WO2006075602A1 - 羊膜を用いたシート状組成物及びその作製方法 - Google Patents
羊膜を用いたシート状組成物及びその作製方法 Download PDFInfo
- Publication number
- WO2006075602A1 WO2006075602A1 PCT/JP2006/300189 JP2006300189W WO2006075602A1 WO 2006075602 A1 WO2006075602 A1 WO 2006075602A1 JP 2006300189 W JP2006300189 W JP 2006300189W WO 2006075602 A1 WO2006075602 A1 WO 2006075602A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- sheet
- cells
- amniotic membrane
- epithelium
- cell
- Prior art date
Links
Classifications
-
- 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
-
- 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
-
- 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
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/40—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing ingredients of undetermined constitution or reaction products thereof, e.g. plant or animal extracts
-
- 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
- A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
-
- 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
- A61L27/3683—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
Definitions
- the present invention relates to a sheet-like composition using amniotic membrane and a method for producing the same.
- the sheet-like composition of the present invention can be used, for example, as a transplant material for reconstructing the ocular surface and skin.
- amniotic membrane is the innermost tissue of the placenta in the mammal's womb, and is a layer of membrane that wraps the fetus.
- amniotic membrane is extremely excellent as a culture substrate for various types of cells.
- cultured cell sheets such as cultured corneal epithelial cell sheet, cultured oral mucosal epithelial sheet, and cultured epidermal cell sheet using amniotic membrane as a carrier are known. (See, for example, Patent Documents 1 to 3).
- Patent Document 1 Pamphlet of International Publication No. 03Z043542 A1
- Patent Document 2 Pamphlet of International Publication No. 03Z092762 A1
- Patent Document 3 International Publication No. 2004Z078225 A1 Pamphlet
- necrotic cells and bacteria When necrotic cells and bacteria are generated, the living body tries to remove them, and cells of the immune system such as Langeron cells, rod cells, macrophages and leukocytes are activated and cause inflammation. In addition, growth factors are released from macrophages, causing angiogenesis. Inflammation can cause redness, tears, eyes, fever, redness, and itching. When angiogenesis occurs in the retinal region, it can lead to vision loss. The occurrence of such physiological effects is very inconvenient in the clinical setting, such as reducing the original therapeutic effect or requiring additional treatment in some cases. It was a big issue when transplanting to the other. On the other hand, the operation of suturing is complicated and requires an operator who requires experience and skill.
- fibrin glue (Tisseal; Baxter, Bolheel; Yi Blood Research Institute, Veriplast; Aventis Pharma) has been mainly used as a material used to adhere a substance to a living body without stitching. It has been used.
- Fibrin glue is a drug that adheres and closes tissues by mimicking the final stage of blood clotting in vivo.
- the surgeon separately purchases fibrin glue as a drug and prepares it during surgery.
- it takes time and labor to prepare a fibrin glue and it is not easy to use it as an adhesive for adhering difficult-to-handle sheets to tissues because it has a property of solidifying relatively easily.
- octopus kombu (Nicomedo alma) exists as a product that bonds a substance to a living body without stitching.
- This product is used as a damage filler and is a pharmaceutical product in which a glue is fixed on one side using a collagen sheet as a carrier. Since the product has already been coated with glue, the adhesion to the tissue can be completed simply by bringing the octopus comb into contact with the affected area, which requires no preparation time and labor.
- what is used as a carrier of octopus kombu Since it is a chemical substance made by reconstituting collagen, it does not directly adhere to living tissues such as amniotic membrane sheets and cultured cell sheets.
- this sheet-like structure has a thickness of several millimeters and is not transparent. Therefore, it cannot be applied to applications that require sufficient transparency after transplantation (for example, reconstruction of the ocular surface).
- the transplanted part is poor in physical space for epithelial tissue reconstruction, etc., so the transplanted material is required to be thin. . In this respect, the above products are not appropriate.
- the present invention provides a sheet-like composition that can be applied to a wide range of uses such as reconstruction of the ocular surface, skin or epidermis, and covering at the time of a burn, and can be transplanted by simple transplantation. Objective.
- the present inventors have decided to use amniotic membrane as a support used in constructing a sheet-like composition. Then, fibrin and thrombin were attached to the surface of the amnion which improves the adhesion and engraftment after transplantation. As a result, the obtained sheet-like composition was very thin and high transparency could be secured. Moreover, it had sufficient strength. On the other hand, when the sheet-like composition was applied to the ocular surface in order to confirm the adhesion and engraftment of the sheet-like composition, rapid adhesion was observed, and good adhesion was maintained over a long period of time. It was done.
- the sheet-like composition constituted by attaching fibrin and thrombin to the surface of the amniotic membrane was excellent in transparency, strength, adhesion and engraftment. That is, it was found that the sheet-like composition satisfies the conditions necessary for application to a wide range of uses. In addition, since it has high adhesiveness and engraftment, it was thought that this could realize a simple transplantation technique without using sutures. If sutures are not necessary, the problems of inflammation and angiogenesis that were problematic when sutures are performed are eliminated. In addition, the labor and time required for transplantation into a living body can be saved, and the range of surgeons who can perform sheet transplantation is expanded, greatly contributing to the development of regenerative medicine. It is expected that On the other hand, since the obtained sheet-like composition has high transparency, it has been found that it can be suitably used for applications requiring high transparency, such as reconstruction of the ocular surface.
- the present invention is based on the above results, and has the following constitutional power.
- a sheet-like composition comprising amniotic membrane with fibrinogen and thrombin attached to the surface.
- the adhesion surface of fibrinogen and thrombin in the amniotic membrane is in a dry state.
- substantially the entire amniotic membrane is in a dry state.
- one embodiment of the present invention is characterized in that aprotune is further attached to the surface of the amniotic membrane.
- amniotic membrane from which the epithelium has been removed can be used as the amniotic membrane.
- the absence of an epithelial component results in a sheet-like composition having further reduced immunogenicity.
- a cell layer that is a living cell-derived cell force is formed on the amniotic membrane, and the surface of the amniotic membrane opposite to the surface on which the cell layer is formed is an adhesion surface for fibrinogen or the like.
- the cultured cell sheet of this embodiment is a transplant sheet having a high therapeutic effect due to the action of contained cells.
- the cells forming the cell layer are, for example, cells derived from the corneal epithelium, conjunctival epithelium, skin epidermis, follicular epithelium, oral mucosa epithelium, iris pigment epithelium, retinal pigment epithelium, airway mucosa epithelium or intestinal mucosa epithelium.
- the adhesion amount of thrombin to the amnion amniotic lcm 2 per 1 G-L mg and child And are preferred. This is because it is possible to reduce inflammation and angiogenesis after transplantation while maintaining high adhesiveness.
- the sheet-like composition of the present invention can be prepared to a thickness of 10 m to 200 m by using amniotic membrane.
- the sheet-like composition of the present invention has adhesiveness to human tissue by attaching thrombin or the like in advance, and good adhesive force can be obtained without suturing at the time of transplantation.
- the present invention also provides a method for producing the above sheet-like composition. Specifically, the following configuration is provided.
- a method for producing a sheet-like composition comprising the steps of (a) preparing an amniotic membrane and (b) attaching fibrinogen and thrombin to the surface of the amniotic membrane.
- step c a drying process is performed.
- the drying treatment is carried out on the entire composition or only the adherent surface of fibrinogen, etc., depending on the intended use.
- the step of attaching aprochon to the surface of the amniotic membrane is performed. It is preferable to perform the step simultaneously with the fibrinogen and thrombin attachment step.
- step a it is preferred to carry out the step of removing the epithelium from the amniotic membrane (step a-1).
- the sheet-like composition is produced using amniotic membrane from which the epithelium has been removed.
- step a-2 a step of drying the amniotic membrane (step a-2) can be performed. That is, in this embodiment, the amniotic membrane is dried during the production process of the sheet-like composition, and used in the subsequent steps.
- step A a step (step A) of forming a cell layer having a biological cell-derived cell force on the amniotic membrane is performed between step a and step b.
- step A a cell layer is formed on the amniotic membrane, and finally a cultured cell sheet is produced.
- FIG. 1 shows an apparatus for culturing oral mucosal epithelial cells and corneal epithelial cells on amniotic membrane. It is sectional drawing which showed the state of these etc. typically.
- Culture insert 2 is allowed to stand in culture dish 1, and 3T3 cell layer 5 is formed on the bottom of culture dish 1.
- the amniotic membrane 3 is allowed to stand on the bottom surface of the culture insert 2 and the oral mucosal epithelial cells and corneal epithelial cells 4 are cultured thereon.
- Reference numeral 6 denotes a culture medium.
- FIG. 2 is a table summarizing experimental results on the relationship between the adhesion amount of adhesive components and adhesiveness. Adhesion is indicated by + (the sheet does not slip or peel off) and-(the sheet slips).
- a first aspect of the present invention relates to a sheet-like composition.
- amniotic membrane is used as one of the main components. Due to the high transparency and toughness of the amniotic membrane, a sheet-like composition excellent in transparency and strength can be constituted.
- the high amniotic membrane biocompatibility and low immunogenicity, it is possible to construct a sheet-like composition that is more excellent in biocompatibility and low in immunogenicity.
- the use of amniotic membrane can be expected to have anti-inflammatory effects, suppression of trace formation, and inhibition of angiogenesis.
- amniotic membrane is also preferable in the case where the sheet-like composition of the present invention includes a cell layer, so that the cell layer is favorably formed. That is, as described later, a sheet-like composition having a cell layer is formed by seeding and culturing predetermined cells on an amniotic membrane as a substrate (support), and the amniotic membrane has cells on it. The ability to adhere and proliferate well enables the use of amniotic membrane to allow good cell adhesion, proliferation, and cell layer formation.
- the amniotic membrane is one from which the epithelium has been removed by reptile treatment or the like. Epithelialization It is also a power to become a sheet-like composition having further reduced immunogenicity due to the absence of the component. In addition, in the amniotic membrane from which the epithelium has been removed, the cells adhere and proliferate better, so that a high quality corneal epithelium-like sheet can be constructed in a shorter time. It is done.
