WO2017077985A1 - 積層化細胞シート組成物を製造する方法、それにより製造される積層化細胞シート組成物及びその製造装置 - Google Patents
積層化細胞シート組成物を製造する方法、それにより製造される積層化細胞シート組成物及びその製造装置 Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
- C12M3/04—Tissue, human, animal or plant cell, or virus culture apparatus with means providing thin layers
Definitions
- 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.
- 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.
- a cell culture dish temperature-responsive culture dish
- 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).
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- cell adhesion substance is a cell adhesion protein, a cell adhesion peptide and / or a cell adhesion polysaccharide.
- the cell adhesive substance is selected from the group consisting of polylysine, collagen, vitronectin, fibrin, fibrinogen and thrombin, fibronectin, thrombospondin, gelatin, laminin, chitosan adhesive, and alginic acid adhesive.
- 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.
- 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 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 method of.
- 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, [1] to [1] [9] The method according to any one of [9].
- 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.
- 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 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.
- the time for laminating the cell sheets is greatly reduced, and the damage to the cells received when laminating the cell sheets is reduced.
- the cell sheet can be laminated, and a laminated cell sheet composition having a thickness can be easily and reproducibly obtained.
- 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
- D It is the figure which immunostained the laminated cell sheet (10 sheets).
- E HE-stained layered cell sheet (15 sheets).
- 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 show observation images in time series (at intervals of about 0.1 seconds), and the green part shows the part where the myocardial 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.
- 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 Schematic diagram showing the states of photographs B to D 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
- 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.
- 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
- FIG. 1 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
- 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
- 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.
- 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.
- 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.
- 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”.
- 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.
- 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.
- the method of the present invention further includes a step of simultaneously loading when applying the stimulus in the above step (3).
- the laminated cell sheet composition which can transplant a lot of cells at once can be manufactured.
- 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.
- 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.
- human, swine for pig treatment, monkey for monkey treatment, and chimpanzee-derived cells for chimpanzee treatment 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.
- the cell type, the number of cells, the ratio of contained cells, etc. used in the present invention may be appropriately selected or adjusted according to the use.
- 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.
- 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
- kidney cells 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.
- 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.
- 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.
- cells collected from the tissue constituting the mucous membrane may be used.
- the mucosa include buccal mucosa, gastric mucosa, intestinal mucosa, olfactory epithelium, oral mucosa and uterine mucosa.
- 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.
- the cells used include, for example, ⁇ cells, ⁇ cells, ⁇ 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.
- the cell used in the present invention may be a cell obtained by mincing a living tissue.
- many types of cells are mixed in cells derived from living tissue.
- 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.
- 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.
- 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.
- 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.
- 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. .
- the number of cells to be seeded for preparing the 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 . It may be 5 ⁇ 10 4 to 8 ⁇ 10 6 pieces / cm 2 , or 0.7 ⁇ 10 4 to 5 ⁇ 10 6 pieces / cm 2 .
- 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.
- 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.
- 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.
- 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.
- FBS fetal bovine serum
- 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.
- 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.
- poly-N-isopropylacrylamide (PIPAAm) is used as a stimulus-responsive polymer, particularly a temperature-responsive polymer.
- PIPAAm poly-N-isopropylacrylamide
- Poly-N-isopropylacrylamide is known as a polymer having a lower critical solution temperature at 31 ° C. If it is in a free state, dehydration occurs in water at a temperature of 31 ° C. or more, 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.
- 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.
- the shape of the cell culture substrate for cell sheet preparation used in the present invention may be, for example, a dish, multiplate, flask, or flat membrane shape.
- 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.
- 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.
- poly-N-acryloylmorpholine polyacrylamide, polydimethylacrylamide, polyethylene glycol, cellulose, etc., which are non-cell-adhesive polymers with low affinity to cells.
- the cell sheet in the present invention does not use a proteolytic enzyme such as dispase and trypsin conventionally used for recovering adherent cells, the protein expressed on the cell surface is hardly damaged. For this reason, the lower surface of the cell sheet peeled off from the cell culture substrate (the surface on the side in contact with the cell culture substrate) is rich in undamaged adhesive proteins, 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 while maintaining 10-40% of the cell-cell desmosome structure. 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.
- a proteolytic enzyme such as dispase and trypsin conventional
- 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
- stacking using a moving jig is mentioned.
- 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.
- the material examples 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.
- 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.
- 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.
- a cell adhesive protein a cell adhesive peptide, a cell adhesive polysaccharide, Other medical adhesives are included.
- 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.
- cell-adhesive polysaccharide examples 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- factors that induce angiogenesis include vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), angiopoietin, platelet-derived growth factor (PDGF), transforming growth factor- ⁇ (TGF- ⁇ ), Matrix metalloproteases (MMP), VE-cadherin, ephrin, plasminogen activator, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), placental growth factor (PlGF) and the like.
- VEGF vascular endothelial growth factor
- FGF fibroblast growth factor
- angiopoietin angiopoietin
- PDGF platelet-derived growth factor
- TGF- ⁇ transforming growth factor- ⁇
- MMP Matrix metalloproteases
- VE-cadherin ephr
- the stacked cell sheets are brought into close contact with each other by applying a load to the cell sheets.
- the clearance gap between a cell sheet and a cell sheet reduces, and the coupling
- 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.
- 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
- 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.
- 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.
- 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.
- 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
- 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 ⁇ m.
- 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.
