WO2021107303A1 - 히알루론산 및 폴리에틸렌글리콜을 포함하여 제조된 세포 시트 및 이의 제조방법 - Google Patents
히알루론산 및 폴리에틸렌글리콜을 포함하여 제조된 세포 시트 및 이의 제조방법 Download PDFInfo
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0068—General culture methods using substrates
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0655—Chondrocytes; Cartilage
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/22—Settling tanks; Sedimentation by gravity
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/20—Small organic molecules
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/30—Synthetic polymers
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/70—Polysaccharides
- C12N2533/80—Hyaluronan
Definitions
- the present invention relates to a method for constructing a cell sheet in a non-adherent state without a support, and to a method for manufacturing a multi-layered cell sheet without a separate lamination step, and to a cell sheet prepared by the method.
- a fold-based cell complex is advantageous.
- the cell structure cultured in a single-layer sheet state has a disadvantage that structural damage can easily occur in the process of handling for cell transplantation, etc., and when the cell sheet is multi-layered with high integrity to secure the thickness of the tissue to be regenerated, There were problems such as requiring additional technology for this purpose.
- HA hyaluronic acid
- PEG polyethylene glycol
- Another object of the present invention is to provide a plate-shaped cell sheet (cell slap) prepared by the above manufacturing method.
- Another object of the present invention is to provide a graft material for cartilage repair, including the plate-shaped cell sheet (cell slap) and a biodegradable polymer material.
- One aspect of the present invention for achieving the above object is a) culturing by sedimenting the cells in a growth medium containing hyaluronic acid; And b) adding a growth medium containing polyethylene glycol; provides a cell sheet manufacturing method comprising a.
- the cell sheet manufactured by the above manufacturing method may be in the form of a plate.
- the "cell sheet (cell slap)" refers to one or more cells arranged in layers, and the shape may be a plate shape, but is not limited thereto.
- the term "multilayer cell sheet” refers to a set of cell sheets formed by stacking one or more cell sheets in a vertical direction.
- “cell sheet” as used herein is used to refer to both a single-layered cell sheet and a multi-layered cell sheet.
- stacking or “forming” means that the thin film layers are stacked layer by layer in the order of creation.
- the laminated structure is not particularly limited as long as it is manufactured by a method known in the art.
- the “hyaluronic acid (HA)” is one of complex polysaccharides composed of amino acids and uronic acid, and is a high molecular compound composed of N-acetylglucosamine and glucuronic acid. It is mainly present in the vitreous body of the eye or the umbilical cord, and plays a role in preventing the penetration of bacteria or poisons.
- hyaluronic acid is a drug used for purposes such as an adjuvant for various ophthalmic surgeries, intra-articular injections, artificial tears, and wound healing.
- Hyaluronic acid is a type of polysaccharide that can contain 300 to 1000 times its own weight in moisture. It is known that the action is excellent, but there is no report on a method for preparing a cell sheet by using it together with PEG as in the present invention.
- the “polyethylene glycol (PEG)” is a kind of surfactant, and it is a chemical component that is used in various ways such as lotions, creams, shampoos, etc. by stably maintaining the product when manufacturing cosmetics and performing a role like glycerin. As in the present invention, there has been no report on a method for preparing a cell sheet by being used together with HA.
- the cells used in the cell sheet may be one or more selected from the group consisting of epidermal cells, fibroblasts, hepatocytes, mesenchymal stem cells, and chondrocytes, and more specifically, may be chondrocytes, but is not limited thereto. does not
- the chondrocytes are derived from the meniscus of rabbits, and a plate-shaped cell sheet (cell slap) was prepared using the chondrocytes, but is not limited thereto.
- the cells used in the cell sheet may be differentiated from human pluripotent stem cells.
- the "human pluripotent stem cells (PSCs)" can be differentiated into all three germ layers constituting the human body, that is, endoderm, mesoderm, and ectoderm.