- amniotic membrane from which the epithelium has been removed can be confirmed by examining that the cells in the amniotic epithelial layer are not contained in the sheet composition. It is particularly preferable to use human amniotic membrane as the amniotic membrane.
- fibrinogen and thrombin are attached to the surface of the amniotic membrane.
- fibrinogen is first specifically hydrolyzed by thrombin to produce fibrin, and then fibrin is polymerized to form stable fibrin. It becomes a lump and exhibits an adhesive action.
- Fibrinogen and thrombin are attached to one or both sides of the amniotic membrane according to the use of the sheet-like composition of the present invention.
- the surface of the amniotic membrane on the side of the chorion that is, the surface opposite to the epithelium side
- the sheet-shaped composition having such a structure it is transplanted to the application site with the side where the epithelium of the amniotic membrane was present facing up.
- the sheet-like composition of the present invention is in an appropriate state in consideration of the purpose of use and the like.
- the sheet-like composition of the present invention includes those to which fibrin or fibrin clots produced due to such causes are attached.
- fibrinogen and thrombin can be prepared using blood such as humans, monkeys, chimpanzees, horses, horses, hidges, and pigs as materials.
- fibrinogen and thrombin recombinants (recombinants) obtained using cultured cells (eg, CHO cells and COS cells) are used. May be used. It is preferable to use fibrinogen and thrombin derived from humans (particularly human-derived thread-reversed forms). It is also an advantageous force in terms of safety, including immunogenicity. In view of the point that stable quality can be used and the problem of infection, it is particularly preferable to use a recombinant.
- fibrinogen and thrombin derived from the blood of a patient (recipient) receiving the transplant of the sheet-like composition of the present invention are particularly preferable to use. This is because there is no risk of inducing immune rejection caused by these adhesive components.
- fibrinogen and thrombin origins of fibrinogen and thrombin are not necessarily the same.
- human blood-derived fibrinogen and sushi blood-derived thrombin can be used in combination.
- the amount of fibrinogen and thrombin attached is not particularly limited.
- the amount of fibrinogen attached can be set in the range of 0.1 mg to 50 mg per 1 cm 2 of amniotic membrane.
- the adhesion amount of thrombin can be set in the range of 0.5 mg to 10 mg per 1 cm 2 of amniotic membrane.
- the adhesive strength is considered first. That is, it is necessary to set the adhesion amounts of these components so that the expected adhesion amount can be obtained. On the other hand, if the amount of fibrinogen or thrombin attached is too large, the problem is that it tends to induce an immune reaction or angiogenesis, depending on the origin of the fibrinogen used.
- angiogenesis due to these components becomes a problem after transplantation, for example, in the case of a sheet-like composition applied to reconstruction of the ocular surface, in order to suppress the initiation of angiogenesis as much as possible, It is preferable to reduce the adhesion amount of these components.
- the adhesion amount of these components By setting the adhesion amount of these components as small as possible, angiogenesis after transplantation can be suppressed and a high therapeutic effect can be expected.
- the amount of fibrinogen attached was about 0.5 mg or more per lcm 2 of amniotic membrane, a good adhesive force to the ocular surface was obtained. .
- a preferable range of the amount of thrombin attached is 1 g to 1 mg per lcm 2 of amniotic membrane, 5 g to 200 g per lcm 2 of amniotic membrane, and a more preferable range is 10 g to 100 g per lcm 2 of amniotic membrane (specifically For example, about 10 g, about g, and about 30 g) can be mentioned.
- aprotun is attached to the amniotic surface in the form of fibrinogen and thrombin.
- Aprotun inhibits the fibrin clot formed by the action of thrombin from being dissolved by plasmin. Therefore, by using aprotune together, the fibrin clots can be prevented from decomposing and the adhesion can be maintained or enhanced.
- aprotinin is not particularly limited.
- aprochons derived from the spleen such as ushi, horse, hidge, pig, monkey, chimpanzee and the like can be used.
- the amount of adhesion is not particularly limited.
- the amount of aprotinin attached can be set in the range of 0.1 KIU to 200 KIU per lcm 2 of amniotic membrane.
- the amount of adhesion was changed by changing the amount of adhesion with aprochon.
- the amount of aprochon was set in the range of 1 KIU to 2 KIU Even so, sufficient adhesion to the ocular surface was obtained.
- fibrin clots are used as adhesives.
- the sheet-like composition of the present invention can obtain a sufficient adhesive force to a living body without using aprotune.
- the fact that the use of aprochon is not necessary means not only that the construction is simplified and it is advantageous in terms of production and cost, but also that there is no need to consider side effects due to the immunogenicity of aprochun itself. To do.
- One embodiment of the sheet-like composition of the present invention comprises a cell layer on the amniotic membrane.
- an adhesive component such as fibrinogen
- fibrinogen is attached to the amnion surface on the side where the cell layer is not formed.
- amniotic membrane from which the epithelium has been removed is used.
- a cell layer is formed on the side where the epithelium was present.
- the cell layer here is composed of biological cell forces.
- the origin of the cells constituting the cell layer is not particularly limited.
- the cell layer is composed of cells derived from corneal epithelium, conjunctival epithelium, skin epidermis, hair follicle epithelium, oral mucosa epithelium, iris pigment epithelium, retinal pigment epithelium, airway mucosa epithelium or intestinal mucosa epithelium.
- the cell layer may be composed of two or more different types of cells.
- hybridized cell layer Such a constitution including two or more types of cells having different origins is also referred to as “hybridization” in the present specification.
- the cell content in the hybridized cell layer is not particularly limited.
- each cell may be scattered or any cell (or multiple types of cells) may be dispersed. It may exist with a certain unity.
- the content rate of each cell may not be uniform over the whole cell layer. Even if the cell layer is a single layer, it may be a multilayer (layered state).
- the types of cells constituting the cell layer are exemplified by the case where the sheet-like composition of the present invention is constructed for corneal epithelial reconstruction. It will be described in detail.
- the type of the first cell is not particularly limited as long as it can form a corneal epithelial-like mucosal epithelial layer.
- Examples of the first cell include cells derived from mucosal epithelium such as oral mucosal epithelium, conjunctival epithelium, nasal mucosal epithelium, or undifferentiated cells capable of constructing such mucosal epithelium (ie, mucosal epithelial stem cells).
- mucosal epithelium such as oral mucosal epithelium, conjunctival epithelium, nasal mucosal epithelium, or undifferentiated cells capable of constructing such mucosal epithelium (ie, mucosal epithelial stem cells).
- derived from or derived from is used for the purpose of specifying the starting material. Therefore, for example, a cell derived from (or derived from) mucosal epithelium means a cell obtained using oral mucosal epithelial cells as a starting material.
- the “undifferentiated cell capable of constructing the mucosal epithelium” refers to a cell having an ability to differentiate into cells constituting the mucosal epithelium.
- undifferentiated cells capable of constructing oral mucosal epithelium are cells that can be separated into oral mucosal epithelial cells.
- Specific examples of undifferentiated sputum cells include precursors or stem cells of cells constituting specific tissues such as oral mucosal epithelium and conjunctival epithelium, or even less differentiated epithelial stem cells.
- the cell layer force that has been observed may include two or more different types of first cells.
- the cell layer may be constructed in a state including cells derived from the oral mucosal epithelium and cells derived from the conjunctival epithelium.
- the "oral mucosal epithelium" in the present invention includes the oral mucosal epithelial part, the lip part, the palate part, the buttocks and the like. That it is a cell derived from the oral mucosal epithelium can be confirmed by using as an index the expression of keratin 4 or keratin 13 specific to the oral mucosal epithelium. Alternatively, the expression of keratin 3 can be used as an index. This keratin 3 is known as one of corneal-specific keratins, but it has been confirmed that it is also expressed in the oral mucosal epithelium. In view of expressing keratin 3, which is a corneal specific keratin, it is preferable to use oral mucosal epithelial cells as a material for preparing a composition for corneal epithelial transplantation.
- the second cell include cells derived from corneal epithelium, conjunctival epithelium, or amniotic epithelium.
- the second cell is preferably a cell derived from corneal epithelium or conjunctival epithelium. This is because the cell layer constructed by cells derived from the ocular surface tissue can have characteristics closer to the corneal epithelium. It is particularly preferred that the second cell is a cell derived from the corneal epithelium. This is because the cell layer has characteristics more similar to those of the corneal epithelium.
- the second cell may be either an autologous cell or another person's cell.
- a cell layer constructed by autologous cells is a cell layer with no or few immune rejection problems. If the cell layer is constructed by cells of other people, it becomes easy to obtain cells as a raw material, so that the viewpoint of its production becomes an advantageous cell layer.
- the cell layer of the present invention may contain two or more different second cells.
- the cell layer may be constructed in a state including cells derived from the corneal epithelium and cells derived from the conjunctival epidermis.
- the fact that the cell layer in the sheet-like composition of the present invention contains cells derived from the corneal epithelium can be confirmed using, for example, the expression of keratin 3 or keratin 12 specific to the corneal epithelium as an index. it can. Alternatively, the expression of keratin 4 can be used as an index.
- the origin can be confirmed by examining the expression of markers or genes specific to the tissues.
- the sheet-like composition of the present invention uses an amniotic membrane as a support, and thus can be constructed in a very thin state.
- the sheet-like composition of the present invention is prepared to have a thickness of, for example, 10 ⁇ to 100 / ⁇ m when the cell layer is not included, and to a thickness of, for example, 20 m to 200 m when the cell layer is included. Can do.
- Such very thinness is also one of the major features of the present invention. By having this feature, it can be applied to various purposes. In particular, it can be applied to the reconstruction of the eye surface using high transparency.
- the state of the sheet-like composition of the present invention is not particularly limited, and may be any of a dry state (for example, a lyophilized state), a semi-dry state, and a wet state (for example, a state in which the solution is infiltrated in a solution). Good.
