WO2016163828A1 - Support polymère permettant d'augmenter la liaison entre les cellules et procédé de culture de cellules l'utilisant - Google Patents

Support polymère permettant d'augmenter la liaison entre les cellules et procédé de culture de cellules l'utilisant Download PDF

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WO2016163828A1
WO2016163828A1 PCT/KR2016/003739 KR2016003739W WO2016163828A1 WO 2016163828 A1 WO2016163828 A1 WO 2016163828A1 KR 2016003739 W KR2016003739 W KR 2016003739W WO 2016163828 A1 WO2016163828 A1 WO 2016163828A1
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cell
cells
polymer
peptide
present
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이근용
이재원
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한양대학교 산학협력단
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Priority claimed from KR1020160043529A external-priority patent/KR20160121780A/ko
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor

Definitions

  • the present invention was made by the task number NRF-2013R1A2A2A03010055 under the support of the Ministry of Science, ICT and Future Planning, the research management specialized organization of the project is the Korea Research Foundation, the research project name is "medium-level researcher support project,” Tissue regeneration using sensitized microgels ”, the host institution is Hanyang University Industry-Academic Cooperation Group, and the research period is from 06.01.
  • the present invention relates to a polymeric cell support that induces binding between cells.
  • tissue engineering and regenerative medicine cell therapy is commonly used to restore damaged tissue and regeneration.
  • various treatments using stem cells have been carried out.
  • simple injection of cells has low engraftment rate and difficulty in controlling differentiation, and many studies have been conducted to maximize such effects.
  • various methods have been developed to maximize the engraftment rate and differentiation capacity of cells in vivo. It is essential to use a support for effective cell delivery and tissue regeneration, and recently, cell-bound support using polymers has been developed.
  • Most of the polymer scaffolds are used to fix the cell binding material in order to increase the cell binding capacity, or to fix the growth factor and growth factor-derived peptides to induce differentiation of cells, but have not reached a satisfactory level.
  • Such conventional polymer scaffolds are focused on enhancing the binding force between cells and extra cellular matrix.
  • none of the cells in vivo exist only by the binding between the extracellular matrix, but exists in an environment that binds to various other cells. Therefore, there is a need for the development of a support that can provide cell-to-cell binding in culturing cells in vitro and in vivo .
  • the inventors have made intensive research efforts to develop polymeric cell scaffolds that provide cell-cell binding.
  • a polymer support conjugated with a cadherin-attached oligopeptide not only the cell-supporting bond is formed, but also the cell-cell binding is induced to be useful for cell culture and stem cell differentiation.
  • Another object of the present invention to provide a cell colonization culture method.
  • Still another object of the present invention is to provide a composition for inducing stem cell differentiation.
  • Another object of the present invention to provide a stem cell culture method for differentiation into chondrocytes.
  • Another object of the present invention to provide a cell composition for implantation in the body.
  • the invention provides a polymeric cell support for forming a cell population comprising:
  • the inventors have made intensive research efforts to develop polymeric cell scaffolds that provide cell-cell binding.
  • a polymer support conjugated with a cadherin-attached oligopeptide not only the cell-supporting bond is formed, but also the cell-cell binding is induced to be useful for cell culture and stem cell differentiation. It was found possible.
  • the polymer cell support of the present invention includes a cDHERIN-attached oligopeptide, thereby forming a bond with CADHERIN protein on the surface of the cultured cell, and inducing cell-cell binding, thereby enabling clustering of cultured cells.
  • CADHERIN protein is a type 1 transmembrane protein, a protein that plays an important role in forming interhesion.
  • biocompatible polymer backbone refers to a structure made of a biocompatible polymer that has affinity upon contact with a cell and does not exhibit a rejection reaction, and is a material of a structure such as a scaffold for conventional cell culture.
  • Various materials known to be used may be used without limitation. However, it is necessary to include a functional group for binding a caherin adherent peptide, or to be surface modified to include such a functional group.
  • the polymer backbone may be prepared in various forms by various methods, and is not particularly limited.
