WO2004073761A1 - 再生角膜内皮細胞シート、製造方法及びその利用方法 - Google Patents
再生角膜内皮細胞シート、製造方法及びその利用方法 Download PDFInfo
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- WO2004073761A1 WO2004073761A1 PCT/JP2004/001975 JP2004001975W WO2004073761A1 WO 2004073761 A1 WO2004073761 A1 WO 2004073761A1 JP 2004001975 W JP2004001975 W JP 2004001975W WO 2004073761 A1 WO2004073761 A1 WO 2004073761A1
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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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- 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
- A61L27/3804—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 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3808—Endothelial cells
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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
- A61L27/3839—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 characterised by the site of application in the body
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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
- A61L27/3895—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 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
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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
- A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
- A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
-
- 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
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0618—Cells of the nervous system
- C12N5/0621—Eye cells, e.g. cornea, iris pigmented cells
-
- 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
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/16—Materials or treatment for tissue regeneration for reconstruction of eye parts, e.g. intraocular lens, cornea
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2539/00—Supports and/or coatings for cell culture characterised by properties
- C12N2539/10—Coating allowing for selective detachment of cells, e.g. thermoreactive coating
Definitions
- Regenerated corneal endothelial cell sheet production method and use thereof
- the present invention relates to a regenerated corneal endothelial cell sheet in the fields of biology, medicine, and the like, a production method, and a treatment method using them.
- Corneal tissue consists of five layers: corneal epithelial layer, Paumann's membrane, corneal stroma, Descemet's membrane, and corneal endothelium layer from the outer surface.
- the innermost corneal endothelium layer is lined against the corneal tissue, and the Descemet's membrane is the basement membrane made of the corneal endothelium adhesion protein.
- the function of corneal endothelial cells is to pump water from the parenchyma to the anterior chamber against the swelling pressure of the corneal stroma, which is believed to be due to the pumping action using Na-K ATP as.
- Human corneal endothelial cells are normally considered not to divide in vivo, and the amount of endothelial cells that have degenerated or dropped out is compensated for by the remaining cells becoming enlarged or moving. At that time, if the number of remaining corneal endothelial cells is too small, the pump function by the corneal endothelium is no longer sufficient, and the corneal tissue expands, causing diseases such as corneal endothelial dysfunction and bullous keratopathy. Become. Treatments for these diseases include the use of soft contact lenses for the treatment of eye pain or the use of hypertonic saline eye ointments or eye drops to remove water from swollen corneal tissue. There is a method, but this method is only symptomatic treatment, and a fundamental treatment was desired.
- keratinized epidermal cells derived from human neonates are cultured in a culture vessel under the condition that a keratinous tissue membrane is formed on the surface of the vessel, and keratin
- a method for producing a transplantable membrane of keratinous tissue, characterized in that the tissue membrane is exfoliated using an enzyme, is described.
- a technique is disclosed in which 3T 3 cells are grown and layered as a feeder layer, and a cell sheet is recovered using dispase, a proteolytic enzyme.
- the method described in the publication has the following drawbacks.
- the affected part transplanted with the cell sheet is easily infected.
- the corneal endothelial cell that is the subject of the present invention is not as strong as the cell-cell connection as skin cells. It had the disadvantage that it could not be peeled off and collected as a sheet.
- a cell culture support in which a substrate is coated with a temperature-responsive polymer having an upper or lower critical solution temperature of 0 to 80 ° C.
- a temperature-responsive polymer having an upper or lower critical solution temperature of 0 to 80 ° C.
- an object of the present invention is to provide a regenerated corneal endothelial cell sheet having good adhesion to ocular tissue.
- Another object of the present invention is to provide a production method and a utilization method thereof.
- corneal endothelial cells are cultured under specific conditions on a cell culture support under specific conditions where the substrate surface is coated with a temperature-responsive polymer, and then the polymer on the substrate surface hydrates the culture solution temperature.
- the regenerated corneal endothelial cell sheet cultured at a temperature is brought into close contact with a specific carrier, and is peeled off together with the carrier while suppressing the contraction of the cell sheet. I found out that I could get it.
- the present invention has been completed based on such findings.
- the present invention provides a regenerated corneal endothelial cell sheet that has good adhesion to an anterior ocular tissue and has a sufficient cell density in terms of function and is in close contact with a carrier.
- the cells are cultured on a cell culture support having a substrate surface coated with a temperature-responsive polymer that dehydrates within a temperature range of 0 to 80 ° C., (1) Let the culture solution temperature be the temperature at which the polymer on the substrate surface is hydrated,
- a method for producing a regenerated corneal endothelial cell sheet is provided.
- the present invention provides a therapeutic method characterized by transplanting the regenerated corneal endothelial cell sheet.
- the present invention provides the above regenerated corneal endothelial cell sheet for treating wounded tissue.
- FIG. 1 shows the regenerated corneal cell sheet peeled from the cell culture support material in Example 4.