- the human pluripotent stem cells are evaluated as very useful materials for clinical trials for the treatment of various diseases, new drug development, toxicity evaluation, disease modeling and early embryogenesis research. .
- the cell sheet may be characterized in that aggregation of cells is suppressed.
- the cell sheet is characterized in that it is formed without a separate scaffold in a non-adherent state, and in the cell sheet thus formed, aggregation of cells is remarkably suppressed, so that spheroids hardly appear. , It was confirmed that the cells maintained the sheet form as they settled on the bottom in the culture solution (Experimental Example 1, FIGS. 2 to 4).
- the "hydrogel” is a material that can contain a large amount of water, and can easily transmit and move substances necessary for cell survival, such as oxygen, water, water-soluble nutrients, polypeptides such as enzymes and cytokines, and waste products. It is a material or form that exists, and generally means that it is biocompatible.
- the shape or form of the hydrogel is not particularly limited as long as it can be incorporated into the cell sheet, but for example, fine particles, granular form, film form, tubular form, disk form, network form, mesh form, porous form, suspended state or dispersed form Various shapes or forms, such as the like, may be used. Among them, hydrogel particles resulting from solidification of an aqueous solution containing colloidal particles are preferable.
- particles made of any material can be used.
- polysaccharides include, but are not limited to, glycosaminoglycans such as hyaluronic acid and chondroitin sulfate, starch, glycogen, agar, pectin, and cellulose.
- collagen and its hydrolyzates gelatin, proteoglycan, fibronectin, vitronectin, laminin, entactin, tenascin, thrombospondin , von Willebrand factor (von Willebrand factor), osteopontin (osteopontin), fibrinogen (fibrinogen) and the like may be mentioned, but is not limited thereto.
- particles made of a material that is biocompatible and degraded by cells in a living body are suitable for the present invention, and most specifically, hyaluronic acid and polyethylene glycol may be used.
- the cell sheet may be composed of multi-layered cells, and each cell layer constituting the multi-layer may be characterized in that it has structural continuity while maintaining the integrity between the cell layers.
- the cell sheet composed of a single layer of cells through 2D culture without a support has the disadvantage that it is easy to tear and is difficult to handle due to its weak strength. There was a limitation in that cell delivery was difficult. In order to solve this problem, attempts have been made to form a multilayer structure to increase cell density, but there are limitations in that the process of forming the multilayer structure is complicated and it is difficult to implement 100% interlayer integrity.
- a multi-layered cell structure is formed by seeding cells at a high concentration, a high number of cells can be delivered per unit area, but the cells seeded at a high concentration are locally aggregated to form spheroids. There was a limitation in that it was impossible to form a cell sheet having one thickness and structural continuity.
- the cell sheet of the present invention has high cell transfer efficiency and is advantageous for tissue regeneration by forming a high-density sheet composed of multi-layered cells without a support (Experimental Example 5, Fig. 6).
- the support is unnecessary in the process of forming the cell sheet, there is no concern about structural deformation occurring during the decomposition of the support, so the structural stability is high, and the cell behavior is controlled through the composition of the medium rather than the coating. It has the advantage of being easy to apply as it is the same as the process for dealing with
- the cell sheet of the present invention induces the formation of a cell sheet under non-adherent conditions, it is not significantly limited in interactions between cells and substrates, and since surface treatment of the culture vessel is unnecessary, culture vessels of various materials can be constructed using 3D printing, etc.
- the cell sheet of the present invention can form a cell structure composed of multi-layered cells without a lamination process, structural stability is increased because it is not a multi-layered structure by lamination, and the lamination process can be omitted, thereby shortening the process and reducing the cell layer It was confirmed that more stable handling was possible by forming thick by itself (Experimental Example 4, FIG. 5).
- Another aspect of the present invention provides a cell sheet prepared by the above method.
- Another aspect of the present invention provides a graft material for tissue repair comprising the cell sheet.
- the tissue may be one or more selected from the group consisting of cartilage, bone, cornea, conjunctiva, adipose tissue, skin, muscle, fascia, tendon, aponeurosis, ligament, joint capsule, bursa, and epithelial tissue, and more specifically, It may mean cartilage, but is not limited thereto.