- the laminated cell sheet of the present invention can be transplanted with a cell sheet having a thickness of 80 ⁇ m or more by one transplantation, and can greatly reduce the burden on the subject.
- 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.
- 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 ⁇ m or more, for example, 85 ⁇ m or more, 90 ⁇ m or more, 95 ⁇ m or more, 100 ⁇ m or more, 110 ⁇ m or more, 120 ⁇ m or more, 130 ⁇ m or more, 140 ⁇ m or more, 150 ⁇ m or more, 160 ⁇ m or more, 170 ⁇ m or more, 180 ⁇ m or more, 190 ⁇ m or more, 200 ⁇ m or more, 210 ⁇ m or more, 220 ⁇ m or more, 240 ⁇ m or more, 250 ⁇ m or more, 260 ⁇ m or more, 270 ⁇ m or more, 280 ⁇ m or more, 290 ⁇ m or more, 300 ⁇ m or more, 400 ⁇ m or more, 500 ⁇ m or more , 600 ⁇ m or more, 700 ⁇ m or more, 800 ⁇ m or more, 900 ⁇ m or more, 1000 ⁇ m or more, or more.
- the average thickness of the laminated cell sheet composition of the present invention is, for example, 80 ⁇ m to 1000 ⁇ m, 80 ⁇ m to 800 ⁇ m, 80 ⁇ m to 600 ⁇ m, 80 ⁇ m to 500 ⁇ m, 80 ⁇ m to 300 ⁇ m, 90 ⁇ m to 1000 ⁇ m, 90 ⁇ m to 800 ⁇ m, 90 ⁇ m to 90 ⁇ m It may be 600 ⁇ m, 90 ⁇ m to 500 ⁇ m, 90 ⁇ m to 300 ⁇ m, 100 ⁇ m to 1000 ⁇ m, 100 ⁇ m to 800 ⁇ m, 100 ⁇ m to 600 ⁇ m, 100 ⁇ m to 500 ⁇ m, 100 ⁇ m to 300 ⁇ m, 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.
- 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.
- the cell sheet further includes means for applying a cell adhesive substance between the cell sheets. Thereby, a cell sheet can be laminated
- 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.
- 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 apparatus is one 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.
- 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.
- 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.
- a buffer a mixture of HANKS and FBS at 1:
- 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
- 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.
- 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. .
- 2 ⁇ L of thrombin is added to the cell sheet side adhered to the gelatin after the second sheet, and 2 ⁇ L 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.
- the cell sheet was cut out from the stamp device together with the gelatin gel using Scalpel, and the laminated tissue was collected.
- HE staining ⁇ Hematoxylin and eosin (HE) staining, immunostaining>
- the collected tissue was fixed in paraffin, sliced at a thickness of 5 ⁇ m, and then the morphology of the laminated tissue was evaluated by HE staining or immunostaining.
- Example 1> (Examination of stamp lamination conditions using fibrin gel)
- any of the examined conditions load: 10, 20, 30 g, gelatin concentration: 60, 65 or 75 mg / mL, fibrin gel amount: 4 ⁇ L) Or 6 ⁇ L, adhesion time: 3 minutes or 5 minutes
- 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.
- 3 (C) to 3 (F) show the results of preparing tissue sections from the laminated cell tissue constructed by this method.
- 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.
- 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).
- Example 3> Pulse synchronization evaluation of laminated myocardial tissue
- 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).
- the tissue in which five cardiomyocyte sheets were laminated according to the present invention was adhered to a ⁇ 60 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.
- OCT optical coherence tomography apparatus
- 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.
- 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.
- the second set of laminated cell sheets was adhered so as to partially overlap the first set of laminated cell sheets (FIG. 6A).
- 4 ⁇ L of fibrin gel was sandwiched between the two laminated cell sheets.
- the pulsatile potential was measured. The potential was measured by the method of Matsuura et al. (The same as the method of Matsuura K. et al., Creation of mouse embryonic stem cell-derived cardiac cell sheets, Biomaterials 32 (2011) 7355-7362.).
- 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.
- 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.
- 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).
- 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.
- UpCell registered trademark
- 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
- the back of a nude rat strain name: F344 / NJcl-rnu / rnu, Claire Japan, Inc. 8-8 weeks old anesthetized (isoflurane) was opened, and two laminated cells were subcutaneously applied. The sheet was transplanted with the gelatin gel separated.
- the transplanted tissue was covered with a silicon sheet (As One, 6-611-01) so that the transplanted site could be identified, and the open back was closed and reared for 2 weeks (FIGS. 8 and 9).
- a silicon sheet As One, 6-611-01
- 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.
- 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 ⁇ m using a microtome to prepare a tissue section.
- tissue sections were prepared, morphological evaluation was performed by Azan staining by a conventional method.
- 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).
- Abbacam, ab7481 Blocking treatment, overnight at 4 ° C with primary antibody (anti-cardiac Toroponin T) diluted 200-fold with PBS, and 2 hours at room temperature 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.
- the fixed area of the transplanted tissue was quantified from the antibody immunostaining image. Sections of 4 cuts were prepared from one tissue at intervals of 300 to 400 ⁇ m, and after immunostaining, a portion that was positive for cardiac troponin T was imageJ (free software, reference: http: //rsb.info.nih. gov / ij / index.html) (FIG. 9E).
- 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).
- the cardiomyocyte sheet produced according to the present invention enhances the blood vessel induction effect after transplantation and has a high therapeutic effect.