- Fig. 2 shows the collagen produced by extracting the human regenerated corneal cell sheet cultured in Example 4 at 1 day (A), 3 days (B), 7 days (C), and 14 days (D). The results of staining IV (upper figure) and fibronectin (lower figure) according to a conventional method are shown.
- FIG. 3 shows the result of immunofluorescent staining of Z011 protein present in the regenerated corneal endothelial cell sheet after 4 days of culture in Example 4.
- FIG. 4 shows the results of staining collagen IV (left figure) and fibronectin (right figure) present in the human regenerated corneal cell sheet in the middle of separation obtained in Example 4 according to a conventional method.
- the arrow in the figure indicates the peeling direction.
- Fig. 5 shows the results of HZE staining of the human regenerated corneal cell sheet detached in Example 4 (left figure), and collagen IV (middle figure) and fibronectin (right figure) present in the cell sheet were stained according to a conventional method. Results are shown.
- FIG. 6 shows a TEM image of the human regenerated corneal cell sheet obtained in Example 4.
- ECM extracellular matrix
- N nucleus
- GC Go 1 gi C omp l ex
- M mitochondria
- EM Endop assic reticu 1 um
- the arrow indicates the cell Shows cell-cell junctions.
- FIG. 7 is present in the surface layer of the corneal endothelial cell sheet shown in Example 5 and Comparative Examples 3 and 4.
- the upper figure shows the result of staining with Cooma ssiebri 1 1 i na tb 1 ue, and the protein present on the surface of the cell sheet.
- the lower figure shows the result of staining with anti-human Zo-1 polyclonal antibody. Indicates.
- T represents the result of the cell sheet peeled off from the cell culture support material of the present invention by low-temperature treatment
- D represents a disperse treatment from a substrate not coated with a commercially available temperature-responsive polymer.
- the obtained cell, S shows the analysis result of the cells detached from the commercially available substrate by the scraper method.
- FIG. 8 shows that the regenerated corneal endothelial sheet obtained in Example 7 was stained with an anti-rabbit NaK ATPase monoclonal antibody and the Na-K ATPase pump site was stained green.
- the results obtained using a confocal microscope after staining the cell nucleus red with propidium iodide are shown.
- a in the upper figure shows the result of observation from the upper surface of the cultured cell sheet, and B shows the result of observation in the thickness direction.
- FIG. 9 A shows the correlation of the number of pumps per cell to the cell density in the human corneal endothelial cell sheet, and B shows the correlation of the number of pumps per unit area to the cell density. Showing gender.
- a typical endothelial cell used in the present invention is a corneal endothelial cell in corneal tissue, but the type is not limited.
- the regenerated corneal endothelial cell sheet means a sheet in which the various cells described above are cultured in a single layer on a culture support and then peeled off from the support.
- the regenerated corneal endothelial cell sheet in the present invention is not damaged by proteolytic enzymes represented by disperse, trypsin and the like during culture. For this reason, the regenerated corneal endothelial cell sheet peeled from the substrate retains the cell-cell desmosome structure, has few structural defects, and has high strength.
- the basement membrane-like protein between the cell and the substrate formed at the time of culture is not damaged by the enzyme. This makes it possible to adhere well to the affected tissue during transplantation. Can be performed efficiently. Specifically, when using a normal proteolytic enzyme such as trypsin, the desmosome structure between cells and cells and the basement membrane-like protein between cells and substrates are almost retained.
- the cells are detached and separated.
- dispase which is a proteolytic enzyme
- the resulting cell sheet is weak in strength because it almost destroys the basement membrane-like protein and the like.
- the cell sheet of the present invention has a desmosome structure and a basement membrane-like protein remaining in a state of 80% or more, and can obtain various effects as described above. .
- the regenerated corneal endothelial cell sheet in the present invention engrafts very well to the anterior ocular tissue which is a living tissue. It was found that this property is realized by suppressing the contraction of the regenerated corneal endothelial cell sheet detached from the support surface. At that time, the contraction rate of the regenerated corneal endothelial cell sheet is desirably 20% or less, preferably 10% or less, more preferably 5% or less, in any length in the sheet. Is preferred. When the length in any direction of the sheet is 20% or more, the detached cell sheet is in a sagging state, and even if it adheres to the living tissue in that state, it does not adhere to the tissue, and is shown in the present invention. However, it is not possible to expect high survival.
- the method of not contracting the regenerated corneal endothelial cell sheet is not limited as long as it does not contract the cell sheet.
- a ring-shaped carrier with the center cut out is brought into close contact with these cell sheets, and the cell sheet is peeled off together with the carrier.
- the carrier used when closely adhering the regenerated corneal endothelial cell sheet is a structure for holding the cell sheet of the present invention so as not to contract, such as a polymer film or a structure molded from the polymer film, A metallic jig or the like can be used.
- the specific materials include polyvinylidene difluoride (PVDF), polypropylene, polyethylene, cellulose and its derivatives, paper, chitin, chitosan, collagen, Ure evening Can be mentioned.