- a dry state for example, a lyophilized state
- a semi-dry state for example, a state in which the solution is infiltrated in a solution.
- a wet state for example, a state in which the solution is infiltrated in a solution.
- the cell layer is included, the cell layer part is preferably in a wet state, and the other part is in a semi-dry state or a dry state, for example.
- the cell layer is in a wet state
- the attachment surface (including fibrinogen etc.) of fibrinogen etc. in the amniotic membrane is in a dry state or semi-dry state
- the other part of the amniotic membrane is in a wet state Can do.
- the sheet-like composition of the present invention can also be provided in a state of being housed in a case such as glass or plastic, or in a state of being packaged using a transparent film or a light shielding sheet.
- the sheet-like composition of the present invention is provided in a sealed state. Also, it is usually provided after sterilization in advance.
- a sheet-like composition may be constructed using a collagen sheet having properties (thinness, transparency, etc.) equivalent to those of amniotic membrane. Since the amniotic membrane is rich in type IV collagen, examples of such collagen sheets include those having a high content of type IV collagen. For example, the sheet can be formed using ushi, pig, or uma collagen.
- the sheet-like composition of the present invention is used as a transplant material for tissue reconstruction.
- Examples of the application area of the sheet-like composition of the present invention include an ophthalmic area, a digestive surgery area, a gynecological area, and a dermatological area. Examples of application sites, target diseases, and uses in each of these areas are shown below.
- a sheet-like composition that does not have a cell layer can be applied to the reconstruction of the sclera and cornea (treatment for pterygium corneal epithelial defects) and prevention of Ryukyu adhesion.
- sheet-like compositions with cell layers are useful for cornea and retina reconstruction (for Steven Jyonson syndrome, thermochemical trauma, pemphigoid, retinal detachment, age-related macular degeneration, glaucoma, retinitis pigmentosa, etc. Treatment).
- a sheet-like composition without a cell layer can be applied to a skin (epidermis) ulcer or a coating at the time of a burn.
- a sheet-like composition comprising a cell layer can be applied to the treatment of epidermal diabetic ulcer (epidermis), epidermolysis bullosa, or burns.
- Another aspect of the present invention relates to a method for producing a sheet-like composition.
- the manufacturing method of the present invention includes the following steps. That is, (a) a step of preparing amniotic membrane and (b) a step of attaching fibrinogen and thrombin to the surface of the amniotic membrane.
- the “amniotic membrane” is a membrane that covers the outermost layers of the uterus and placenta in mammals, and is composed of a basement membrane and an epithelial layer formed on a collagen-rich parenchyma. It is preferable to use human amniotic membrane as the amniotic membrane.
- human amnion can be collected by force such as human fetal membrane and placenta obtained as a postpartum at the time of delivery.
- a human amniotic membrane can be prepared by treating and purifying an integral body consisting of human fetal membrane, placenta and umbilical cord obtained immediately after delivery.
- a known method such as the method described in JP-A-5-5698 can be employed. That is, the amniotic membrane is peeled off from the fetal membrane obtained at the time of delivery, the remaining tissue is removed by physical treatment such as ultrasonic washing and enzyme treatment, and the human amniotic membrane can be prepared through an appropriate washing step.
- the human amniotic membrane thus prepared can be stored frozen until use. Freezing of the human amniotic membrane can be performed, for example, at ⁇ 80 ° C. in a liquid in which DMEM (Dulbecco's modified Eagle's medium) and glyceride are mixed in an equal volume ratio.
- DMEM Denbecco's modified Eagle's medium
- the operability can be improved by cryopreserving, and the antigenicity can be expected to decrease.
- amniotic membrane can be used as it is, it is preferable to use a material obtained by removing the epithelium from the amniotic membrane by reptile treatment or the like. Removal of the epithelium reduces antigenicity. For example, after thawing human amnion frozen as described above, it is treated with EDTA or proteolytic enzyme to loosen the adhesion between cells, and the epithelium is replated using a cell scraper, etc. A removed human amniotic membrane can be prepared.
- the amniotic membrane is previously dried.
- Adhesive components such as fibrinogen can be better adhered by using the amniotic membrane in a mulberry state.
- the drying process include freeze drying, air drying, vacuum drying, and reduced pressure drying. Of these, lyophilization is preferred. This is because the flexibility of the amniotic membrane is difficult to decrease in the case of freeze-drying.
- the adhesion of fibrinogen and thrombin to the surface of the amniotic membrane is performed individually or simultaneously.
- the attachment method is not particularly limited. Ingredients to be adhered as an example of adhesion method And a method of immersing the amniotic membrane in a solution of the component to be adhered.
- fibrinogen itself (or thrombin itself) or fibrinogen (or thrombin) dissolved in a suitable solvent and added to the surface of the amniotic membrane (sprayed) to fibrinogen (or (Thrombin) can be attached to the surface of the amniotic membrane.
- a mixture of these two components is prepared, and fibrinogen and thrombin are simultaneously adhered to the amniotic membrane surface by coating, dropping, or the like using the mixture.
- fibrinogen and thrombin are simultaneously adhered to the amniotic membrane surface by coating, dropping, or the like using the mixture.
- a fibrinogen solution is prepared. Specifically, fibrinogen is dissolved in a solvent (solvent) such as ethanol (for example, 94% ethanol) to a desired concentration.
- a solvent such as ethanol (for example, 94% ethanol)
- alcohols such as absolute ethanol, isopropanol, and methanol, and acetone can be used as the solvent.
- a thrombin solution separately by the same procedure.
- ethanol for example, 99.5% ethanol
- alcohols such as absolute ethanol, isopropanol and methanol, acetone, and the like can be used.
- a fibrinogen solution and a thrombin solution are typically performed uniformly over the entire area of the amniotic membrane surface. (E.g., multiple spots at intervals like spots) This can be done in the area or only in the surrounding area), or with the amount of adhesion added.
- the force at which fibrinogen and thrombin are simultaneously attached may be attached in separate steps. That is, fibrinogen attachment and thrombin attachment may be performed in two steps. However, it is preferable to perform the adhesion operation in one step using a mixture of fibrinogen and thrombin because the operation can be simplified and fibrinogen and thrombin can be adhered in a more uniform dispersion state. ⁇
- Fibrinogen and thrombin can be prepared from blood according to a conventional method.
- Recombinant fibrinogen or the like can also be used, and in this case, it can be prepared by a conventional method from a culture solution or cell disruption solution of an appropriate cultured cell.
- Commercially available fibrinogen or the like may be used.
- human-derived fibrinogen can be purchased from Baxter.
- human-derived thrombin can be obtained from Baxter.
- caprotun may be attached to the surface of the amniotic membrane. That is, in this embodiment, the step of attaching aprotune (step b-1) is further performed.
- Aprochung can be attached by the same means and procedure as fibrinogen and the like. That is, aprochon can be attached to the amniotic membrane surface by application, dripping, spraying, or dipping using aprotonic solution.
- the caprotun solution can be prepared by dissolving caprotun in a sodium chloride solution (eg, 0.85% solution), potassium chloride solution, calcium chloride solution, magnesium chloride solution, and the like.
- Aprotune can be prepared from the spleen of ushi according to a conventional method.
- Recombinant aprotune can also be used, and in this case, it can be prepared by a conventional method from a culture solution or cell disruption solution of an appropriate cultured cell.
- a commercially available aprotune may be used.
- aprochun derived from Ushi can be purchased from Bayer Yakuhin.
- step of attaching caprotun it is also possible to carry out the step of attaching caprotun alone. Preferably, it is carried out simultaneously with the step of attaching fibrinogen and thrombin.
- Adhesion This is a culm that simplifies the operation of attaching the minute.
- fibrinogen, thrombin, and aprotune can adhere to the amnion surface in a more uniformly dispersed state. For example, by preparing a mixture of fibrinogen, thrombin, and aprotune and applying it, these three components can be attached simultaneously to the amniotic membrane. The order of mixing these three components is not particularly limited.
- Attachment of fibrinogen or the like is performed on one or both sides of the amniotic membrane.
- the epithelial side the presence of epithelium is present
- the epithelial side regardless of the presence or absence of epithelium in the amniotic membrane.
- the adhesion of fibrinogen or the like is performed on the surface opposite to the V-plated side (that is, the chorionic side).
- drying treatment general drying treatment methods such as air drying, vacuum drying, reduced pressure drying and freeze drying can be employed.
- a cell layer using living cells is formed on the amniotic membrane.
- This cell layer is formed prior to the operation of attaching thrombin or the like to the surface of the amniotic membrane. That is, in this embodiment, after forming a cell layer on the amniotic membrane, an adhesive component such as thrombin is attached to the surface of the amniotic membrane (the surface on the side where the cell layer is not formed).
- the step of forming the cell layer can be performed by the following procedure.
- an appropriate living body-derived cell is prepared (step of preparing a living body-derived cell).
- cells suitable for the use of the finally obtained sheet-like composition are used.
- skin epidermal cells including stem cells and precursor cells
- hair follicle epithelial cells including stem cells and precursor cells
- corneal epithelial cells including stem cells and progenitor cells
- mucosal epithelial cells for the purpose of regeneration of mucosal epithelial tissue.
- Their stem cells and progenitor cells are preferably used.
- mucosal epithelial cells include oral mucosal epithelial cells, intestinal mucosal epithelial cells, and airway mucosal epithelial cells.
- Corneal epithelial cells can be obtained from corneal limbal tissue force.
- endothelium cells such as corneal limbal tissue force are peeled and removed, and the conjunctiva is excised to produce a single cell suspension. This is stored in a nitrogen tank and then rapidly thawed at 37 ° C to prepare the corneal epithelial cell suspension.
- Subculture if necessary.
- EpiLife TM (Cascade), MCDB153 medium (Nissui Pharmaceutical Co., Ltd.), which is a serum-free medium, or a medium produced by modifying the amino acid composition of these mediums, etc. should be used. Can do.