- 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.
- 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).
- 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).
- 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).
- the thickness of the cell sheet that can be transplanted at one time is less than 80 ⁇ m.
- the laminated cell sheet of the present invention is a 10-layer laminated cell sheet having a thickness of 200 ⁇ m 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 ⁇ m can be engrafted without necrosis after transplantation.
- 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.
- 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.
- 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.
- the cell adhesive substance applying means 17 is 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.
- 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.
- the base member 15 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.
- the mounting table 16 is 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.
- 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.
- 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.
- a culture dish for example, it is a light source (for example, LED) that gives a light stimulus.
- 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 from the cell culture substrate 20 as a cell sheet, and the cultured cell moving jig 10 Paste to.
- 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.
- 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.
- 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.
- a laminated cell sheet composition can be produced.
- 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. .
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Abstract
Description
(2)刺激応答性培養基材上の第2シート状細胞群の上面、及び/又は、該培養細胞移動治具に付着した該第1細胞シートの下面、に細胞接着性物質を塗布する工程、
(3)該培養細胞移動治具に付着した該第1細胞シートの下面を、該第2シート状細胞群の上面に接触させ、該第2シート状細胞群が該刺激応答性培養基材から剥離する刺激を与え、剥離した第2細胞シートを該第1細胞シートの下面に接着させる工程、を含む、積層化細胞シート組成物を製造する方法。
(4)刺激応答性培養基材上の第3シート状細胞群の上面、及び/又は、該工程(3)で得られた該培養細胞移動治具に付着した積層化細胞シート組成物の下面、に細胞接着性物質を塗布する工程、
(5)該培養細胞移動治具に付着した該積層化細胞シート組成物の下面を、該第3シート状細胞群の上面に接触させ、該第3シート状細胞群が該刺激応答性培養基材から剥離する刺激を与え、剥離した第3細胞シートを該積層化細胞シート組成物の下面に接着させる工程、
(6)該工程(4)~(5)を任意の回数繰り返す工程、
を含む、[1]又は[2]に記載の方法。
刺激応答性培養基材上の第2シート状細胞群の上面、及び、該培養細胞移動治具に付着した該第1細胞シートの下面、に細胞接着性物質を塗布する工程であって、
該第2シート状細胞群の上面に塗布する細胞接着性物質がフィブリノーゲン、及び、該第1細胞シートの下面に塗布する細胞接着性物質がトロンビンである、或いは、
該第2シート状細胞群の上面に塗布する細胞接着性物質がトロンビン、及び、該第1細胞シートの下面に塗布する細胞接着性物質がフィブリノーゲンである、[1]~[5]のいずれか1項に記載の方法。
刺激応答性培養基材上の第3シート状細胞群の上面、及び、該工程(3)で得られた該培養細胞移動治具に付着した積層化細胞シート組成物の下面、に細胞接着性物質を塗布する工程であって、
該第3シート状細胞群の上面に塗布する細胞接着性物質がフィブリノーゲン、及び、該積層化細胞シート組成物の下面に塗布する細胞接着性物質がトロンビンである、或いは、
該第3シート状細胞群の上面に塗布する細胞接着性物質がトロンビン、及び、該積層化細胞シート組成物の下面に塗布する細胞接着性物質がフィブリノーゲンである、[3]~[6]のいずれか1項に記載の方法。