- the close contact refers to a state in which the cell sheet does not shift or move on the carrier at the boundary surface between the cell sheet and the carrier so that the cell sheet does not contract. Even if they are in close contact, they may be in close contact via a liquid (for example, a culture solution or other isotonic solution) existing between the two.
- a liquid for example, a culture solution or other isotonic solution
- the shape of the carrier is not particularly limited.
- a part of the carrier cut out to the same extent as the transplant site or larger than the transplant site is used.
- the cell sheet is fixed only to the peripheral part of the cutout, and it is only necessary to apply the cell sheet in the cutout part to the transplantation site, which is convenient.
- the force that the corneal endothelial tissue is located in the innermost layer of the corneal tissue, and the other side of the cell sheet that is in contact with the support of the cell sheet are fixed to the jig, and the corneal endothelial tissue is fixed as it is. It can be transplanted by inserting into the tissue and leaving the cell sheet.
- the shape of the jig at that time is not particularly limited.
- a device provided with a suction port is easy to operate and convenient.
- the high engraftability to living tissue which is a feature of the regenerated corneal endothelial cell sheet in the present invention, is realized under specific culture conditions. That is, the cell sheet of the present invention can be obtained by culturing after seeding corneal endothelial cells on the surface of the support, but after 10 days after the cells become confluent (full state) on the surface of the support.
- the regenerated corneal endothelial cell sheet in the present invention is a high-density cell sheet having the original function of corneal endothelial tissue.
- the cell density in the 2 5 0 0 mm 2 or more preferably rather is 2 7 0 0 mm 2 or more, further not less 2 9 0 0 / mm 2 or more were found to be good preferable . If it is less than 2500 / mm 2 , a sufficient pump function cannot be realized, and high functionality, which is one of the features of the present invention, cannot be expected.
- the regenerated corneal endothelial cell sheet in the present invention is a cell sheet having a sufficient pumping function.
- the number of pump sites (Na ZK AT Pase pump sites) at that time is 3.4 X 10 9 mm 2 or more, preferably 3.8 X 10 9 pieces / mm 2 or more, and 4.2 It was found that X 10 9 pieces / mm 2 or more was preferable. 3.4 X 10 9 pieces Zmm 2 or less cannot exhibit a sufficient pumping function, and high functionality, which is one of the features of the present invention, cannot be expected.
- the regenerated corneal endothelial cell sheet in the present invention is a high-density cell sheet that can adhere extremely well to a living tissue and can sufficiently function as a corneal endothelial tissue. It was not obtained.
- the temperature-responsive polymer coated on the substrate is hydrated or dehydrated by changing the temperature, and the temperature range is 0 ° C to 80 ° C. It was found that the temperature was preferably 10 ° C to 50 ° C, more preferably 20 ° C to 45 ° C. If the temperature exceeds 80 ° C, the cells may die, which is not preferable. On the other hand, if it is lower than ⁇ , the cell growth rate is generally extremely reduced or the cells are killed, which is also not preferable.
- the temperature-responsive polymer used in the present invention may be either a homopolymer or a copolymer.
- a polymer described in Japanese Patent Application Laid-Open No. 2-211 885 is cited. Specifically, for example, it can be obtained by homopolymerization or copolymerization of the following monomers.
- monomers that can be used include (meth) acrylamide compounds ((meth) acrylamide means acrylic amide and methacrylamide. The same shall apply hereinafter.), ⁇ - (or ⁇ , ⁇ -di) alkyl substituted (meta ) Acrylamide derivatives or vinyl ether derivatives are listed, and in the case of copolymers, any two or more of them can be used.
- copolymerization with monomers other than the above monomers may be used. It is also possible to crosslink within a range that does not impair the original properties of the polymer.
- the base material to be coated substances such as glass, modified glass, polystyrene, polymethylmethacrylate, etc., which are usually used for cell culture, and other substances that can generally be given form, for example, polymer compounds other than those mentioned above , Ceramics Can be used.
- the method for coating the temperature-responsive polymer on the support is not particularly limited, but may be, for example, the method described in JP-A-2-211865. That is, such coating is performed by applying the electron beam irradiation (EB), r-ray irradiation, ultraviolet irradiation, plasma treatment, corona treatment, organic polymerization reaction, coating, kneading, etc. It can be carried out by physical adsorption or the like.
- EB electron beam irradiation
- r-ray irradiation ultraviolet irradiation
- plasma treatment corona treatment
- organic polymerization reaction organic polymerization reaction
- coating kneading, etc. It can be carried out by physical adsorption or the like.
- the coating amount of the temperature-responsive polymer is preferably in the range of 0.4 to 3.0 g / cm 2 , preferably 0.7 to 2.8 gZcm 2 , more preferably 0.9 to 2.5 gZcm. 2 .
- the coating amount is less than 0.4 gZcm 2 , the cells on the polymer are difficult to detach even when stimulated, and the working efficiency is remarkably deteriorated. Conversely, if it is 3. O ⁇ gZcm 2 or more, it is difficult for cells to adhere to that region, and it becomes difficult to attach cells sufficiently.
- the form of the support in the present invention is not particularly limited, and examples thereof include a dish, a multiplate, a flask, and a cell insert.
