell moving jig;
An apparatus for producing a laminated cell sheet composition, comprising: a cell adhesive substance applying means for applying a cell adhesive substance to the cell sheet on the stimulus-responsive culture substrate. The cultured cell moving jig is brought into contact with the upper surface of the first sheet-like cell group on the stimulus-responsive culture substrate placed on the mounting table, and the stimulus-responsive culture substrate is placed on the stimulus-responsive culture substrate by the stimulation means. Giving a stimulus to peel off the first sheet-like cell group, and attaching the obtained first cell sheet to the cultured cell moving jig;
Applying the cell adhesive substance supplied by the cell adhesive substance applying means to the upper surface of the second sheet-like cell group placed on the mounting table and / or the lower surface of the first cell sheet,
The lower surface of the first cell sheet attached to the cultured cell moving jig is brought into contact with the upper surface of the second sheet-like cell group, and the second sheet-like cell on the stimulus-responsive culture substrate by the stimulating means. 15. The method according to claim 14, wherein the second cell sheet obtained is quickly laminated on the first cell sheet by applying a load to the cultured cell moving jig at the same time as applying a stimulus for detaching the group. The manufacturing apparatus of the laminated cell sheet composition of description.