- Oral mucosal epithelial cells include cells present in the root of the tooth (inner oral mucosal epithelial cells), lip cells, palate cells, buttocks cells, and the like. Of these, the intraoral mucosal epithelial cells are particularly preferred because of their high proliferative ability and low antigenicity. Oral mucosal epithelial cells can be collected by excising the area where the target cells are present with a scalpel or by cleaving. In the oral marginal mucosal epithelial cells, the oral mucosal epithelium adhering to the tooth extraction can be separated from the enamel cement transition and collected. In order to remove impurities such as connective tissue, treatment with enzymes such as dispase or trypsin or filter treatment is preferred!
- Intestinal mucosal epithelial cells are collected from colonoscopic intestinal epithelial biopsy tissue or open surgery Sometimes collected in the usual way.
- epidermal cells can be excised from the tissue by lazer capture microdissection.
- the technique of the present invention is also applied to a biological tissue sheet produced using all human digestive tract epithelial cells of the esophagus, stomach, duodenum, small intestine, and large intestine.
- human gastrointestinal epithelium is damaged by ulcers or inflammation, bone marrow-derived cells play a rescue role in response to emergency situations, and the epithelium is repaired.
- Gastrointestinal epithelial cells though some, are made by bone marrow.
- the significance of the present invention can be regarded as equivalent to that using corneal epithelial cells.
- the number of epithelial cells made from bone marrow which is usually only about a few thousand, increases from 50 times to 100 times in the process of healing ulcers (wounds) inside the gastrointestinal tract due to gastric ulcer, colitis, etc. It has been found that 1 in 10 gastrointestinal epithelial cells are derived from the bone marrow.
- Gastrointestinal mucosal epithelial cell-derived tissue tissue prepared here is used for intractable ulcers and inflammation of enteric diseases such as severe intestinal infections, ulcerative colitis, Crohn's disease, Behcet's disease, etc. In the sense of promoting the regeneration of the intestinal epithelium, it is considered to be extremely meaningful. Expected to be useful for intestinal allergy
- Airway mucosal epithelial cells are easily obtained from biopsy tissue of the airway mucosa, and in the same way as described above, treatment with an enzyme such as dispase trypsin or filtering is performed to remove impurities such as connective tissue. Is preferred. Airway mucosal epithelial cells play an important role in the pathogenesis of various infectious diseases through the biosynthesis and release of j8 defensin. The role of airway mucosal epithelium is high in asthma and allergic diseases! Providing a biological tissue sheet produced from the airway mucosal epithelial cells according to the present invention to the airway mucosa that has undergone tissue damage goes beyond the emergency response and leads to the provision of an artificial airway. In particular, the immunosuppressive action of the sheet prepared on the amniotic membrane is beneficial.
- oral mucosal epithelial cells and intestinal mucosal epithelial cells are preferably subjected to treatment with an enzyme such as dispase trypsin or filter treatment to remove impurities such as connective tissue after collection of the tissue.
- an enzyme such as dispase trypsin or filter treatment to remove impurities such as connective tissue after collection of the tissue.
- the living body-derived cells are also prepared with a person (recipient) ability to receive transplantation of the living tissue sheet.
- the donor of the living cell and the recipient of the living transplant sheet are the same A person is preferred.
- the prepared biological cells are seeded on the amniotic membrane (step of seeding the biological cells on the amniotic membrane) and then subjected to culture (step of culturing and proliferating the seeded biological cells) ).
- amniotic membrane from which the epithelium has been removed it is particularly preferable to use amniotic membrane from which the epithelium has been removed.
- the reduction of antigenicity can be expected by removing the epithelium, and the target cell layer can be formed well by removing unnecessary cells in advance.
- the amniotic membrane from which the epithelium has been removed it is preferable to seed the living body-derived cells on the side of the surface exposed by removing the epidermis (that is, the basement membrane side). This is because this surface side is rich in type IV collagen, and it is considered that the seeded living cells proliferate and stratify well.
- a hybridized cell layer may be formed by using two or more types of cells.
- a specific method for forming the cell layer in such a case will be described in detail below, taking as an example the case of producing a sheet-like composition for corneal epithelial reconstruction.
- oral mucosal epithelial cells, conjunctival epithelial cells, nasal mucosal epithelial cells, other mucosal epithelial cells, or any one of these mucosal epithelia is used as one of the cell types (first cells) used to form the cell layer.
- An undifferentiated cell that can be constructed is preferably used.
- corneal epithelial cells, conjunctival epithelial cells, or amniotic epithelial cells are preferably used as the cell type (second cell) used for forming the cell layer together with the first cells. These cells are collected from the living tissue in which they are present.
- a part of the tissue in which the target cells are present is collected with a scalpel, etc., and then processed into a cell suspension (suspension) through treatment such as connective tissue removal and cell separation. .
- a cell suspension sustained-term evolution
- Two or more different types of cells may be used as the first cell.
- two or more different types of cells may be used for the second cells.
- stem cells have been suggested in the oral mucosal epithelium suitable as a collection source of the first cells, and it is considered that differentiation induction into cells forming an epithelial cell layer is easy.
- using oral mucosal epithelial cells is easy to collect, can collect a large amount of cells, and even when treating patients with binocular disease, prepare transplant materials using their own cells. It has advantages such as being able to do so. Especially for patients who cannot collect corneal epithelial cells. The advantage of being able to apply my cell-derived transplantation material is expected to greatly eliminate clinically important rejection problems.
- Oral mucosal epithelial cells include cells present in the root of the tooth (inner oral mucosal epithelial cells), lip cells, palate cells, buttocks cells, and the like. Of these, the intraoral mucosal epithelial cells are particularly preferred because of their high proliferative ability and low antigenicity. Oral mucosal epithelial cells can be collected by excising the area where the target cells are present with a scalpel or by cleaving. In the oral marginal mucosal epithelial cells, the oral mucosal epithelium adhering to the tooth extraction can be separated from the enamel cement transition and collected. In order to remove impurities such as connective tissue, treatment with enzymes such as dispase or trypsin or filter treatment is preferred!
- Oral mucosal epithelial cells collected from the oral cavity other than the patient scheduled to be transplanted with the sheet-like composition prepared according to the present invention can also be used, but considering immune rejection, oral mucosal epithelial cells from the patient's own oral cavity Is preferably collected and subjected to culture.
- the oral mucosa has a high proliferative capacity, and usually wounds are healed by taking antibiotics for a few days after surgery, disinfecting with isodine, and so on.
- the corneal epithelial cells of another person can be preferably used as the second cells.
- Such corneal epithelial cells can be obtained from, for example, an infectious disease-free donor eyeball from an eye bank (Northwest eye bank, etc.).
- Cells that can be used as the second cell are not limited to corneal epithelial cells, and conjunctival epithelial cells, amniotic epithelial cells, and the like may be used.
- a corneal epithelial cell which is a cell constituting the corneal epithelium in a living body, or a conjunctival epithelial cell present in the vicinity thereof is employed, a sheet-like composition that better reproduces the characteristics of the corneal epithelium can be constructed. It is conceivable that.
- a corneal epithelial cell was used as the second cell, a cell layer similar to the corneal epithelium could be constructed. This fact supports the above prediction and confirms that corneal epithelial cells are particularly suitable as the second cells.
- amniotic epithelial cells were used as the second cells, it was possible to form a cell layer that well reproduced the characteristics required for the cornea. This fact indicates that amniotic epithelial cells can also be suitably used as the second cells.
- the ability to use own cells as the second cells If cells of another person are used, the cells can be obtained more easily. For example, even when a sheet-like composition is prepared for treatment of a binocular patient, corneal epithelial cells as second cells can be obtained.
- first cell and the second cell (hereinafter collectively referred to as "first cell etc.") prepared respectively are seeded on the amniotic membrane and subjected to culture.
- first and second cells prepared in the form of a cell suspension are dropped onto the amniotic membrane and cultured.
- the seeding of the first cell and the seeding of the second cell are performed at the same time (the term "simultaneous" here is literally the same time as well as placing a substantial time interval after one seeding. If the other cells are seeded at different timings, for example, the second cells are seeded after several minutes to several hours after the seeding of the first cells. Good.
- the seeding time By shifting the seeding time in this way, for example, a region rich in cells derived from the first cells is localized and a cell layer is constructed, and the structure of the cell layer and the characteristics associated therewith are not changed. Can be adjusted.
- the ratio of the first cells and the like to be seeded is not particularly limited, but typically, approximately the same number of the first cells and the second cells are seeded.
- the number of the first cells the number of the second cells S3: 7, 5: 5
- Comparison of the cases of 7: 3 showed that there was no clear difference between them in terms of cell proliferation and stratification (data not shown).
- the first and second cells proliferate to form a cell layer (it is thought that at least some of the cells are separated during this process).
- a step of bringing the surface layer of the cell layer into contact with air is performed. This step is also referred to as air-lifting in this specification. This step is performed for the differentiation of the cells forming the cell layer and the induction of the barrier function.
- the surface of the culture solution is lowered by temporarily removing a part of the culture solution using a dropper, pipette, etc., thereby exposing the outermost layer of the cell layer to the outside of the culture solution. It can be carried out. Or lift the cell layer together with the amniotic membrane, It can also be carried out by temporarily exposing the layer from the culture medium surface. Furthermore, air may be sent into the culture solution using a tube or the like, and air may be brought into contact with the uppermost layer of the cell layer. From the viewpoint of ease of operation, it is preferable to carry out the method by lowering the surface of the culture solution and exposing the outermost layer of the cell layer.
- the time for performing this step that is, the time for contacting the outermost layer of the layered cell layer with air varies depending on the state of the cell and the culture conditions, but is, for example, about 3 days to 2 weeks, preferably within 1 week. More preferably, it is within 3 days.
- a corneal epithelium-like cell layer in which the first cells and the like are stratified is formed on the amniotic membrane.
- the sheet-like composition thus obtained can be used as a transplant material (substitute for corneal epithelium) for patients with damaged or deficient cornea along with amniotic membrane used as a culture substrate for the first cells and the like.