- the medium temperature is not particularly limited as long as it is performed at a temperature at which the polymer coated on the substrate surface is dehydrated. However, it goes without saying that culturing in a low temperature range where cultured cells do not proliferate or in a high temperature range where cultured cells die is inappropriate. Culture conditions other than temperature are not particularly limited as long as conventional methods are followed.
- the medium to be used may be a medium to which a serum such as a known urine fetal serum (FCS) is added, or a serum-free medium to which such a serum is not added.
- FCS urine fetal serum
- the cultured regenerated corneal endothelial cell sheet is brought into close contact with the carrier, and the temperature of the support material to which the cells are attached is coated with the support substrate.
- the temperature of the support material to which the cells are attached is coated with the support substrate.
- separation may be performed smoothly by applying a water flow between the cell sheet and the support.
- the sheet can be peeled off in the culture medium in which the cells have been cultured, or in other isotonic solutions, and can be selected according to the purpose.
- Poly (N-isopropylpropylamine) as a temperature-responsive polymer This will be explained with reference to the above example.
- Poly (N-isopropylacrylamide) is known as a polymer with a lower critical solution temperature at 31 ° C.
- the polymer on the substrate surface will be dehydrated in the same way, but the polymer surface will be hydrophobic and coated because the polymer chain is coated and fixed on the substrate surface. become.
- the polymer on the substrate surface hydrates but the polymer chain is coated and fixed on the substrate surface so that the substrate surface is hydrophilic.
- the hydrophobic surface is an appropriate surface on which cells can attach and proliferate, and the hydrophilic surface becomes a surface on which cells cannot adhere, and also cools cells or cell sheets in culture. It will be peeled off only.
- the cell sheet of the present invention contains cells at a high density.
- the production method is not particularly limited. However, since corneal endothelial cells do not proliferate rapidly and with high density, for example, the cells are subcultured in advance several times to reach a predetermined total number of cells. A method of seeding all the cells in a predetermined area. At that time, in order to increase the cell concentration in the cell dispersion liquid, it may be concentrated by centrifugation, or the number of cells per unit area may be increased by reducing the culture area of the substrate.
- the substrate used for cell subculture is not particularly limited. For example, when cultured on an adhesive protein such as collagen IV, collagen I, collagen EL laminin, fibronectin, or matrigel, the morphology of corneal endothelial cells Is convenient without breaking down.
- the number of cells per unit area at the time of seeding is preferably 200 or more Zmm 2 or more, preferably 2 300 or more / mm 2 or more, more preferably 2500 or more Zmm 2 or more. . When it is less than 200 mm 2 , it becomes difficult to make the cell density of the obtained regenerated corneal endothelial cell sheet 2500 or more Zmm 2 or more.
- the cell sheet after the cell sheet is applied to the affected area, the cell sheet may be peeled off from the carrier.
- peel it off For example, a method of wetting the carrier to weaken the adhesion between the carrier and the cell sheet, or using a knife such as a scalpel, scissors, laser light, or plasma wave. It may be used or may be cut.
- a knife such as a scalpel, scissors, laser light, or plasma wave. It may be used or may be cut.
- the cell sheet may adhere to an extra area other than the affected area. Avoidable and convenient.
- the method for fixing the regenerated corneal endothelial cell sheet and the living tissue shown in the present invention is not particularly limited, and the cell sheet and the living tissue may be sutured, or the regenerated corneal endothelium as shown in the present invention. Since the cell sheet quickly engrafts with the living tissue, the cell sheet attached to the affected part does not need to be sutured to the living body side.
- the cell culture support is tapped or shaken, the medium is stirred using a pipette, the cell sheet and the substrate A method of applying a water flow between the two may be used alone or in combination.
- the cultured cells may be detached and collected by washing with an isotonic solution or the like as necessary.
- the use of the regenerated corneal endothelial cell sheet shown in the present invention is not limited at all, but is effective for, for example, corneal endothelial dysfunction and bullous keratopathy.
- the regenerated corneal endothelial cell sheet obtained by the above method is extremely superior in that it is non-invasive at the time of exfoliation and has higher functionality than those obtained by the conventional method.
- the clinical application as an endothelial sheet is strongly expected.
- the regenerated corneal endothelial cell sheet of the present invention has a high engraftability with living tissue, unlike conventional transplantation sheets, and so engrafts in living tissue very quickly. This is considered to be an extremely effective technology that improves the treatment efficiency of the affected area and reduces the burden on the patient.
- the cell culture support used in the method of the present invention can be used repeatedly.
- the culture dish is washed with ion-exchanged water to remove residual monomers and PI PAAm not bound to the culture dish, dried in a clean bench, and sterilized with ethylene oxide gas to support the cell culture. Obtained material.
- the coating amounts of PI PAAm were found to be 1.6 ug / cm 2 (Example 1) and 1.8 g / cm 2 (Example 2), respectively.