[13] 前記積層化細胞シート組成物の平均の厚さが、80μm以上である[12]に記載の積層化細胞シート組成物。
該細胞シートを付着させるための培養細胞移動治具と、
該培養細胞移動治具を取り付けて、該培養細胞移動治具に荷重をかけるためのアームと、
該刺激応答性培養基材上の該細胞シートに細胞接着性物質を塗布する細胞接着性物質塗布手段と、を備えた、積層化細胞シート組成物の製造装置。
該載置台に載置された第2シート状細胞群の上面、及び/又は、該第1細胞シートの下面に、該細胞接着性物質塗布手段によって供給される該細胞接着性物質を塗布し、
該培養細胞移動治具に付着した該第1細胞シートの下面を、該第2シート状細胞群の上面に接触させ、該刺激手段によって該刺激応答性培養基材上の該第2シート状細胞群が剥離される刺激を与えると同時に、該培養細胞移動治具に荷重をかけて、得られる第2細胞シートを該第1細胞シートに迅速に積層することを特徴とする、[15]に記載の積層化細胞シート組成物の製造装置。
(1)刺激応答性培養基材上の第1シート状細胞群の上面に、培養細胞移動治具を接触させ、該刺激応答性培養基材上の第1シート状細胞群を剥離させる刺激を与えて、剥離した第1細胞シートを該培養細胞移動治具に付着させる工程、
(2)刺激応答性培養基材上の第2シート状細胞群の上面、及び/又は、該培養細胞移動治具に付着した該第1細胞シートの下面、に細胞接着性物質を塗布する工程、
(3)該培養細胞移動治具に付着した該第1細胞シートの下面を、該第2シート状細胞群の上面に接触させ、該第2シート状細胞群が該刺激応答性培養基材から剥離する刺激を与え、剥離した第2細胞シートを該第1細胞シートの下面に接着させる工程。
(4)刺激応答性培養基材上の第3シート状細胞群の上面、及び/又は、該工程(3)で得られた該培養細胞移動治具に付着した積層化細胞シート組成物の下面、に細胞接着性物質を塗布する工程、
(5)該培養細胞移動治具に付着した該積層化細胞シート組成物の下面を、該第3シート状細胞群の上面に接触させ、該第3シート状細胞群が該刺激応答性培養基材から剥離する刺激を与え、剥離した第3細胞シートを該積層化細胞シート組成物の下面に接着させる工程、
(6)該工程(4)~(5)を任意の回数繰り返す工程、
を含んでいる。
刺激応答性培養基材に刺激を与え、該刺激応答性培養基材から細胞シートを剥離させるための刺激手段を具備した載置台と、
該細胞シートを付着させるための培養細胞移動治具と、
該培養細胞移動治具を取り付けて、該培養細胞移植治具に荷重をかけるためのアームと、
該刺激応答性培養基材上の該細胞シートに細胞接着性物質を塗布する細胞接着性物質塗布手段と、を備え、
該載置台に載置された刺激応答性培養基材上の第1シート状細胞群の上面に、該培養細胞移動治具を接触させ、該刺激手段によって該刺激応答性培養基材上の該第1シート状細胞群が剥離される刺激を与えて、得られる第1細胞シートを該培養細胞移動治具に付着させ、
該載置台に載置された第2シート状細胞群の上面、及び/又は、該第1細胞シートの下面に、該細胞接着性物質塗布手段によって供給される該細胞接着性物質を塗布し、
該培養細胞移動治具に付着した該第1細胞シートの下面を、該第2シート状細胞群の上面に接触させ、該刺激手段によって該刺激応答性培養基材上の該第2シート状細胞群が剥離される刺激を与えると同時に、該細胞移動治具に荷重をかけて、得られる第2細胞シートを該第1の細胞シートに迅速に積層することを特徴とする積層化細胞シート組成物の製造装置、が挙げられる。
本実施例において、別途特別に表記が無い限り、以下の動物、細胞、試薬等を使用した。
・ラット(生後1日、日本医科学動物資材研究所社、Hsd:Sprague Dawley SD(系統名))
・HepG2(ATCC社、HB-8065)
・温度応答性培養皿(UpCell(登録商標))(セルシード社)
・ラット心筋細胞培養液(medium199 40%+HANKS balanced salt Solution 54%+FBS6%+penicillin-streptomycin solution1%+166mmol/L NaCl、1.0 mmol/L NaH2PO4、0.8 mmol/L MgSO4、26.2mmol/L NaHCO3、0.9mmol/L CaCl2、5mmol/L glucose)
・HepG2細胞培養液(DMEM+FBS10%+penicillin-streptomycin solution1%)
・FBS(ジャパンバイオシーラム社、S1650-500)
・ハンクス平衡塩溶液(ナカライテスク社、17460-15)
・DMEM(SIGMA、D6429)
・penicillin-streptomycin solution(Life Technologies、15140-122)
・4%パラホルムアルデヒド固定液(武藤化学社、3311-1)
・50%水酸化ナトリウム溶液(和光純薬工業社、197-12375)
・抗ラット心筋トロポニンT(サーモフィッシャーサイエンティフィック社、MS-295-P1)
・CD31抗体 mouse anti ratPECAM-1[CD31](ミリポア社、MAB1393)
・2次染色抗体 Alexa Fluor488 goat anti-mouse(Life technolodies、A11017)
・2次染色抗体 Alexa Fluor594 goat anti-rabbit(Life technolodies、A11037)
・核染色ProLong Gold antifade reagent with DAPI(Life technolodies、P36935)
・Anti-fibronectin抗体(abcam、ab2413)
・フィブリンゲル(フィブリノーゲン+トロンビン)(帝人ファーマ社、ボルヒール組織接着用)
・コラーゲン溶液(高研社、コラーゲン酸性溶液I-AC 5mg/mL)
・ゼラチン(SIGMA社、G1890-100G)
<使用した機器>
・スタンプデバイス(参考:Ngo T.X.et al.Endothelial cell behavior inside myoblast sheets with different thickness.Biotechnol Lett(2013) 35:1001-1008)