- the amniotic membrane is transplanted into the corneal epithelial defect portion so that it is on the eyeball side.
- culture of living body-derived cells is performed in the presence of supporting cells.
- Supporting cells are also called feeder cells and supply growth factors and the like into the culture medium.
- cell proliferation efficiency is improved.
- 3T3 cells Stem 3T3 cells, mouse NIH3T3 cells, 3T3J2 cells, etc.
- mouse NIH3T3 cells it is preferable to use mouse NIH3T3 cells as support cells from the viewpoints of proliferation efficiency and ease of handling.
- inert gas can also be performed by radiation treatment or the like.
- the cell density of the support cells for example, about 1 X 10 2 or ZCM 2 or more, preferably about 1 X 10 2 pieces Zc m 2 ⁇ about 1 X 10 7 cells / cm 2, more preferably about 1 X 10 It can be from 3 pcs / cm 2 to about 1 X 10 5 pcs / cm 2 .
- the number of supporting cells to be used is, for example, 1/10 3 times to 1 X 10 2 times, preferably 1/10 2 to 1 times the total number of living cells. Culturing can be performed under such conditions.
- the number of supporting cells is small, the growth rate of living cells is lowered, and if the number is too small, good growth and stratification of living cells cannot be obtained. on the other hand, If the number of supporting cells is too large, the proliferation rate of living cells is inevitably reduced, which is not preferable.
- the isolation membrane a well-known one having a pore size from which feeder cells cannot pass can be appropriately selected and used.
- pore size is about 0.4 m ⁇
- a 3.0 m membrane can be used.
- the material of the separator is not particularly limited, and may be polyester or the like in addition to polycarbonate. Such separators are commercially available and can be easily manned.
- inactivated support cells are seeded and cultured in a container such as a petri dish (first container) to form a support cell layer on the surface of the container.
- a second container having a bottom surface formed of a separator is placed in the first container.
- the position of the second container is adjusted so that the bottom surface of the second container is in the culture medium.
- the amniotic membrane is placed on or attached to the bottom surface of the second container, that is, the isolation membrane.
- biological cells are seeded on the amniotic membrane and cultured.
- the amniotic membrane is placed or attached in advance to the bottom surface of the second container (for example, the amniotic membrane from which the epithelium has been removed is placed on the bottom surface of the second container, and in this state, a drying treatment is performed once), and this second container
- the container may be placed in a first container seeded with feeder cells, and biological cells may be seeded on the amniotic membrane and cultured.
- D MEM Dynamic Eagle's medium
- Nom F12 medium Hyam's F12 medium
- a medium containing FBS, growth factors, antibiotics, etc. is used. be able to.
- FBS FBS (10%), insulin (5 mg / ml), cholera toxin (0.1 nM),
- Examples include a mixed medium (mixing volume ratio 1: 1) of DMEM and Ham's F12 medium supplemented with epidermal growth factor (EGF) (10 ng / ml) and penicillin streptomycin (50 IU / m 1).
- EGF epidermal growth factor
- triodothyronine eg, 2 nM
- glutamine eg, 4 mM
- transferrin eg, 5 mg / ml
- adenine eg, 0.18 mM
- Z or Hyde mouth cortisone eg, DMEMZ Ham F12 mixed medium supplemented with 0.4 mg / ml
- Culture of biological cells may be performed in the absence of heterologous animal cells.
- “in the absence of heterologous animal cells” means that animal cells that are heterologous to the living cells are not used as conditions for culturing the living cells.
- human cells eg, human skin epidermis cells or human corneal epithelial cells
- cells of animal species other than humans such as mice and rats
- the culture medium co-existing
- foreign-derived components including the foreign cells themselves
- the transplant material ie, the sheet-like composition
- the medium used for culturing living body-derived cells is not particularly limited as long as the cells are allowed to grow.
- MCDB153 medium Nisui Pharmaceutical Co., Ltd.
- EpiLife TM Cascade
- medium prepared by modifying the amino acid composition of these mediums DMEM (Dulbecco's modified Eagle's medium) commonly used for epithelial cell growth )
- Ham's F12 medium can be used.
- a serum-free medium that does not contain a protein derived from a different animal.
- a medium supplemented with growth factors, antibiotics and the like may be used.
- a serum-free culture method as the culture method in the present invention.
- the culture may be carried out in a medium containing serum.
- serum derived from the same species human-derived serum when cultivating human biological cells
- autologous serum is used. It is preferable to use it.
- autologous serum that eliminates the risk of causing immune rejection. It is possible to change the culture conditions in the middle of the culture step for the purpose of good growth of living cells.
- bio-derived cells such as cell density of about 1 X 10 3 cells ZCM 2 or more, preferably about 1 X 10 3 cells / cm 2 ⁇ about 1 X 10 7 cells / cm 2, more preferably about 1 X 10 4 seeds / cm 2 to about 1 ⁇ 10 6 seeds / cm 2 are seeded on the amniotic membrane.
- the amniotic membrane is placed on a human fibroblast-containing collagen matrix prepared in advance, and then biological cells are seeded on the amniotic membrane and cultured. That is, in this embodiment, after culturing human fibroblasts in a collagen gel (Step B), and placing the amniotic membrane on the collagen gel, the biological cells are seeded or placed on the amniotic membrane. Step (Step C) is performed.
- the sheet-like composition produced by such a procedure will contain biological cells grown on amniotic membrane placed on a collagen gel containing human fibroblasts.
- the sheet-like composition of this embodiment can also be used as a transplant material after removing the collagen matrix. It can also be used as a transplant material for each collagen matrix.
- Collagen gel functions as a culture substrate for human fibroblasts.
- the type of collagen used as a raw material for the collagen gel is not particularly limited, and type I collagen, type III collagen, type IV collagen, and the like can be used. A mixture of a plurality of types of collagen can also be used.
- These collagens can be extracted and purified from the skins of animals such as pigs, sushi, and hedges, and connective tissues such as soft bones by acid solubilization, alkali solubilization, enzyme solubilization, etc. it can.
- a so-called atherocollagen in which the telopeptide is removed by treatment with a degrading enzyme such as pepsin or trypsin.
- Collagen derived from amnion may be used as a material for the collagen gel.
- the term “derived from amniotic membrane” means that it is widely obtained using amnion as a starting material.
- the origin of the human fibroblasts contained in the collagen gel is not particularly limited, and any tissue can be used as long as it produces collagen. For example, a force such as skin tissue or oral mucosa tissue was also prepared. Human fibroblasts can be used.
- human fibroblasts are prepared by the following procedure. The skin is collected, and then the dermis is peeled off from the skin. Shred the dermis and attach it to the I-type collagen coat dish. After static culture, pass human fibroblasts that migrate from the dermis. The cells are detached from the bottom of the dish, and the cell suspension is prepared and seeded in a cell culture dish. If appropriate, the cells are stored frozen (eg, stored in liquid nitrogen). On the other hand, a neutralized collagen solution is prepared using type I collagen (see Examples described later). Add this to a culture vessel (eg culture insert) and let it stand at room temperature for about 10 minutes to gel.
- a culture vessel eg culture insert
- the logarithmically growing human fibroblasts cultured in advance by the above-mentioned method are mixed with this gel and gelled again. Then, static culture is performed.
- a collagen matrix containing human fibroblasts is obtained by the above procedure. This ingenuity provides a collagen matrix that has the necessary strength and can be loaded with an amniotic layer or living body cells, and forms the basis of the present invention.
- a separately prepared amniotic membrane is placed (adhered) on the collagen matrix. Thereafter, the cells are seeded and cultured according to the above-described procedure.
- amniotic membrane sheet with an adhesive component-attached epithelium was prepared by the following procedure.
- Amniotic membranes were collected at the time of cesarean section in the operating room after giving sufficient informed consent with the obstetrician and gynecologist in advance for pregnant women scheduled for cesarean section without systemic complications. The operation was careful of cleanliness, and a special gown was worn after hand washing according to the surgical operation. Before delivery, a clean bat for collecting amnion and physiological saline for washing were prepared. After delivery, the placenta tissue was transferred to a vat and the amnion tissue was manually detached from the placenta. The adhesion between the amniotic membrane and the placenta was strong! The part was excised with scissors.
- the amniotic process was performed in the order of (1) washing, (2) trimming, and (3) storage. In all processes, use containers and equipment that should be sterilized, which should be used in a clean draft, and use petri dishes, etc. that have been sterilized and discarded (disposable). Eve was used. Blood components adhering to the collected amniotic membrane were removed while washing with physiological saline, and further washed with a sufficient amount of physiological saline (0.005% ofloxacin added). Next, the amniotic membrane was transferred to a phosphate buffer solution (PBS) in a petri dish and divided into about 4 ⁇ 3 cm sizes using scissors. After the division, the preservation solution was further immersed in several petri dishes, and amniotic membranes in good condition were selected.
- PBS phosphate buffer solution
- Each 1cc of the stock solution was put into a 2cc sterilized cryotube, collected, washed and selected one by one, and then stored in a refrigerator at _80 ° C.
- 50% sterilized glycerol in DMEM (Dulbecco'S Modofied Eagle Medium: GIBCOBR L) was used as a preservation solution.
- the stored amniotic membrane was used for 3 months and incinerated when the expiration date passed.
- Liquids A, B, and aprotune prepared in 1-1 were mixed and pipetted thoroughly (mixed liquid). Next, after dripping this mixture so that it almost spreads over the entire surface (chorionic side) of the dried amniotic membrane prepared in 1-2-4. (Fibrinogen adhesion amount: 5.5 mg / cm 2 , The adhering amount of thrombin: 0.13 mg (1.4 U) / cm 2 and the adhering amount of caprotun: 1.4 KIU / cm 2 ) were dried under reduced pressure at room temperature for 2 hours.
- the frame was removed from the dried amniotic membrane, transferred to a two-layered bag with polyamide nylon on the outside and polyethylene on the inside, and vacuum packed using a household vacuum pack device (Frame Nova, Magic Pack).
- the vacuum-packed amniotic membrane thus obtained was sterilized by irradiation with gamma rays (about 25 kGy).