- corneal endothelial tissue was collected from the periphery of the white rabbit cornea under deep anesthesia by a conventional method, and the corneal endothelial cells were cultured for 5 passages according to a conventional method using a flask coated with collagen IV ( Medium used: DMEM, 10% FCS, 37 ° C, 10% CO 2 ). As a result, 4 ⁇ 10 6 corneal endothelial cells were finally recovered.
- the cultured cells are cut out in a circular shape with a diameter of 1. 8 cm. 2. Covered with a carrier molded from a 3 cm polypinidene difluoride (PVDF) membrane, the medium is gently aspirated, and the cell culture support By incubating the whole material at 20 minutes for 30 minutes and cooling, the cells on any cell culture support material were detached with the overlying carrier.
- the obtained cell sheet was sufficiently strong as a single sheet having a shrinkage rate of 5% or less.
- the cell density in the obtained cell sheet was 3000 cells / mm 2 .
- low temperature treatment was performed under the condition of incubation at 20 ° C. for 30 minutes.
- “low temperature treatment” is not limited to these temperatures and times.
- a preferable temperature condition for the “low temperature treatment” in the present invention is 0 ° C. to 30 ° C., and a preferred treatment time is 2 minutes to 1 hour.
- the regenerated corneal endothelial cell sheet obtained in Examples 1 and 2 was transplanted into a white rabbit lacking the corneal endothelial tissue part. At that time, it is in contact with the support of the regenerated corneal endothelial cell sheet. The other side was sucked and fixed with a jig having a suction surface with the same curvature as that of the corneal endothelial tissue in the living body, and the carrier was excised with a scalpel. The regenerated corneal endothelial cell sheet was pressed against the wound using a jig, the jig was aspirated, and allowed to adhere for 15 minutes.
- Example 3 the regenerated corneal endothelial cell sheet and the living body were not sutured. Finally, according to a conventional method, the cut corneal tissue was sutured to the eyeball. After 3 weeks, when the affected area was observed, the regenerated corneal endothelial cell sheet in both Example 2 satisfactorily adhered to the eyeball, and no swelling of the cornea was observed.
- Example 3
- an acrylamide monomer containing N, N-methylenbisacrylamide (lwt% / acrylamide monomer) on top of this was dissolved in isopropyl alcohol to 5 wt%.
- Apply 0.1 ml of the solution put on a metal mask with a diameter of 1.8 cm, and irradiate the electron beam with an intensity of 0.25 MGy as it is.
- acrylamide polymer (PAAM) was immobilized.
- the culture dish is washed with ion-exchanged water to remove residual monomers and PA Am not bound to the culture dish, dried in a clean bench, and sterilized with ethylene oxide gas to obtain the cell culture support material. Obtained.
- corneal endothelial tissue was collected from the white rabbit cornea periphery under deep anesthesia, and finally subcultured 4 times to obtain 7.6 X 10 5 Corneal mesothelial cells could be recovered.
- all these cells were seeded on the cell culture support material and continued to culture for 3 weeks.
- a carrier molded from a polyvinylidene difluoride (PVDF) film having a diameter of 2.3 cm cut out into a circular shape having a diameter of 1.8 cm is placed on the cultured cells in the same manner as in Example 1.
- PVDF polyvinylidene difluoride
- the obtained cell sheet was sufficiently strong as one sheet having a shrinkage rate of 5% or less.
- the cell density in the obtained cell sheet was 2800 cells / mm 2 .
- the regenerated corneal endothelial cell sheet thus obtained was deficient in the corneal endothelial tissue as in Example 1. Transplanted into white rabbits. Three weeks later, when the affected area was observed, the regenerated corneal endothelial cell sheet satisfactorily engrafted in the eyeball, and no swelling of the cornea was observed. Comparative Example 1
- a corneal endothelial cell sheet was produced in the same manner as in Example 2, except that a corneal endothelial cell sheet was prepared in Example 2 and the cell sheet was detached and contracted without using a carrier. The shrinkage at that time was 42%.
- the corneal endothelial cell sheet obtained in the same manner as in Example 2 was transplanted to a rabbit which had a corneal endothelial tissue defect.
- the corneal endothelial cell sheet was slightly poorly engrafted in the eye and swelling of the cornea was also observed. Comparative Example 2
- Example 4 An attempt was made to produce a regenerated corneal endothelial cell sheet in the same manner as in Example 3, except that the corneal endothelial cells were cultured in Example 3 and the period from the culturing to the cell culture support was 9 days later. As in Example 3, the regenerated corneal endothelial cell sheet was attempted to be peeled off, but could only be partly peeled, which was insufficient as a cell sheet.
- the corneal endothelial cells collected from the periphery of the human cornea by the same method as in Example 3 were applied to the cell culture support material obtained by placing the metal mask having a diameter of 1.8 cm in Example 3 Example 3
- the cells were seeded on the cell culture support material and cultured for 4 weeks. After culturing, the cell culture support material is incubated for 30 minutes at 20 ° C for 30 minutes while using a polyvinylidene difluoride (P VD F) carrier and cooled, so that the endothelial cells on the cell culture support material are cooled.