  • the polymer backbone of the present invention may be prepared, for example, in the form of microspheres, and for this, a water-in-oil emulsion method may be used. See the embodiments on this specification for specific methods.
  • cadherin adherent oligopeptide refers to an oligopeptide sequence capable of forming a bond with a cadherin protein of a cultured cell, wherein the oligopeptide sequence forms a bond with a cadherin protein on a cell. It will be obviously understood that the effects of the present invention are significantly exerted only if so. Identification of oligopeptides that form bonds with Cadherin proteins can be carried out through a variety of methods known in the art, such as two-hybrid analysis or three-hybrid analysis (US Pat. No. 5,283,317; Zervos et. al., Cell 72, 223-232, 1993; Madura et al., J.
  • an oligopeptide that binds to the cadrerin protein can be screened by using the cadrerin protein as a bait protein.
  • Two-hybrid systems are based on the modular nature of the transcription factors composed of cleavable DNA-binding and activation domains. For simplicity, this assay uses two DNA constructs.
  • a cadrine-encoding polynucleotide is fused to a DNA binding domain-encoding polynucleotide of a known transcription factor (eg, GAL-4).
  • a DNA sequence encoding an oligopeptide (“prey” or “test material”) of interest is fused to a polynucleotide encoding the activation domain of the known transcription factor. If bait and prey interact in in vivo to form a complex, the DNA-binding and activation domains of the transcription factors are contiguous, which triggers transcription of the reporter gene (eg, luciferase). As a result, the expression of the reporter gene can be detected, which indicates that the prey can bind with the cadrerin protein, which means that the prey can be used as the cadrerin adherent oligopeptide of the present invention.
  • the reporter gene eg, luciferase
  • the bond between the polymer backbone and the catherine attachable oligopeptide of the present invention is an amide bond formed between the biocompatible polymer backbone described above and the catherine attachable oligopeptide described above.
  • Cadherin adherent oligopeptides can be associated with the polymeric backbone using N-terminal or C-terminal amino or carboxyl groups.
  • the polymer backbone of the present invention is required to include a functional group capable of forming an amide bond with a cadmin attached oligopeptide.
  • the biocompatible polymer backbone of the present invention comprises a carboxyl group, hydroxyl group or amino group for forming the amide bond.
  • the polymer backbone of the present invention not only includes a carboxyl group or a hydroxyl group capable of binding to the amino group of the caherin-adhering oligopeptide, or an amino group capable of bonding to the carboxyl group of the caherin-adhering oligopeptide, It is understood to include surface modified to include carboxyl, hydroxyl, or amino groups.
  • amide bond formation between carboxyl group and amino group is more generally known
  • amide bond formation between hydroxyl group and amino group is also known by using an organometallic catalyst (Kim Ki-Chul et al., Such as amide using an organometallic catalyst from an alcohol and an amine).
  • an organometallic catalyst Kim Ki-Chul et al., Such as amide using an organometallic catalyst from an alcohol and an amine.
  • the polymer backbone of the present invention may include a carboxyl group or hydroxyl group, or a surface modified to include a carboxyl group or hydroxyl group.
  • the polymer backbone of the present invention may form an amide bond with the N-terminal amino group of the cadherin-attached oligopeptide, or form a bond with the C-terminal carboxyl group of the cadherin-attached oligopeptide via a linker. It is possible.
  • Linkers capable of forming bonds between carboxyl groups in the present invention are specifically, for example, diamine, divinylsulfone, 1,4-butanediol, diglycidyl ether (BDDE) and glutaraldehyde, Carbodiimide, hydroxysuccinimide, imidoester, maleimide, haloacetyl, disulfide, hydroazide, or alkoxyamine may be used, but is not limited thereto.
  • the polymeric backbone of the present invention is any one or more selected from the group consisting of alginic acid polymers, chitosan polymers and hyaluronic acid polymers.