- the sterilized amniotic membrane was stored at room temperature in a vacuum-packed state until just before use (adhesive component-attached amnion sheet with epithelium).
- a sheet in which the adhesive component was adhered to the amniotic membrane from which the epithelium had been removed (hereinafter, also referred to as “adhesive component-attached epidermis-free amniotic membrane sheet”) was prepared by the following procedure.
- the amniotic membrane collected and stored by the above procedure (1 2-1 to 1 2-3.) was thawed at room temperature, and then thoroughly washed with a sterilized phosphate buffer (PBS) in a petri dish. After washing, the cells were stored in a 0.02% EDTA solution (Nacalai tesque) for 2 hours at 37 ° C, and then the epithelium was mechanically cleaved using a cell scraper (cell scraper, Nunc USA). The amnion without epithelium obtained in this way is dried and coated with an adhesive component according to the above procedure (1-2-4. And 1-2-5). Adhesion amount: 0.5 mg / cm 2 , Thrombin adhesion amount: 12 ⁇ g / cm 2 , Aprotune adhesion amount: 0.12 KIU / cm 2 ) Obtained.
- PBS sterilized phosphate buffer
- Adhesion component adhesion Cell layer-forming amniotic membrane sheet (1) Adhesive adhesion cultured cornea sheet
- a sheet in which a cell layer derived from corneal epithelial cells is formed on the amniotic membrane (without epithelium), and an adhesive component adheres to the surface of the amniotic membrane (hereinafter also referred to as “adhesive component-attached cultured cornea sheet”) was prepared by the following procedure.
- the amniotic membrane collected and stored by the above procedure (1 2-1 to 1 2-3.) was thawed at room temperature, and then thoroughly washed with a sterilized phosphate buffer (PBS) in a petri dish. After washing, the cells were stored in a 0.02% EDTA solution (Nacalai tesque) for 2 hours at 37 ° C, and then the epithelium was mechanically capped using a cell scraper (Nunc USA). The amnion containing no epithelium thus obtained was used as a substrate for the following cell culture.
- PBS sterilized phosphate buffer
- a corneal limbal tissue piece of about 5 ⁇ 10 mm was collected.
- the collected tissue pieces were immersed twice in a phosphate buffer solution (PBS) containing 50 IU / ml penicillin streptomycin and gentacin for 30 minutes at room temperature.
- the tissue was then immersed in phosphate buffer (PBS) containing 1.2 U dispase (Nacalai tesque) for 1 hour at 37 ° C, followed by 0.05% trypsin-EDTA solution (GIBCOBRL) for 15 minutes.
- the cells were separated by immersion treatment. Enzyme activity was stopped by immersion in DMEM containing 10% ushi fetal serum (FBS). Thereafter, excess tissue was removed with a 60 m cell 'filter, and corneal epithelial cells were isolated (corneal epithelial cell suspension).
- 3T3 cells NIH-3T3 cells
- 3T3 cells were used as co-cultured cells (supporting cells).
- 3 ⁇ 3 cells cultured in advance and confluent in a 75F flask (BD Falcon) were immersed in 0.05% mitomycin C solution for 2 hours to suppress the growth activity of 3T3.
- PBS phosphate buffer
- a 3T3 cell suspension was prepared by treating with 0.05% trypsin EDTA solution.
- corneal epithelial cells were co-cultured with the 3T3 cells subjected to the above treatment in the following procedure.
- a culture apparatus a 6-well culture dish (Corning, NY) and a culture insert (culture insertion container) (polycarbonate, average pore size 3.0 m, Corning, NY) were used.
- the amnion substrate was left standing with the skin side up and allowed to dry at room temperature for 10 minutes. Thereafter, the corneal epithelial cell suspension was seeded on the culture insert with the amnion attached to a cell density of about 1 ⁇ 10 4 Zcm 2 .
- FIG. 1 is a schematic cross-sectional view showing a state during culture.
- Culture insert 2 is allowed to stand in culture dish 1, and 3T3 cell layer 5 is formed on the bottom of culture dish 1.
- the amniotic membrane 3 is allowed to stand on the bottom surface of the culture insert 2 and the corneal epithelial cells 4 are cultured thereon.
- Symbol 6 is a medium.
- the medium includes DMEM / Ham F12 mixed medium (mixing volume ratio 1: 1), 10% FBS, insulin (5 mg / ml), cholera toxin (0.1 nM), penicillin streptomycin (50 IU / ml) and human recombinant epithelial cell growth factor (EGFXlOng / ml) added.
- the air lifting method is a method in which the liquid surface of the culture medium reaches the cell layer surface formed on the amniotic membrane and the surface of the cell layer is exposed to the air. During the submerge, the culture medium was changed every other day, and after air lifting, the culture medium was changed every day.
- the culture cornea sheet obtained as a result of the above was taken out of the culture solution, and then the surface opposite to the cell layer forming side (chorionic membrane side) was made into a semi-dried state by air drying by blowing air.
- the surface of the amniotic membrane in a semi-dried state is immersed in a mixture of fibrinogen, thrombin, and aprotune (prepared in the same procedure as 12-2-5), and then air-dried to obtain a semi-dried state. did.
- the cell culture surface was kept moist.
- Adhesive component adhesion Cell layer-forming amniotic membrane sheet (2) Adhesive component adhesion cultured epidermis sheet
- a sheet in which a cell layer derived from epidermal keratinocytes is formed on an amniotic membrane (without epithelium) and an adhesive component adheres to the surface of the amniotic membrane (hereinafter also referred to as an “adhesive component-attached cultured cornea sheet”) is Prepared by procedure.
- the amniotic membrane collected and stored by the above procedure (1 2-1 to 1 2-3.) was thawed at room temperature, and then thoroughly washed with a sterilized phosphate buffer (PBS) in a petri dish. After washing, the cells were stored in a 0.02% EDTA solution (Nacalai Tesque) for 2 hours at 37 ° C, and then the epithelium was mechanically cleaved using a cell scraper (Nunc USA). The amniotic membrane containing no epithelium thus obtained was used as a substrate for the following cell culture.
- PBS sterilized phosphate buffer
- the peeled epidermis is washed with DMEM, then with PBS, and then immersed in a 0.25% trypsin solution and treated at 37 ° C for 10 minutes. Transfer the epidermis to a plastic petri dish containing a trypsin neutralization solution, loosen the epidermis with tweezers, and transfer to a 50 ml sterile tube. PBS is added to prepare the epidermal keratinocyte suspension. Count the number of cells and centrifuge at 1000 rpm for 5 minutes to precipitate the cells.
- the supernatant is aspirated and the cells are suspended in MC DB153 medium, which is a serum-free medium, and 2 to 3 X 10 6 cells per 10 mm per 100 mm collagen-coated petri dish (Asahi Techno Glass, Type I collagen-coated dish; 4010-010) Seed in culture medium . Change the culture medium the next day, and then change the culture medium every other day. Subculture when cell density reaches about 70-80%.
- the peeled dermis is washed with DMEM, and then cut into small pieces with a scalpel on one side.
- the cut dermis is closely attached to the type I collagen-coated dish at an interval of about 1 cm.
- the cells Collect the remaining cells using PBS, precipitate the cells by centrifugation at 1000 rpm for 5 minutes, aspirate the supernatant, add DMEM medium containing 10% fetal bovine serum, and adjust the cell suspension. Inoculate in a cell culture dish. Passage cell density should be approximately 1: 3. The cells are stored frozen as appropriate.
- the cryopreservation solution is 10% glycerol, 20% FCS, 70% DMEM, and is stored in liquid nitrogen.
- Type I collagen solution (cell matrix type 1A: 3 mg / ml: Nitta Gelatin): 0.1N NaOH: 1 volume, 8 times concentration for 6 volumes DMEM: 1 volume, 20% FCS / DMEM: 10 volumes
- a neutral collagen solution (final collagen concentration: lmg / ml) at 4 ° C, add lml to a 24mm diameter force insert (Corning-Costar), and let stand at room temperature for 10 minutes. Make it gelled. 5xl0 5 cells were grown in logarithmic culture and used as fibroblasts in the logarithmic growth phase (cells cultured by the outgrowth method from the remaining dermis treated with dispase and exfoliated from the epidermis and then passaged 5-10).
- the bottom surface of the collagen gel is in close contact with the membrane, but the upper part of the collagen gel contracts to a thickness of 2 to 3 mm.
- Wash the preserved amniotic membrane (amniotic membrane prepared by the same procedure as in 1-3-1 above, with the epithelium removed) twice with PBS and then once with the culture medium for keratinocytes. Transfer the amniotic membrane to the culture insert with the parenchyma side facing down, and attach it to the collagen gel using tweezers. Extend so that wrinkles cannot be made with tweezers, and attach the periphery of the amniotic membrane to the side wall of the culture insert. CO incubation
- 1-5- Detach the keratinocytes prepared in step 2 using trypsin ⁇ EDTA and collect. l Centrifuge for 5 minutes, remove the supernatant, and resuspend the cells to a concentration of 2 million cells / 0.25 ml. Inoculate 0.25 ml of cell suspension on the amniotic membrane inside the culture insert and transfer to a CO incubator to ensure that the keratinocytes adhere to the amniotic membrane.
- Air exposure is performed 3 days after seeding epidermal cells on the amniotic membrane. Place sterilized filter paper in a maintenance container for air exposure, and add the layering medium to the extent that the filter paper is immersed (approximately 9 ml). Carefully remove the culture medium inside the culture insert, transfer the culture insert onto filter paper,
- the stratification medium is prepared as follows.