・顕微鏡(ニコン社、ECLIPSE,E800)
・共焦点顕微鏡(オリンパス社、FV1200)
・光干渉断層撮影装置(OCT、パナソニックヘルスケア社、参考:Kobayashi M.,et al.,Real-time,noninvasive optical coherence tomography of cross-sectional living cell-sheets in vitro and in vivo. JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS vol.103(6),1267-1273,Aug.2015)
心筋細胞シートは、Sakaguchiらの方法(Sakaguchi K.et al.,In Vitro Engineering of Vascularized Tissue Surrogates,Scientific Reports,3,1316,2013.)に従って作製した。具体的には、生後1日の仔ラットより回収した心臓をミンス(mince)して心筋細胞を回収した。回収した細胞をバッファー(HANKSとFBSを1:1で混合したもの)で洗浄し、ラット心筋培養液に懸濁した。心筋細胞は、温度応答性の24wellプレート培養皿(UpCell(登録商標)、セルシード社)に90万cells/wellの濃度で播種し、0.5mLのラット心筋培地中で4日間、5%CO2、飽和水蒸気下の37℃インキュベーターで培養した。その後、20℃で保温することとで、細胞シートとして回収した。
ピペッティング法による細胞シートの積層は、Haraguchiらの方法(Haraguchi Y.et al.,Fabrication of functional three-dimensional tissues by stacking cell sheets in vitro,Nature Protocol,7,850,2012.)に従って実施した。具体的には、培養液に浮遊させた細胞シートを、細胞培養液ごとピペットで吸引し、別の細胞シート上に吸引した培養液を排出することで細胞シートを積層した。
スタンプ法による細胞シートの積層は、基本的にはHaraguchiらの方法(Haraguchi Y.et al.,Fabrication of functional three-dimensional tissues by stacking cell sheets in vitro,Nature Protocol,7,850,2012.)に従い、一部改変して行った(図1及び図2)。具体的には、粉末状のゼラチンを65mg/mlの濃度でハンクス平衡塩溶液に溶かし、pHを7.4付近になるように50%水酸化ナトリウム溶液を加えた。pH調整をしたゼラチン溶液をスタンプデバイスの型に気泡が入らないようにゆっくりと流し込み、4℃で30分間静置することでゼラチンゲル付きスタンプデバイスを完成させた。完成したスタンプデバイスを、培養液を抜いた24wellプレート培養皿上のシート状細胞群に乗せ、上からスタンプデバイスと合わせて合計30gの荷重がかかるようにおもりを乗せ、30分間氷上で静置した。1枚目のシート状細胞群とゼラチンを接着させたら、2枚目以降はゼラチンに付着した細胞シート側にトロンビン2μLを、24wellプレート培養皿上のシート状細胞群側にフィブリノーゲン2μLをピペットマンにより添加し、1枚目と同様に上から合計30gの荷重をかけて積層させた。目的の枚数まで積層が完了したら、スカルペルを使用してゼラチンゲルごとスタンプデバイスから細胞シートを切り出し、積層組織を回収した。
回収した組織をパラフィン固定し5μmの厚さで薄切した後、HE染色や免疫染色をすることで積層組織の形態を評価した。
塩酸でpH3に調整した塩酸水を用いてコラーゲン溶液を10倍希釈した後、温度応答性24wellプレート培養皿に300μL加え30分間静置した。その後、コラーゲン溶液を除去して2時間風乾させ、コラーゲンコーティングを施した。その後、そこにHepG2細胞を20万cells/wellの濃度で播種して3日間37℃インキュベーターで培養する。なお培養液にはDMEM(1%penicillin-streptomycin solution、10%FBSを含む)を使用した。スタンプ積層に関しては前述の心筋細胞シートの積層と同様の方法で行った。
(フィブリンゲルを用いたスタンプ積層条件の検討)
スタンプデバイスとフィブリンゲルを用いた細胞シート積層化方法の条件を検討した結果、検討したいずれの条件(荷重:10、20、30g、ゼラチン濃度:60、65又は75mg/mL、フィブリンゲル量:4μL又は6μL、接着時間:3分又は5分)においても、従来の方法では30分以上を必要としていた積層化時間を短縮することが可能であった。その中でも、表1に示した条件が最も再現よく心筋細胞シートを積層化することが可能であった。本発明によって、これまで、2層目以降の細胞シートの積層時間が30分以上必要であったが、5分以下に短縮可能となった。
(細胞生存率の評価)
生後1日の仔ラットの心臓から回収した細胞を90万cells/wellの濃度で温度応答性培養皿(24well)に播種し、37℃ CO2インキュベーターで4日間培養した。その後、ゼラチンを形成したスタンプデバイスを、培養液を抜いた細胞の上に置き、30gの荷重をかけながら30分静置させ、1枚目の細胞をスタンプデバイスに接着させた。2枚目の積層は、従来の30分かけて積層する方法と、間にフィブリンゲルを加えることで接着時間を5分にする本発明の積層方法により、準備した。2つの方法で2層の積層が完了した後、ゼラチンごと2層の細胞シートを60mm dishに移動させ、5分 37℃インキュベーターで培養することでゼラチンゲルを溶解して取り除いた。2層の細胞シートを回収した後、トリプシン酵素処理を行って細胞シートを個々の細胞へとバラバラにし、トリパンブルー染色を使用して死細胞、生細胞をカウントした。コントロールには、積層前の細胞を準備し、同様の処理をした後に死細胞、生細胞をそれぞれカウントした。
細胞の生存率は、以下の式で算出した(図4)。
(積層心筋組織の拍動同期評価)