- the cells used in the cell sheet may be differentiated from human pluripotent stem cells into cells of a desired tissue.
- tissue repair may be used as a concept including both regeneration and healing of tissues, and regeneration reconstructs the damaged area with the same tissue composition as the damaged cells.
- healing refers to a fibroproliferative response that patches the damaged tissue.
- the term "for tissue repair” is not limited to the type of use for repair of damaged tissue, and includes not only cartilage damage, muscle or tendon damage, but also cosmetic filler use, cosmetic implant use, etc. can mean
- cartilage and soft tissues do not regenerate or heal themselves after being damaged, methods for promoting tissue growth are not suitable for application, unlike other tissues, and transplantation is known to be effective.
- the "graft material for tissue repair” of the present invention is a tissue that is damaged using physical properties when the tissue is damaged, such as cartilage, bone, cornea, conjunctiva, adipose tissue, skin, muscle, fascia, tendon, aponeurosis, ligament, joint capsule, bursa, etc.
- tissue that is damaged using physical properties when the tissue is damaged, such as cartilage, bone, cornea, conjunctiva, adipose tissue, skin, muscle, fascia, tendon, aponeurosis, ligament, joint capsule, bursa, etc.
- the site refers to a composition that treats the damage of the defect or dent site and restores the original volume, and if the composition is used for the above purpose, it is not limited to the embodiment and can be applied with appropriate changes.
- the cell sheet of the present invention is composed of a high concentration of cells without including a separate support and has excellent therapeutic and regenerative effects on the transplanted tissue site (FIG. 6), a tissue repair graft material comprising the cell sheet may indicate high tissue regeneration efficiency.
- the cell sheet manufacturing method of the present invention is characterized in that it is possible to form a cell sheet in a non-adherent state without a support, and can be detached and handled without a separate coating treatment. In addition, it is possible to manufacture a cell sheet capable of implanting a high number of cells per unit area without going through a separate lamination process or multi-layering process.
- FIG. 1 schematically shows a method for preparing a cell sheet using hyaluronic acid (HA) and PEG (poly-ethylene glycol).
- HA hyaluronic acid
- PEG poly-ethylene glycol
- Example 2 shows the results of confirming the degree of cell aggregation of the cell sheets prepared by the manufacturing methods of Example 1 and Comparative Examples 1 to 3 after culturing for 24 hours.
- Example 3 shows the results of observing the number of agglomerates (nodules) in Example 1 and Comparative Examples 1 to 3;
- Figure 5 shows the process of attaching the cell sheet of Example 1 to PLA (Poly Lactic Acid) mesh.
- FIG. 6 shows the results of induced cartilage differentiation for 2 or 3 weeks after attaching the cell sheet to the PLA mesh in a sandwich form, and confirming the shape of the formed tissue and whether the matrix is formed through histological staining (A: Example 1 A cell sheet of, B: a cell sheet seeded at high concentration on the surface of a collagen gel sheet).
- Example 1 Manufacturing method of cell sheet (Cell slap) using HA and PEG
- FIG. 1 A method for preparing a cell sheet using hyaluronic acid (HA) and polyethylene glycol (PEG) is briefly illustrated in FIG. 1 .
- HA hyaluronic acid
- PEG polyethylene glycol
- cell binding was prevented by coating the bottom surface with poly-HEMA (poly(2-hydroxyethyl methacrylate)) in a rectangular well closed on all sides.
- poly-HEMA poly(2-hydroxyethyl methacrylate)
- chondrocytes derived from rabbit meniscus between 3 and 5 passages were placed in a growth medium containing 1 mg/ml hyaluronic acid (HA) at 2 ⁇ 10 6 /100 ⁇ L. It was seeded with a concentration and cultured for 2.5 hours to induce the cell layer to sink uniformly.