- Dulbecco's modified ME M medium: F-12 medium 1: 1, calcium concentration; 1.95 mM, monoethanolamine; 0.1 mM, 0-ph phosphoethanolamine; 0.1 mM, insulin; 5 ug / ml, hydrocortisone; 0.4 ug / ml, L— glutamine; 4 mM, Adenin; 0.18mM, transfferin; 5ug / ml, selenious acid; 53nM, triiodothyronine; 20p M, serine; lmM, choline chloride; 0.64mM, linoleic acid; 2ug / ml, FCS; 2%
- the cultured skin sheet obtained as a result of the above operation easily peels off the dish bottom surface force or collagen matrix force. Since the conventional technology causes the sheet to shrink, it is necessary to use a chitin membrane (peskitin W) as a support and often breaks, but the above method produces a strong sheet, Since no sheet shrinkage is observed, the use of a support becomes unnecessary.
- peskitin W chitin membrane
- the cultured epidermis sheet was prepared by the above method, the epidermis became 5 to 8 layers on the 7th day after exposure to air, the formation of stratum corneum was observed, and the structure was almost the same as normal human skin.
- the histological findings of the stratified keratinocytes are a single basal cell-like cell and a 5-8 layer stratification on it, and a differentiated cell structure is observed.
- the cultured epidermis sheet can be used about 4 weeks after the skin is collected, and the surface area of the culture surface increases several thousand times in the calculation.
- the prepared cultured skin sheet can be frozen by using a small amount of a preservation solution with or without a carrier. Specifically, it is first stored frozen in an 80 ° C freezer and then stored the next day in a 150 ° C ultra-low temperature freezer. When stored at 150 ° C, it can be stored in the form of a sheet for a long period of time, and a sufficient therapeutic effect is actually obtained. In addition, it is possible to store at 4 ° C using a storage solution used for storage of biological tissues. In this case, it is desirable to add an antioxidant.
- Collagen matrix strength After removing the peeled cultured skin sheet, the surface opposite to the cell layer forming side was made into a semi-dried state by air-drying with air blowing. Next, the surface of the amniotic membrane in a semi-dried state is immersed in a mixture of fibrinogen, thrombin, and aprotune (prepared in the same procedure as 1-2-5), and then air-dried to obtain a semi-dried state. did. During this series of operations, the cell culture surface was kept moist.
- a sheet-like composition adheresive component-attached cultured skin sheet in which an adhesive component adhered to the surface of the amniotic membrane on the side opposite to the side on which the cell layer was formed was obtained.
- Adhesion component adhesion The adhesion of the epithelial amniotic sheet (1-2-5) was examined using porcine sclera as the application site.
- Tissues from pig eyes slaughtered for food were used.
- the conjunctiva was excised with scissors from the porcine eye to expose the sclera (bearing state).
- the abnormal conjunctiva is usually peeled off and the conjunctiva removal site is covered with normal conjunctiva.
- Pig eyes treated as described above are widely used as clinical models of pterygium. It's being used.
- Adhesive component adhesion-amniotic membrane sheet with epithelium (1-2-5) was used as the transplantation sheet.
- the application method of the transplantation sheet was as follows. First, the water on the surface of the sclera of the pig eye subjected to the above treatment was wiped with a cotton swab to make the tissue semi-dry. In addition, it was based on the extent that fine water droplets were not recognized as judged visually. On this sclera, the amniotic membrane sheet with the adhesion component adhesion epithelium prepared in 1-2-5 was placed in a dry state with the adhesion component adhesion surface side down.
- the sheet was lightly pressed with sushi, and the entire area was brought into contact with the sclera while being careful not to introduce bubbles.
- the adhesive component adhering to the sheet is moistened by the weak water remaining on the sclera, and the sheet and the tissue are bonded.
- Displacement test We examined whether or not the sheet was offset by pressing it with a cotton swab and applying force in the horizontal direction.
- the sheet had good adhesiveness without slipping or peeling.
- the amount of fibrinogen in the table is the amount deposited per lcm 2 of the sheet. Also, + in the table indicates that the sheet has not slipped or peeled off, and 1 indicates that the sheet has shifted (even if it is slightly shifted).
- the adhering component ratio (fibrinogen adhering amount: thrombin adhering amount: aprotun adhering amount) was adjusted so that the thrombin adhering amount and the aprotun adhering amount were adjusted.
- the fibrinogen adhesion amount is 0.5 mg / cm 2 (thrombin adhesion amount: 12 ⁇ g / cm 2 , aprotune adhesion amount: 0.12 KIU / cm 2 ) or more, the sheet A very good adhesion is observed that does not dislodge or peel off the tissue. In other words, it became clear that sufficient adhesion could be obtained if the fibrinogen adhesion amount was 0.5 mg / cm 2 2-2.
- Adhesion component adhesion The adhesion and action of the epithelium-free amnion sheet (1-3-1.) Were examined using the rabbit sclera as the application site.
- the rabbit was anesthetized, and the conjunctiva of the eye was resected with scissors to expose about 1 X lcm, and the sclera was exposed (bearing state) to create a pterygium model.
- water and blood on the sclera surface were wiped with a cotton swab to make the sclera surface semi-dry.
- the guideline was that the bleeding stopped and the water content could not be discerned visually.
- the adhesive component-attached epithelium-free amnion sheet prepared in 1-3-1 was placed in a dry state with the adhesive component-attached surface side down.
- the sheet was lightly pressed with sushi, and the entire area was brought into contact with the sclera while being careful not to introduce bubbles.
- the rabbits were given an antibacterial agent (Taribit ointment) and a steroid agent (Linderon ointment) once a day.
- this sheet adhered to the sclera immediately after transplantation, and the sheet (amniotic membrane) was transferred in the first week after transplantation. Epithelialization occurred in all areas of the vegetation. On the other hand, angiogenesis and inflammation were not recognized. In the 2nd and 4th weeks, the sheet remained adhered and the state of epithelialization was maintained. At 2 and 4 weeks, angiogenesis and inflammation were not observed. In addition, the results of immunostaining revealed that fibrinogen was biodegraded in the 4th week. On the other hand, the amniotic membrane remained on the sclera. In addition, normal epithelialization was confirmed on the amniotic membrane, and the conjunctival covering was normal.
- Adhesion component adhesion The adhesion and action of the cultured cornea sheet (1-4 6.) were examined using the rabbit cornea as the application site.
- the cornea and conjunctival epithelium were removed to a thickness of 100 m by using a talent knife 4 mm outside the ring. This operation eliminates epithelial cells including corneal epithelial stem cells, and it can be considered that artificial ocular surface stem cell exhaustion is reproduced.
- the adhesive component adhesion-cultured corneal sheet was transplanted to a region slightly inside from the annulus. After surgery, antibacterial agents (Taribit ointment) and steroids (Linderon ointment) were applied once on Z day.
- Adhesion component adhesion The adhesiveness and action of the cultured skin sheet (1-5-8) can be examined with the following procedure using the rabbit skin as the application site.
- the epidermis of the part is peeled off.
- the adherent component adhesion-cultured skin sheet (1-5-8) is transplanted. After transplanting, lightly hold the sheet and bring the whole area into contact. After the operation, the antibacterial agent is applied externally once a day. By observing the adhesive state after transplantation and the change in the adhesive state over time, the adhesion and action of this sheet Can be evaluated.
- the sheet-like composition provided by the present invention can be used as a transplant material for tissue reconstruction.
- Application areas of the sheet composition of the present invention include ophthalmic areas, digestive surgery areas, gynecological areas, and dermatological areas.
- the sheet-like composition of this invention is excellent in adhesiveness. Therefore, it can be expected to adhere to the surrounding tissue in a short time after application and maintain a good adhesion state for a long time. Therefore, a high therapeutic effect can be expected without using suturing or the like. Depending on the application target, even higher adhesive strength is required, and even when it is considered appropriate to use suturing etc., it can be handled with relatively simple means, reducing the burden on doctors and patients. .
- step A includes the following steps:
- (A-1) a step of preparing biological cells
- a step of culturing and proliferating the seeded biological cells (A-3) A step of culturing and proliferating the seeded biological cells.
- step A includes the following steps:
- (A-1) a step of preparing biological cells
- a step of culturing and proliferating the seeded biological cells (A-3) A step of culturing and proliferating the seeded biological cells.
- Step A-3 is performed in the absence of a heterologous animal cell.
- Step A-3 is performed using a serum-free medium.
- Step A-3 The production according to any one of (1) to (4) above, wherein Step A-3 is performed using a medium containing only serum derived from the recipient as a serum component.
- the living body-derived cell is a cell derived from corneal epithelium, conjunctival epithelium, skin epidermis, hair follicle epithelium, epithelium of oral mucosa, iris pigment epithelium, retinal pigment epithelium, airway mucosa epithelium or intestinal mucosa epithelium. And the manufacturing method in any one of said (1)-(6).