心筋細胞シートは拍動していることが知られ、また積層させることで2枚の心筋細胞シートの拍動が同期することも確認されている。そこで、本手法により構築した立体心筋組織が拍動を行っているか、また拍動が同期するのかをOCTと明視野顕微鏡による観察により確かめた(図5)。
(積層心筋組織の拍動電位の同期評価)
フィブリンゲル用いて積層した心筋細胞シート間において、拍動電位が同期するかどうかを確認した。生後1日の仔ラットの心臓から回収した細胞を90万cells/wellの濃度で温度応答性培養皿(24well)に播種し、37℃ CO2インキュベーターで4日間培養した。その後、ゼラチンを形成したスタンプデバイスを、培養液を抜いた細胞の上に置き、30gの荷重をかけながら30分間静置させ、1枚目の細胞をスタンプデバイスに接着させた。この方法で2層の心筋細胞シートを積層させたものを2セット作製したら、1セット目を電位測定用の特殊な培養皿に接着させ、37℃ CO2インキュベーターで5分間培養し、ゼラチンを溶解させて取り除いた。次に2セット目の積層化細胞シートを1セット目の積層化細胞シートに一部が重なるように接着させた(図6(A))。なお、この際に2つの積層化細胞シートの間には4μLのフィブリンゲルを挟み込んだ。37℃ CO2インキュベーターで5分間培養し、ゼラチンを溶解させて取り除いた後、拍動電位の測定を行った。なお、電位測定はMatsuuraらの方法(Matsuura K.et al.,Creation of mouse embryonic stem cell-derived cardiac cell sheets,Biomaterials 32(2011)7355-7362.)と同様の方法で行った。
(積層心筋組織内における血管網評価)
これまでの研究から立体細胞組織を生体外で培養するためには、血管網の構築が必要不可欠であることが分かっている。そこで本手法により構築した積層組織内における血管網の様子を血管内皮細胞特異的な抗原であるCD31を用いて抗体免疫染色することで確かめた。本発明により2枚スタンプ積層したラット心筋細胞シートを4%パラホルムアルデヒドで15分間固定した後、PBSで10%希釈したヤギ血清で2時間ブロッキングをした。その後PBSで100:1に希釈したCD31抗体で4℃オーバーナイトして反応させた後、PBSで100:1に希釈した2次染色抗体 Alexa Fluor488 goat anti-mouse(Life technolodies、A11017)を1時間、室温で反応させた。最後に細胞核をPBSで300:1に希釈したHoechst33258で15分間染色し、共焦点顕微鏡により観察した。
(高速スタンプ積層組織の移植評価)
生後1日の仔ラットの心臓から回収した細胞を90万cells/wellの濃度で温度応答性培養皿(UpCell(登録商標)、24well)に播種し、37℃ CO2インキュベーターで4日間培養した。その後、ゼラチンを形成したスタンプデバイスを、培養液を抜いた細胞の上に置き、30gの荷重をかけながら30分間静置させ、1枚目の細胞をスタンプデバイスに接着させた。2枚目以降の積層からは、再び30分間かけて積層する方法と、細胞シートと細胞シートとの間にフィブリンゲルを加えることで接着時間を5分間にして積層する方法によって積層化細胞シートを準備した。2つの方法で5層まで積層を行った。なお、2つの方法で5層積層した細胞シートの完成時間が同時になるように、積層の始める時間を調整した。積層が完了したら、直ちに8~12週齢の麻酔(イソフルラン)をかけたヌードラット(系統名:F344/NJcl-rnu/rnu、日本クレア社)の背中を開き、皮下側に2つの積層化細胞シートをゼラチンゲルごと切り離して置くようにして移植した。その後、移植した部位が特定できるように、移植した組織の上からシリコンシート(アズワン社、6-611-01)を被せ、開いた背中を閉じて2週間飼育した(図8、図9)。移植2週間後同様に背中を開き、移植した部位を回収し、4%パラホルムアルデヒドに24時間浸けて組織を固定した。組織を固定後、1×PBSで洗浄し最後にパラフィン置換してパラフィンブロックを作製した。パラフィンブロックを作製後、ミクロトームを用いて5μmの厚さで薄切し、組織切片を作製した。組織切片を作製したら慣例的な方法によりアザン染色をして形態評価を行った。また、組織切片をキシレンとエタノールを使って脱パラフィン処理を行い、賦活化(Target Retrieval Solution 10×concentrate(Dako、S1699))溶液にて処理を行った後、室温で1時間のNormal Goat Serum(アバカム社、ab7481)にてブロッキング処理、PBSで200倍に希釈した1次抗体(anti-cardiac ToroponinT)で4℃オーバーナイト、PBSで200倍に希釈した2次抗体(Alexafluor488)で2時間室温反応、Hoechst33258で15分室温反応させ封入剤で封入することで、抗体免疫染色切片を作製して共焦点顕微鏡により観察した。
(スタンプ積層技術によるHepG2細胞シートの積層化)
フィブリンゲルを用いた新規スタンプ積層技術の検討は全てラット心臓由来細胞シートを用いて行われた。そこで他細胞種の細胞シートにも本手法が適用可能かを検討するためHepG2細胞シート(ヒト肝癌由来細胞)を用いてラット心筋細胞シートと同様に積層実験を行い、組織切片を作製し形態を評価した。コラーゲンコーティングした温度応答性培養皿にHepG2細胞を播種して3日間培養することでHepG2細胞シートを作製し、新規スタンプ積層技術により積層した結果を図7に示した。図7の結果から厚みのあるHepG2細胞組織が構築されている様子が確認でき、他細胞種においても本発明の積層技術が適用できる可能性が示唆された。
(積層化細胞シート組成物(10層)の移植評価)
実施例6と同様、フィブリンゲルを使用した方法によって、生後1日の仔ラットの心臓から回収した細胞を用いて10層の積層化細胞シートを作製した。また、得られた積層化細胞シートを実施例6と同様の方法でヌードラットへ移植した。移植2週間後の移植部位周辺より組織切片を作製し、免疫染色法によって心筋トロポニンT(緑、心筋細胞の位置を示す)及びCD31(赤、血管内皮細胞の位置を示す)を染色し、同時にHoechst33258にて細胞核を染色した。得られた組織切片を共焦点顕微鏡で観察した(図13)。