- HA hyaluronic acid
- the growth medium is ⁇ -MEM (alpha-Minimum Essential Medium, Gibco Invitrogen) medium 5% fetal bovine serum (FBS, Lonza), 1% penicillin / streptomycin (penicillin / streptomycin, Welgene) , 2mM L-glutamine (Welgene), 10 -8 M dexamethasone (Sigma Aldrich) and 10 -4 M ascorbic acid (ascorbic acid, Sigma Aldrich) was added.
- FBS fetal bovine serum
- FBS fetal bovine serum
- penicillin / streptomycin penicillin / streptomycin, Welgene
- 2mM L-glutamine Welgene
- 10 -8 M dexamethasone Sigma Aldrich
- 10 -4 M ascorbic acid ascorbic acid, Sigma Aldrich
- Example 1 was prepared by culturing by adding a growth medium containing 1% polyethylene glycol (PEG).
- PEG polyethylene glycol
- the cell sheet of Comparative Example 1 was prepared in the same manner as in Example 1 except that hyaluronic acid (HA) and polyethylene glycol (PEG) were not added except that all conditions were the same as in Example 1.
- HA hyaluronic acid
- PEG polyethylene glycol
- the cell sheet of Comparative Example 2 was prepared using the preparation method of Example 1, but using a growth medium containing hyaluronic acid (HA) was maintained in the same manner as in Example 1, and after culturing for 2.5 hours, polyethylene glycol (PEG) A cell sheet of Comparative Example 2 was prepared by adding a growth medium not containing
- the cell sheet of Comparative Example 3 was prepared by maintaining the same conditions as in Example 1, but using a growth medium not added with hyaluronic acid (HA) when initially seeding rabbit meniscus-derived chondrocytes.
- HA hyaluronic acid
- Example 1 The manufacturing methods of Example 1 and Comparative Examples 1 to 3 are briefly illustrated in FIG. 1 .
- Comparative Example 1 which did not contain both HA and PEG, a large number of low-density spheroids were formed similarly to general spheroid formation conditions after 24 hours of culture, and Comparative Example 2 containing HA at the time of cell seeding In the case of , it was observed that the tendency to form relatively dense spheroids was high, and in the case of Comparative Example 3, in which only the PEG-containing medium was added without HA, aggregation of cells occurred in disorder to form huge spheroids. .
- Example 1 in which cells were seeded under conditions containing HA and a medium containing PEG was added, aggregation of cells was minimally suppressed and spheroids did not appear, and the cells were deposited as they settled to the bottom in the culture medium. It was confirmed that the shape was maintained to maintain a relatively uniform thickness.
- the PEG contained in the medium exhibits repulsive force against cells, so the force to minimize the interface between the PEG and cells contained in the medium. This occurs and it appears that the shape of the cell slap is maintained.
- the strength is weak, so it is easy to tear and it is difficult to handle without a separate handling tool.
- the process is not only very complicated, but also 100% interlayer integration It was difficult to realize the performance, and it was impossible to form a cell sheet with uniform thickness and structural continuity.
- the cell sheet of the present invention is formed under non-adherent conditions without a support, and is composed of multi-layered cells without a separate lamination process and is formed to be sufficiently thick.
- the cell sheet of Example 1 is PLA (Poly Lactic Acid) The process of attaching to the mesh was demonstrated (FIG. 5).
- the collagen-coated PLA mesh was attached around the front and back to form a complex for cartilage differentiation.
- Example 1 since the cell sheet of Example 1 was formed in a non-adherent condition without a separate support, it was possible to separate a uniform cell sheet having a constant thickness only with a cell scraper.
- the separated cell sheet was formed to be sufficiently thick with multi-layered cells, it was possible to handle without tearing only with tweezers without a separate handling tool.
- the cell sheet produced by the method for manufacturing the cell sheet of the present invention is formed in a non-adherent condition without a support, and is structurally stable by forming a multi-layered cell structure by itself without a separate lamination process, as well as a uniform thickness and structural continuity. was confirmed to have
- the cell sheet is attached to the PLA mesh in a sandwich form, and whether the cells on both surfaces of the mesh are combined with each other to be integrated into a single tissue and when cartilage differentiation The matrix-forming effect was confirmed.