- P VD F polyvinylidene difluoride
- the sheet was peeled off.
- the obtained endothelial cell sheet was sufficiently strong as a single sheet having a shrinkage rate of 5% or less.
- FIG. 1 shows the resulting endothelial cell sheet with the carrier removed. It can be seen that the human regenerated corneal endothelial cell sheet is suspended in the culture support material.
- FIG. 1 Human regenerated corneal endothelial cell sheet in culture 1 day after culture (A), 3 days after (B), 7 Figure 2 shows the results of staining of collagen IV (upper figure) and fibronectin (lower figure), which were taken out after 14 days (C) and 14 days later (D), respectively, according to a conventional method. It can be seen that collagen IV and fibronectin accumulate as the number of culture days increases.
- the Z01-1 protein present in the regenerated corneal endothelial cell sheet was immunofluorescently stained.
- Figure 3 It can be seen that this protein is localized between cells, and from this result, the regenerated corneal endothelial cell sheet obtained from the present invention forms a bond between cells, and the formation is peeled off. It can be seen that it remains without being destroyed.
- Fig. 4 shows the results of staining collagen IV (left figure) and fibronectin (right figure) present in the human regenerated corneal endothelial cell sheet in the middle of detachment according to a conventional method.
- the regenerated corneal endothelial cell sheet obtained as shown in this figure had collagen IV and fibronectin.
- Fig. 5 shows the results of HZE staining of the human regenerated corneal endothelial cell sheet after detachment (left figure), and results of staining for collagen IV (middle figure) and fibronectin (right figure) present in the cell sheet according to conventional methods.
- the human regenerated corneal endothelial cell sheet obtained in the present invention is a single layer as in the normal state in vivo. Collagen IV showed that the endothelial cell sheet was in contact with the support. It can be seen that fibronectin is localized between cells.
- FIG. 6 shows the results of observation of a regenerated corneal cell sheet prepared in the same manner with 2% dartalaldehyde, and then stained with osmium acid with a transmission electron microscope.
- the obtained regenerated corneal cell sheet had a tissue similar to that in vivo.
- Example 1 corneal endothelial cells collected from the periphery of the human cornea by the same method as in Example 3 are seeded on the cell culture support material in the same manner as in Example 3. The culture was continued for 4 weeks. After incubation, polyvinylidene difluor The endothelial cell sheet on the cell culture support material was detached by incubating the whole cell culture support material at 20 ° C for 30 minutes and cooling without using a ride (PVDF) carrier. Proteins present on the surface layer of the obtained endothelial cell sheet and Zo-1 protein involved in cell-cell binding were extracted according to a conventional method and confirmed by SDS-PAGE. The result is shown as T in FIG.
- PVDF ride
- Example 5 the culture of human corneal endothelial cells was continued for 4 weeks in the same manner except that the culture was performed on a commercially available culture substrate not covered with a temperature-responsive polymer.
- the dispase treatment which is a conventional method, is used to remove the cells (Comparative Example 3), and the other is physically separated using a rubber scraper. (Comparative Example 4) was performed.
- the cells obtained by each method were extracted in the same manner as in Example 5 by extracting the proteins present on the surface layer of the endothelial cell sheet and the ZO-1 protein involved in cell-cell binding according to conventional methods. It was confirmed by the law.
- Example 3 The number of pumps per cell before and after exfoliation in Example 3 was calculated from the total amount of ouabain bound to the cell sheet and the cell density of the cell sheet.
- the number of pumps before exfoliation was 3.5 X 10 6
- the number of pumps after peeling was 3.5 ⁇ 10 6 pieces.
- Example 3 The corneal endothelial cells collected from the periphery of the human cornea by the same method as in Example 3 were applied to the cell culture support material obtained by placing a metal mask having a diameter of 1.8 cm in Example 3.
- Example 3 In the same manner as above, the cells were seeded on the cell culture support material and cultured for 4 weeks.
- the human regenerated corneal endothelium sheet before peeling was stained with an anti-rabbit Na—K ATP as monoclonal antibody, and the Na—K ATP as pump site was stained green. At that time, the cell nuclei were stained red with p op op i um i od i d e.
- the results obtained using the confocal microscope are shown in FIG.
- a in the upper figure shows the result of observation from the upper surface of the cultured cell sheet, and B shows the result of observation in the thickness direction.
- B shows the result of observation in the thickness direction.
- Example 6 Cells using the human corneal endothelial cells, were cultured operating in the same manner as in Example 6 except that the cultured to a cell density in the cell sheet is 575 pieces Roh mm 2 ⁇ 3070 amino ZMM 2, Example 6 In the same way as above, the number of Na _K ATPase pump sites was measured by measuring the amount of 3 H-labeled ouabain bound in a liquid scintillation county. Na—K ATPa se pump site number and cell density From the above, the number of pumps per cell of the regenerated corneal endothelial cell sheet exfoliated was calculated. The obtained results are shown in FIG.