本発明の積層化細胞シート組成物を製造するための装置を例示する。図12は、本発明の細胞シート積層化装置1を示す斜視図である。10は、細胞シートを保持する培養細胞移動治具10である。この培養細胞移動治具10は、アーム11先端下部に着脱可能である。培養細胞移動治具10の細胞シートと接触する細胞付着には、細胞シートを捕捉できる機能を有するものであればよく、例えば、材料としては、ポリビニリデンジフルオライド(PVDF)、シリコーン樹脂、ポリビニルアルコール、ウレタン、セルロース及びその誘導体、キチン、キトサン、コラーゲン、ゼラチン、フィブリンゲル等を用いることができる。これにより、細胞シートを貼り付けて持ち上げることができる。アーム11は、(四角柱形状の)基部材の上部に垂直平面内で揺動可能に取り付けられている。アーム11の揺動回転軸12を挟んで図中左側には培養細胞移動治具10と、図中右側には押圧力(重さ)を計測する押圧力測定手段13を備える。基部材15の下部は蛇腹シール14でシールされている。その基部材の下部には、基部材15を水平面内のX方向及びY方向に移動可能にする(図示しない)駆動手段と基部材をZ方向に昇降動作させる昇降駆動手段を備える。
10 培養細胞移動治具
11 アーム
12 揺動回転軸
13 横圧力測定手段
14 蛇腹シール
15 基部材
16 載置台
16a 回転軸
17 細胞接着性物質塗布手段
18 刺激供与手段
20 細胞培養基材
30 基台
31 搬出入口
Claims (15)
- (1)刺激応答性培養基材上の第1シート状細胞群の上面に、培養細胞移動治具を接触させ、前記刺激応答性培養基材上の第1シート状細胞群を剥離させる刺激を与えて、剥離した第1細胞シートを前記培養細胞移動治具に付着させる工程、
(2)刺激応答性培養基材上の第2シート状細胞群の上面、及び/又は、前記培養細胞移動治具に付着した前記第1細胞シートの下面、に細胞接着性物質を塗布する工程、
(3)前記培養細胞移動治具に付着した前記第1細胞シートの下面を、前記第2シート状細胞群の上面に接触させ、前記第2シート状細胞群が前記刺激応答性培養基材から剥離する刺激を与え、剥離した第2細胞シートを前記第1細胞シートの下面に接着させる工程、
を含む、積層化細胞シート組成物を製造する方法。 - 前記工程(3)の前記刺激を与える時に、さらに同時に荷重する、請求項1に記載の方法。
- 前記工程(3)で得られた、前記培養細胞移動治具に付着した積層化細胞シート組成物に対し、
さらに、
(4)刺激応答性培養基材上の第3シート状細胞群の上面、及び/又は、前記工程(3)で得られた前記培養細胞移動治具に付着した積層化細胞シート組成物の下面、に細胞接着性物質を塗布する工程、
(5)前記培養細胞移動治具に付着した前記積層化細胞シート組成物の下面を、前記第3シート状細胞群の上面に接触させ、前記第3シート状細胞群が前記刺激応答性培養基材から剥離する刺激を与え、剥離した第3細胞シートを前記積層化細胞シート組成物の下面に接着させる工程、
(6)前記工程(4)~(5)を任意の回数繰り返す工程、
を含む、請求項1又は2に記載の方法。 - 前記細胞接着性物質が、細胞接着性タンパク質、細胞接着性ペプチド及び/又は細胞接着性多糖類である、請求項1~3のいずれか1項に記載の方法。
- 前記細胞接着性物質が、ポリリジン、コラーゲン、ビトロネクチン、フィブリン、フィブリノーゲン及びトロンビンの混合物、フィブロネクチン、トロンボスポンジン、ゼラチン、ラミニン、キトサン系接着剤、アルギン酸系接着剤からなる群から選択される1又は2以上の物質である、請求項1~3のいずれか1項に記載の方法。
- 前記工程(2)が、
刺激応答性培養基材上の第2シート状細胞群の上面、及び、前記培養細胞移動治具に付着した前記第1細胞シートの下面、に細胞接着性物質を塗布する工程であって、
前記第2シート状細胞群の上面に塗布する細胞接着性物質がフィブリノーゲン、及び、前記第1細胞シートの下面に塗布する細胞接着性物質がトロンビンである、或いは、
前記第2シート状細胞群の上面に塗布する細胞接着性物質がトロンビン、及び、前記第1細胞シートの下面に塗布する細胞接着性物質がフィブリノーゲンである、
請求項1~5のいずれか1項に記載の方法。 - 前記工程(4)が、
刺激応答性培養基材上の第3シート状細胞群の上面、及び、前記工程(3)で得られた前記培養細胞移動治具に付着した積層化細胞シート組成物の下面、に細胞接着性物質を塗布する工程であって、
前記第3シート状細胞群の上面に塗布する細胞接着性物質がフィブリノーゲン、及び、前記積層化細胞シート組成物の下面に塗布する細胞接着性物質がトロンビンである、或いは、
前記第3シート状細胞群の上面に塗布する細胞接着性物質がトロンビン、及び、前記積層化細胞シート組成物の下面に塗布する細胞接着性物質がフィブリノーゲンである、
請求項3~6のいずれか1項に記載の方法。 - 前記刺激応答性培養基材が、温度応答性培養基材であって、前記刺激が、下限臨界溶解温度以下又は上限臨界溶解温度以上の温度である、請求項1~7のいずれか1項に記載の方法。
- 前記刺激応答性培養基材が、ポリ-N-イソプロピルアクリルアミドが少なくともその培養面の一部に被覆された培養基材である、請求項1~8のいずれか1項に記載の方法。
- 前記培養細胞移動治具が、細胞接着性タンパク質、細胞接着性ペプチド又は親水性ポリマーの1種又は2種以上からなる細胞付着部を有する培養細胞移動治具である、請求項1~9のいずれか1項に記載の方法。
- 前記積層化細胞シート組成物が、心筋細胞、肝細胞、線維芽細胞、筋芽細胞、膵細胞、腎細胞、血管内皮細胞、上皮細胞からなる群から選択される1又は2以上の細胞を含む、請求項1~10のいずれか1項に記載の方法。
- 請求項1~11のいずれか1項に記載の方法により得られる積層化細胞シート組成物。
- 前記積層化細胞シート組成物の平均の厚さが、80μm以上である請求項12に記載の積層化細胞シート組成物。
- 刺激応答性培養基材に刺激を与え、前記刺激応答性培養基材から細胞シートを剥離させるための刺激手段を具備した載置台と、
前記細胞シートを付着させるための培養細胞移動治具と、
前記培養細胞移動治具を取り付けて、前記培養細胞移動治具に荷重をかけるためのアームと、