- cartilage differentiation was induced for 2 or 3 weeks. Then, the shape of the formed tissue and whether the matrix was formed was confirmed through histological staining (FIG. 6).
- the tissues were dehydrated by sequentially adapting to 70%, 80%, 90%, and 100% alcohol, and then placed in xylene to adapt and then mounted with a plastic mounting solution (mountant). .
- the nucleus is stained with Weigert's hematoxylin for 10 minutes, washed with distilled water for 10 minutes, and Vieverich Crimson Acid Red Fluxin
- the cytoplasm was stained with an aqueous solution of (Biebrich scarlet acid red fuchsin) for 5 minutes.
- fix the staining with 1% acetic acid dehydrate the tissue by acclimating it to 70%, 80%, 90%, and 100% alcohol in turn, and put it in xylene to adapt it, and then put it in a plastic mounting solution (Permount, Fisher Chemical). All reagents were products of Sigma Aldrich unless otherwise indicated.
- the tissue formed from the cell sheet of Example 1 penetrated into the PLA mesh located in the center and then combined with each other to form a single tissue, confirming that the formation of cartilage-specific matrix was excellent did.
- the cell sheet formed using the collagen gel sheet had a significantly lower degree of penetration or movement of cells seeded on the collagen surface into the PLA mesh, resulting in two separate layers with the PLA mesh as the boundary. was formed.
- the degree of cartilaginous matrix formation was very low, and it was confirmed that the cell-seeded collagen gel sheet continued to remain (trichrome staining result).
- the cell sheet of the present invention prepared including HA and PEG is formed without a separate support such as a collagen gel sheet, has a high cell density, has high cell delivery efficiency, is advantageous for tissue regeneration, and has perfect integrity was confirmed to exhibit high structural continuity with
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Abstract
Description
Claims (7)
- a) 히알루론산이 포함된 성장배지에서 세포를 침강시켜 배양하는 단계; 및b) 폴리에틸렌글리콜이 포함된 성장배지를 추가하는 단계;를 포함하는, 세포 시트 제조방법.
- 제1항에 있어서,상기 세포는 표피세포, 섬유아세포, 간세포, 중배엽성 줄기세포 및 연골세포로 구성된 군에서 선택된 하나 이상인 것인, 세포 시트 제조방법.
- 제1항에 있어서,상기 세포시트는 판(plate)형인 것인, 세포 시트 제조방법.
- 제1항에 있어서,상기 세포시트는 세포의 응집이 억제된 것인, 세포 시트 제조방법.
- 제1항 내지 제4항 중 어느 한 항의 제조방법으로 제조된, 세포 시트.
- 제5항의 세포시트를 포함하는, 조직 수복용 이식재.
- 제6항에 있어서,상기 조직은 연골, 골, 각막, 결막, 지방조직, 피부, 근육, 근막, 건, 건막, 인대, 관절낭, 점액낭 및 상피조직으로 이루어진 군에서 선택되는 하나 이상인 것인, 조직 수복용 이식재.