- a in the figure shows the correlation of the number of pumps per cell to the cell density
- B in the figure shows the correlation of the number of pumps per unit area to the cell density. From Fig. A, it was found that the number of pumps per cell decreased as the cell density increased, and from Fig. B, the number of pumps per unit area increased as the cell density increased. It was revealed that the number of pump sites of the present invention was reached by setting the cell density to 2500 Zmm 2 . Comparative Example 5
- the culture dish is washed with ion-exchanged water to remove residual monomer and PAAm that is not bound to the culture dish, dried in a clean bench, and sterilized with ethylene oxide gas.
- coated part in the support was obtained.
- the regenerated corneal endothelium sheet before exfoliation, and collagenase treatment and exfoliation were performed in the same manner as in Example 4, and then again a commercially available 3.5 cm cell culture culture dish (FALCON 3001).
- FALCON 3001 a commercially available 3.5 cm cell culture culture dish
- the number of pumps per cell before and after detachment was calculated.
- the number of pumps before detachment was 3.5 ⁇ 10 6
- the number of pumps after detachment was 1.5 ⁇ 10 6 .
- the cell damage that occurred during detachment was significant. Industrial applicability
- the regenerated corneal endothelial cell sheet obtained by the present invention has extremely high engraftment properties in living tissues and is highly functional. For example, clinical applications such as corneal endothelial disease treatment are strongly expected. did Therefore, the present invention is extremely useful in the fields of medicine, biology, etc., such as cell engineering and medical engineering.
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Abstract
Description
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Priority Applications (5)
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US10/546,275 US9981064B2 (en) | 2003-02-20 | 2004-02-20 | Regenerated corneal endothelial cell sheets, processes for producing the same, and methods of using the same |
KR1020057015310A KR101228251B1 (ko) | 2003-02-20 | 2004-02-20 | 재생 각막 내피 세포 시트, 제조 방법 및 그 이용 방법 |
JP2005502785A JP4335873B2 (ja) | 2003-02-20 | 2004-02-20 | 再生角膜内皮細胞シート、製造方法及びその利用方法 |
EP04713172.7A EP1600177B1 (en) | 2003-02-20 | 2004-02-20 | Endothelial cell sheet for cornea regeneration, method of producing the same and method of using the same |
CN2004800046423A CN1753696B (zh) | 2003-02-20 | 2004-02-20 | 再生角膜内皮细胞片、制造方法及其使用方法 |
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JP2003-089300 | 2003-02-20 | ||
JP2003089300 | 2003-02-20 |
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EP (1) | EP1600177B1 (ja) |
JP (1) | JP4335873B2 (ja) |
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002010349A1 (en) * | 2000-07-21 | 2002-02-07 | Cellseed Inc. | Cultured epidermal cell sheet, laminated cultured skin sheet and process for producing the same |
JP2003038170A (ja) * | 2001-07-26 | 2003-02-12 | Mitsuo Okano | 前眼部関連細胞シート、3次元構造体、及びそれらの製造法 |
Family Cites Families (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4842599A (en) * | 1986-10-28 | 1989-06-27 | Ann M. Bronstein | Prosthetic cornea and method of implantation therefor |
AU6242298A (en) | 1997-01-17 | 1998-08-07 | Celadon Science, Llc | Methods for promoting healing of corneal resurfacing wounds |
EP1264877B1 (en) | 2000-03-16 | 2013-10-02 | Cellseed Inc. | Cell cultivation-support material, method of cocultivation of cells and cocultivated cell sheet obtained therefrom |
ATE495766T1 (de) | 2000-07-21 | 2011-02-15 | Cellseed Inc | Herzmuskel-ähnliche zellschicht, dreidimensionales konstrukt, herzmuskel-ähnliches gewebe und verfahren zur herstellung |
EP1600498B1 (en) | 2003-02-06 | 2012-08-15 | Cellseed Inc. | Substrates for high-density cell arrays and preparation processes and uses thereof |
KR101203434B1 (ko) | 2003-02-06 | 2012-11-21 | 가부시키가이샤 셀시드 | 전안부 관련 세포 시트, 3 차원 구조체, 및 그들의 제조법 |