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002594396A CA2594396A1 (en) | 2005-01-14 | 2006-01-11 | Sheet-shaped composition utilizing amnion and method of preparing the same |
JP2006552926A JPWO2006075602A1 (ja) | 2005-01-14 | 2006-01-11 | 羊膜を用いたシート状組成物及びその作製方法 |
EP06711538A EP1847277A4 (en) | 2005-01-14 | 2006-01-11 | SHEET-LIKE COMPOSITION USING AN AMNIOS AND PREPARATION METHOD THEREOF |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2005-008381 | 2005-01-14 | ||
JP2005008381 | 2005-01-14 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2006075602A1 true WO2006075602A1 (ja) | 2006-07-20 |
Family
ID=36677627
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2006/300189 WO2006075602A1 (ja) | 2005-01-14 | 2006-01-11 | 羊膜を用いたシート状組成物及びその作製方法 |
Country Status (7)
Country | Link |
---|---|
US (1) | US20080113007A1 (ja) |
EP (1) | EP1847277A4 (ja) |
JP (1) | JPWO2006075602A1 (ja) |
KR (1) | KR20070093991A (ja) |
CN (1) | CN101102800A (ja) |
CA (1) | CA2594396A1 (ja) |
WO (1) | WO2006075602A1 (ja) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010084970A1 (ja) | 2009-01-23 | 2010-07-29 | 国立大学法人大阪大学 | 標的細胞誘導用フィーダー細胞 |
JPWO2010143711A1 (ja) * | 2009-06-11 | 2012-11-29 | 一般財団法人化学及血清療法研究所 | 創傷被覆材 |
JP2019176942A (ja) * | 2018-03-30 | 2019-10-17 | 株式会社 ジャパン・ティッシュ・エンジニアリング | 移植材料の製造方法 |
Families Citing this family (23)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2338442B1 (en) | 2003-12-11 | 2013-01-30 | Isto Technologies Inc. | Particulate cartilage system |
US8512730B2 (en) | 2004-07-12 | 2013-08-20 | Isto Technologies, Inc. | Methods of tissue repair and compositions therefor |
WO2007025290A2 (en) | 2005-08-26 | 2007-03-01 | Isto Technologies, Inc. | Implants and methods for repair, replacement and treatment of joint disease |
US8372437B2 (en) | 2006-08-17 | 2013-02-12 | Mimedx Group, Inc. | Placental tissue grafts |
US8658851B2 (en) * | 2006-10-20 | 2014-02-25 | Keracure, Inc. | Devices with cells cultured on flexible supports |
US8163549B2 (en) | 2006-12-20 | 2012-04-24 | Zimmer Orthobiologics, Inc. | Method of obtaining viable small tissue particles and use for tissue repair |
WO2008128075A1 (en) | 2007-04-12 | 2008-10-23 | Isto Technologies, Inc. | Compositions and methods for tissue repair |
US9358320B2 (en) | 2008-04-25 | 2016-06-07 | Allosource | Multi-layer tissue patches |
US9480549B2 (en) | 2008-04-25 | 2016-11-01 | Allosource | Multi-layer tissue patches |
US20110129520A1 (en) * | 2008-04-25 | 2011-06-02 | Simon Bogdansky | Anti-Adhesion Barrier Wound Dressing Comprising Processed Amniotic Tissue and Method of Use |
US10130736B1 (en) | 2010-05-14 | 2018-11-20 | Musculoskeletal Transplant Foundation | Tissue-derived tissuegenic implants, and methods of fabricating and using same |
US8883210B1 (en) | 2010-05-14 | 2014-11-11 | Musculoskeletal Transplant Foundation | Tissue-derived tissuegenic implants, and methods of fabricating and using same |
US9352003B1 (en) | 2010-05-14 | 2016-05-31 | Musculoskeletal Transplant Foundation | Tissue-derived tissuegenic implants, and methods of fabricating and using same |
CN102266587B (zh) * | 2011-07-20 | 2013-12-04 | 山东省眼科研究所 | 一种含杯状细胞的重组眼结膜上皮膜片的制备方法 |
US10245306B2 (en) | 2012-11-16 | 2019-04-02 | Isto Technologies Ii, Llc | Flexible tissue matrix and methods for joint repair |
US20140178343A1 (en) | 2012-12-21 | 2014-06-26 | Jian Q. Yao | Supports and methods for promoting integration of cartilage tissue explants |
US10179191B2 (en) | 2014-10-09 | 2019-01-15 | Isto Technologies Ii, Llc | Flexible tissue matrix and methods for joint repair |
WO2016187413A1 (en) | 2015-05-21 | 2016-11-24 | Musculoskeletal Transplant Foundation | Modified demineralized cortical bone fibers |
MX2019014503A (es) * | 2017-06-05 | 2020-01-23 | Mayo Found Medical Education & Res | Metodos y materiales para cultivar, proliferar, y diferenciar celulas madre. |
CN107865816A (zh) * | 2017-11-08 | 2018-04-03 | 郑楠 | 一种羊膜基膜制备方法、单层胶联羊膜面膜及其制备方法 |
US11161204B1 (en) | 2020-04-10 | 2021-11-02 | Amnio Technology Llc | Allograft optimization system |
CN111450323A (zh) * | 2020-04-29 | 2020-07-28 | 天津百和至远医疗技术有限公司 | 一种即粘组织补片及其制备方法 |
CN111657269B (zh) * | 2020-06-22 | 2022-02-08 | 镇江雷音再生医学科技有限公司 | 纤维蛋白胶用于smile来源人角膜透镜保存前的保护处理方法 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH07265406A (ja) * | 1994-04-01 | 1995-10-17 | Terumo Corp | フィブリンゲル |
JP2001514050A (ja) * | 1997-08-28 | 2001-09-11 | バクスター・アクチエンゲゼルシャフト | フィブリノーゲンに基づく組織接着剤 |
WO2004078225A1 (ja) * | 2003-02-26 | 2004-09-16 | Amniotec Inc. | 羊膜由来医用材料、及びその作製方法 |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE60120676T2 (de) * | 2000-04-27 | 2007-05-24 | Jui-Fang Ray Tsai | Verfahren zur entwicklung von epithelialen stammzellen |
TWI290055B (en) * | 2002-03-14 | 2007-11-21 | Tissuetech Inc | Amniotic membrane covering for a tissue surface and devices facilitating fastening of membranes |
AU2003211971A1 (en) * | 2002-04-30 | 2003-11-17 | Amniotec Inc. | Corneal endothelium-like sheet and method of constructing the same |
AU2005231781A1 (en) * | 2004-03-31 | 2005-10-20 | Cook Incorporated | ECM-based graft material |
-
2006
- 2006-01-11 CN CNA2006800022451A patent/CN101102800A/zh active Pending
- 2006-01-11 WO PCT/JP2006/300189 patent/WO2006075602A1/ja active Application Filing
- 2006-01-11 CA CA002594396A patent/CA2594396A1/en not_active Abandoned
- 2006-01-11 KR KR1020077015648A patent/KR20070093991A/ko not_active Application Discontinuation
- 2006-01-11 US US11/795,302 patent/US20080113007A1/en not_active Abandoned
- 2006-01-11 EP EP06711538A patent/EP1847277A4/en not_active Withdrawn
- 2006-01-11 JP JP2006552926A patent/JPWO2006075602A1/ja active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH07265406A (ja) * | 1994-04-01 | 1995-10-17 | Terumo Corp | フィブリンゲル |
JP2001514050A (ja) * | 1997-08-28 | 2001-09-11 | バクスター・アクチエンゲゼルシャフト | フィブリノーゲンに基づく組織接着剤 |
WO2004078225A1 (ja) * | 2003-02-26 | 2004-09-16 | Amniotec Inc. | 羊膜由来医用材料、及びその作製方法 |
Non-Patent Citations (3)
Title |
---|
DUCHESNE B. ET AL.: "Use of Human Fibrin Glue and Amniotic Membrane Transplant in Corneal Perforation", CORNEA, vol. 20, no. 2, 2001, pages 230 - 232, XP002998660 * |
See also references of EP1847277A4 * |
SZURMAN P. ET AL: "Sutureless amniotic membrane fixation with fibrin glue for ocular surface reconstruction", INVEST. OPHTHALMOL. VIS. SCI., vol. 45, 2004, pages E-ABSTRACT 3917, XP002998659, Retrieved from the Internet <URL:http://abstracts.iovs.org/cgi/content/abstract/45/5/3917> * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010084970A1 (ja) | 2009-01-23 | 2010-07-29 | 国立大学法人大阪大学 | 標的細胞誘導用フィーダー細胞 |
JPWO2010143711A1 (ja) * | 2009-06-11 | 2012-11-29 | 一般財団法人化学及血清療法研究所 | 創傷被覆材 |
JP5675607B2 (ja) * | 2009-06-11 | 2015-02-25 | 一般財団法人化学及血清療法研究所 | 創傷被覆材 |
JP2019176942A (ja) * | 2018-03-30 | 2019-10-17 | 株式会社 ジャパン・ティッシュ・エンジニアリング | 移植材料の製造方法 |
JP6990139B2 (ja) | 2018-03-30 | 2022-02-03 | 株式会社 ジャパン・ティッシュ・エンジニアリング | 移植材料の製造方法 |
Also Published As
Publication number | Publication date |
---|---|
KR20070093991A (ko) | 2007-09-19 |
US20080113007A1 (en) | 2008-05-15 |
JPWO2006075602A1 (ja) | 2008-06-12 |
EP1847277A4 (en) | 2008-03-19 |
EP1847277A1 (en) | 2007-10-24 |
CA2594396A1 (en) | 2006-07-20 |
CN101102800A (zh) | 2008-01-09 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2006075602A1 (ja) | 羊膜を用いたシート状組成物及びその作製方法 | |
JP5109030B2 (ja) | シート状組成物 | |
US20080039940A1 (en) | Biological Tissue Sheet, Method Of Forming The Same And Transplantation Method By Using The Sheet | |
JP5795166B2 (ja) | 生命工学による組織コンストラクト並びに生産及び使用のための方法 | |
RU2645473C2 (ru) | Тканевые конструкции, полученные с помощью биоинженерии, и способы их получения и применения | |
US20140220102A1 (en) | Ectocornea-like sheet and method of constructing the same | |
JPWO2004078225A1 (ja) | 羊膜由来医用材料、及びその作製方法 | |
US20110293666A1 (en) | Bioengineered Tissue Constructs and Methods for Production and Use | |
JP2006507851A (ja) | コラーゲンバイオ繊維、ならびにその調製方法および使用 | |
EP1782846A1 (en) | Corneal epithelial sheet and process for producing the same | |
WO2003092762A1 (fr) | Feuille de type endothelium corneen et procede permettant de la produire | |
US20200078492A1 (en) | Process for obtaining a functional dermal substitute of decellurized amniotic membrane from the placenta combination with keratinocytes and its use as an agent for tissue regeneration of the skin | |
WO2007029676A1 (ja) | 生体組織シート及びその作製方法 | |
WO2006090696A1 (ja) | 上皮が除去された羊膜を調製する方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
WWE | Wipo information: entry into national phase |
Ref document number: 2006552926 Country of ref document: JP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2594396 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1020077015648 Country of ref document: KR |
|
WWE | Wipo information: entry into national phase |
Ref document number: 200680002245.1 Country of ref document: CN |
|
WWE | Wipo information: entry into national phase |
Ref document number: 11795302 Country of ref document: US |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2006711538 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 2006711538 Country of ref document: EP |