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CN202080081520.3A CN114761543A (zh) | 2019-11-25 | 2020-06-11 | 包含透明质酸及聚乙二醇来制备的细胞片层及其制备方法 |
US17/779,186 US20220409770A1 (en) | 2019-11-25 | 2020-06-11 | Cell sheet comprising hyaluronic acid and polyethylene glycol, and method for producing same |
EP20892125.4A EP4067477A4 (en) | 2019-11-25 | 2020-06-11 | CELL SHEET COMPRISING HYALURONIC ACID AND POLYETHYLENE GLYCOL, AND METHOD FOR PRODUCING SAME |
JP2022530685A JP2023503467A (ja) | 2019-11-25 | 2020-06-11 | ヒアルロン酸及びポリエチレングリコールを含んで製造された細胞シート及びその製造方法 |
CA3159454A CA3159454A1 (en) | 2019-11-25 | 2020-06-11 | Cell sheet comprising hyaluronic acid and polyethylene glycol, and method for producing same |
AU2020393233A AU2020393233B2 (en) | 2019-11-25 | 2020-06-11 | Cell sheet comprising hyaluronic acid and polyethylene glycol, and method for producing same |
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KR1020190152197A KR102228619B1 (ko) | 2019-11-25 | 2019-11-25 | 히알루론산 및 폴리에틸렌글리콜을 포함하여 제조된 세포 시트 및 이의 제조방법 |
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CN (1) | CN114761543A (ko) |
AU (1) | AU2020393233B2 (ko) |
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EP1857126B1 (en) * | 2005-02-28 | 2018-10-24 | CellSeed Inc. | Cultured cell sheet, production method thereof, and application method thereof |
JP2013116045A (ja) * | 2010-03-08 | 2013-06-13 | Osaka Univ | 三次元細胞集合体の作製方法およびそれに用いる細胞培養用三次元ゲル担体並びに三次元細胞集合体 |
JP6189747B2 (ja) * | 2011-02-28 | 2017-08-30 | 株式会社セルシード | ヒアルロン酸産生能を有する細胞シート及びその製造方法 |
EP3409763A4 (en) * | 2016-01-29 | 2019-10-30 | National Cerebral and Cardiovascular Center | CELLULAR AMAS, CELL STRUCTURE AND THREE DIMENSIONAL TISSUE BODY |
JP6823874B2 (ja) * | 2016-04-22 | 2021-02-03 | 国立大学法人東北大学 | 細胞・ナノ薄膜複合体の製造方法 |
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JP2018501845A (ja) * | 2014-12-11 | 2018-01-25 | イーティーエッチ チューリッヒ | 軟骨修復のための移植片足場及びその製造方法 |
KR20170099033A (ko) * | 2016-02-22 | 2017-08-31 | 중앙대학교 산학협력단 | 기계적 안정성이 증대된 세포시트 |
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FENG YU, XIAODONG CAO, YULI LI, LEI ZENG, BO YUAN, XIAOFENG CHEN: "An injectable hyaluronic acid/PEG hydrogel for cartilage tissue engineering formed by integrating enzymatic crosslinking and Diels–Alder "click chemistry"", POLYMER CHEMISTRY, vol. 5, no. 3, 1 January 2014 (2014-01-01), pages 1082 - 1090, XP055477947, ISSN: 1759-9954, DOI: 10.1039/C3PY00869J * |
See also references of EP4067477A1 |
STEVEN R CALIARI, BURDICK JASON A: "A practical guide to hydrogels for cell culture", NATURE METHODS, NATURE PUB. GROUP, NEW YORK, vol. 13, no. 5, New York, pages 405 - 414, XP055392866, ISSN: 1548-7091, DOI: 10.1038/nmeth.3839 * |
YANG RUIRUI, XU CAIXIA, WANG TAO, WANG YUANQI, WANG JINGNAN, QUAN DAPING, DENG DAVID Y. B.: "PTMAc-PEG-PTMAc hydrogel modified by RGDC and hyaluronic acid promotes neural stem cells' survival and differentiation in vitro", RSC ADVANCES, vol. 7, no. 65, 1 August 2017 (2017-08-01), pages 41098 - 41104, XP055815360, DOI: 10.1039/C7RA06614G * |
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KR102228619B1 (ko) | 2021-03-17 |
AU2020393233A1 (en) | 2022-06-09 |
US20220409770A1 (en) | 2022-12-29 |
AU2020393233B2 (en) | 2024-06-27 |
EP4067477A1 (en) | 2022-10-05 |
EP4067477A4 (en) | 2024-04-03 |
CA3159454A1 (en) | 2021-06-03 |
CN114761543A (zh) | 2022-07-15 |
JP2023503467A (ja) | 2023-01-30 |
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