EP1602383B1 (en) | 2003-02-06 | 2015-05-27 | Cellseed Inc. | Cell sheets for ectocornea formation, method of producing the same and method of using the same |
KR101228251B1 (ko) | 2003-02-20 | 2013-01-30 | 가부시키가이샤 셀시드 | 재생 각막 내피 세포 시트, 제조 방법 및 그 이용 방법 |
US9587222B2 (en) | 2003-08-01 | 2017-03-07 | Cellseed Inc. | Three-dimensional tissue structure |
EP1731030B1 (en) | 2004-03-04 | 2017-06-21 | CellSeed Inc. | Method of constructing animal having cancer cells transplanted thereinto |
JP4827729B2 (ja) | 2004-04-25 | 2011-11-30 | 株式会社セルシード | 培養歯根膜細胞シート、製造方法及びその利用方法 |
JPWO2006093153A1 (ja) | 2005-02-28 | 2008-08-07 | 株式会社セルシード | 培養細胞シート、製造方法及びその利用した組織修復方法 |
EP1857126B1 (en) | 2005-02-28 | 2018-10-24 | CellSeed Inc. | Cultured cell sheet, production method thereof, and application method thereof |
US9598668B2 (en) | 2008-10-14 | 2017-03-21 | Cellseed Inc. | Temperature-responsive cell culture substrate and method for producing the same |
WO2010134606A1 (ja) | 2009-05-22 | 2010-11-25 | 学校法人 東京女子医科大学 | 胚性幹細胞或いは人工多能性幹細胞の分化誘導方法 |
IN2012DN01268A (ja) | 2009-08-02 | 2015-05-15 | Univ Tokyo Womens Medical | |
JPWO2011024963A1 (ja) | 2009-08-27 | 2013-01-31 | 学校法人東京女子医科大学 | 直鎖型温度応答性高分子が固定化された温度応答性細胞培養基材、及びその製造方法 |
-
2004
- 2004-02-20 KR KR1020057015310A patent/KR101228251B1/ko active IP Right Grant
- 2004-02-20 WO PCT/JP2004/001975 patent/WO2004073761A1/ja active Application Filing
- 2004-02-20 CN CN2004800046423A patent/CN1753696B/zh not_active Expired - Lifetime
- 2004-02-20 JP JP2005502785A patent/JP4335873B2/ja not_active Expired - Lifetime
- 2004-02-20 EP EP04713172.7A patent/EP1600177B1/en not_active Expired - Lifetime
- 2004-02-20 US US10/546,275 patent/US9981064B2/en active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002010349A1 (en) * | 2000-07-21 | 2002-02-07 | Cellseed Inc. | Cultured epidermal cell sheet, laminated cultured skin sheet and process for producing the same |
JP2003038170A (ja) * | 2001-07-26 | 2003-02-12 | Mitsuo Okano | 前眼部関連細胞シート、3次元構造体、及びそれらの製造法 |
Non-Patent Citations (4)
Title |
---|
KIKUCHI AKIHIKO ET AL.: "Two-dimensional manipulation of confluently cultured vascular endothelial cells using temperature-responsive poly(N-isopropylacylamide)-grafted surfaces", J. BIOMATER. SCI. POLYMER. EDN., vol. 9, no. 12, 1998, pages 1331 - 1348, XP000971027 * |
NISHIDA KOJI: "Tissue engineering for cornealepithelium and corneal endothelium", BAIOMATERIARU - JOURNAL OF JAPANESE SOCIETY FOR BIOMATERIALS, vol. 20, no. 4, 15 July 2002 (2002-07-15), pages 259 - 268, XP002903782 * |
See also references of EP1600177A4 * |
YAMATO MASAYUKI ET AL.: "Creation of cell sheet engineerin", BAIOMATERIARU - JOURNAL OF JAPANESE SOCIETY FOR BIOMATERIALS, vol. 21, no. 1, 15 January 2003 (2003-01-15), pages 46 - 52, XP002903783 * |
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WO2007083685A1 (ja) * | 2006-01-19 | 2007-07-26 | Senju Pharmaceutical Co., Ltd. | 生体内で細胞増殖可能な角膜内皮製剤 |
JP5255846B2 (ja) * | 2006-01-19 | 2013-08-07 | 千寿製薬株式会社 | 生体内で細胞増殖可能な角膜内皮製剤 |
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JP2008011766A (ja) * | 2006-07-05 | 2008-01-24 | Mitsuo Okano | 細胞培養支持体 |
WO2011021706A1 (ja) | 2009-08-19 | 2011-02-24 | 国立大学法人東北大学 | 角膜移植用シート |
JP2012157445A (ja) * | 2011-01-31 | 2012-08-23 | Kawamura Institute Of Chemical Research | 角膜内皮細胞欠損治療用ゲルフィルム |
US9347041B2 (en) | 2011-07-15 | 2016-05-24 | Osaka University | Method for preparing corneal endothelial cell |
JP5946046B2 (ja) * | 2012-12-27 | 2016-07-05 | 新田ゼラチン株式会社 | ヒト角膜内皮細胞シート |
JP2015029464A (ja) * | 2013-08-02 | 2015-02-16 | 大日本印刷株式会社 | 細胞シート移送のための方法および器具 |
JP2015029462A (ja) * | 2013-08-02 | 2015-02-16 | 大日本印刷株式会社 | 細胞シートの製造および移送のための細胞培養器具 |
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US20070148137A1 (en) | 2007-06-28 |
JPWO2004073761A1 (ja) | 2006-06-01 |
CN1753696B (zh) | 2010-04-28 |
KR20060006003A (ko) | 2006-01-18 |
EP1600177A1 (en) | 2005-11-30 |
EP1600177B1 (en) | 2016-05-25 |
KR101228251B1 (ko) | 2013-01-30 |
CN1753696A (zh) | 2006-03-29 |
US9981064B2 (en) | 2018-05-29 |
EP1600177A4 (en) | 2011-03-02 |
JP4335873B2 (ja) | 2009-09-30 |
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