前記刺激応答性培養基材上の該細胞シートに細胞接着性物質を塗布する細胞接着性物質塗布手段と、を備えた、積層化細胞シート組成物の製造装置。 - 前記載置台に載置された前記刺激応答性培養基材上の第1シート状細胞群の上面に、前記培養細胞移動治具を接触させ、前記刺激手段によって前記刺激応答性培養基材上の該第1シート状細胞群が剥離される刺激を与えて、得られる第1細胞シートを前記培養細胞移動治具に付着させ、
前記載置台に載置された第2シート状細胞群の上面、及び/又は、前記第1細胞シートの下面に、前記細胞接着性物質塗布手段によって供給される前記細胞接着性物質を塗布し、
前記培養細胞移動治具に付着した該第1細胞シートの下面を、前記第2シート状細胞群の上面に接触させ、前記刺激手段によって前記刺激応答性培養基材上の前記第2シート状細胞群が剥離される刺激を与えると同時に、前記培養細胞移動治具に荷重をかけて、得られる第2細胞シートを前記第1細胞シートに迅速に積層することを特徴とする、請求項14に記載の積層化細胞シート組成物の製造装置。
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111467579A (zh) * | 2020-04-03 | 2020-07-31 | 北京臻溪谷医学研究中心(有限合伙) | 基于合金内核的治疗动脉瘤与血管狭窄用干细胞支架及其制法 |
WO2021004933A1 (en) | 2019-07-10 | 2021-01-14 | Kunz Helmuth Heinrich | Methods for deriving autologous and hypoimmunogenic hair follicle containing sheets in vitro |
JPWO2021065395A1 (ja) * | 2019-10-01 | 2021-04-08 | ||
JP2021531807A (ja) * | 2018-07-31 | 2021-11-25 | ロキット ヘルスケア インク. | 多層細胞シートの製造方法、及び、これを用いて製造された多層細胞シート |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014192909A1 (ja) * | 2013-05-31 | 2014-12-04 | iHeart Japan株式会社 | ハイドロゲルを組み込んだ積層化細胞シート |
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-
2016
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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Non-Patent Citations (3)
Title |
---|
HARAGUCHI Y. ET AL.: "Fabrication of functional three-dimensional tissues by stacking cell sheets in vitro", NAT. PROTOC., vol. 7, no. 5, 2012, pages 850 - 858, XP055291922 * |
KIKUCHI T. ET AL.: "Automatic fabrication of 3- dimensional tissues using cell sheet manipulator technique.", BIOMATERIALS, vol. 35, no. 8, 2014, pages 2428 - 2435, XP028814366 * |
MATSUSAKI M. ET AL.: "Fabrication of cellular multilayers with nanometer-sized extracellular matrix films.", ANGEW. CHEM. INT. ED., vol. 46, no. 25, 2007, pages 4689 - 4692, XP055382303 * |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2021531807A (ja) * | 2018-07-31 | 2021-11-25 | ロキット ヘルスケア インク. | 多層細胞シートの製造方法、及び、これを用いて製造された多層細胞シート |
EP3831419A4 (en) * | 2018-07-31 | 2022-04-20 | Rokit Healthcare Inc. | METHOD FOR MAKING A MULTILAYER CELL SHEET, AND MULTILAYER CELL SHEET MADE BY MEANS OF THE SAME METHOD |
JP7228672B2 (ja) | 2018-07-31 | 2023-02-24 | ロキット ヘルスケア インク. | 多層細胞シートの製造方法、及び、これを用いて製造された多層細胞シート |
WO2021004933A1 (en) | 2019-07-10 | 2021-01-14 | Kunz Helmuth Heinrich | Methods for deriving autologous and hypoimmunogenic hair follicle containing sheets in vitro |
JPWO2021065395A1 (ja) * | 2019-10-01 | 2021-04-08 | ||
WO2021065395A1 (ja) * | 2019-10-01 | 2021-04-08 | 国立大学法人大阪大学 | フィブリンシートの製造方法 |
JP7222567B2 (ja) | 2019-10-01 | 2023-02-15 | 国立大学法人大阪大学 | フィブリンシートの製造方法 |
CN111467579A (zh) * | 2020-04-03 | 2020-07-31 | 北京臻溪谷医学研究中心(有限合伙) | 基于合金内核的治疗动脉瘤与血管狭窄用干细胞支架及其制法 |
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