  • the “alginic acid polymer” of the present invention is a polymer polymer represented by Formula 1, and is a polymer suitable for forming a bond with a cadmin adherent oligopeptide of the present invention including a carboxyl group.
  • the "chitosan polymer” of the present invention is a polymer polymer represented by the formula (2), and includes a hydroxyl group and an amino group, and is a polymer suitable for forming a bond with the cardherin adherent oligopeptide of the present invention.
  • the "hyaluronic acid polymer” of the present invention is a polymer represented by the formula (3), and includes a hydroxyl group and a carboxyl group, and is a polymer suitable for forming a bond with the cardherin adhesion oligopeptide of the present invention.
  • a polymer polymer surface-modified to include a carboxyl group, hydroxyl group or amino group can be suitably used.
  • a method described in the prior art can be used without limitation, and specifically, introducing a carboxyl group to the surface of the polymer polymer by graft using acrylic acid (AAc), for example, It is possible.
  • the caherin adherent oligopeptide of the present invention is an LRP5 peptide consisting of SEQ ID NO: 1 or HAV peptide consisting of SEQ ID NO: 2.
  • LRP5 peptide of the present invention is a sequence of the first part of the sequence list as a part of the amino acid sequence of the low-density lipoprotein receptor-related protein 5 (LRP5) that binds effectively to the cardherin protein.
  • LRP5 peptide is a sequence of the amino acid sequence of a specific site of the cardinin protein directly participating in the tight junction (tight junction) of the cell consisting of the second sequence of the sequence listing. The inventors anticipated that the effect of the present invention would be caused by caherin adhesion, independent of sequence specificity, and demonstrated this using two sequences of caherin attachment peptides that were not related to each other.
  • the LRP5 peptide or HAV peptide of the present invention forms a bond with the cardherin protein of the cell.
  • Cadherin protein is a protein that plays an important role of directly participating in the intercellular adhesion (adhesion), as described above, the present inventors target the cadherin protein oligopeptides, specifically examples For example, using the LRP5 peptide and / or HAV peptide, a polymer cell support capable of forming a cell population was prepared.
  • the cell support of the present invention further comprises (c) an integrin adherent oligopeptide bound to the above-described polymer backbone.
  • the "integrin” of the present invention is a transmembrane receptor that acts as a bridge of cell-cell and extracellular matrix (ECM) interactions.
  • ECM extracellular matrix
  • the term "integrin adherent oligopeptide” refers to a peptide strand consisting of approximately 2-20 amino acids that form a bond with the aforementioned integrins.
  • the cell population is more compact in spherical form. It was confirmed that it was formed.
  • the integrin adherent oligopeptides of the invention are RGD, PSHRN, FHRRIKA, YIGSR, KGD, RHD, NGR, SDGR, KQAGDV, LDV, DGEA, YGYYGDALR, FYFDLR, DALR, DLR, RLD, KRLDGS, IDA, IDAPS and REDV.
  • PSHRN (Benoit and Anseth, 2005), FHRRIKA (Rezania and Healy, 1999), YIGSR (Massia and Hubbell, 1990), KGD (Plow et al, 1985; Scarborough et al 1993), RHD (Ghiso et al 1992, Saporito-Irwin & van Nostrand 1995), NGR, SDGR (Yamada & Kennedy 1987), KQAGDV (Kloczewiak et al, 1984; Lam et al 1987), LDV (Guan & Hynes 1990, Mold et al 1990), DGEA (Staatz et al 1991), YGYYGDALR, FYFDLR (Underwood et al 1995), DALR, DLR, RLD (Altieri et al, 1993; Koivunen et al, 1995), KRLDGS (Alfieri et al 1993), LDV, IDA, IDAPS and REDV (Mould) et al,
  • the present invention provides a cell colonization culture method comprising the step of culturing the cells in contact with the cell support described above.
  • a feeder cell layer for cell attachment.
  • the polymer cell support of the present invention acts like a feeder cell layer, and not only forms a cell-supporter bond, but also induces a cell-cell bond to form cells in a cell population. It can be cultured. There is no particular limitation on the cells that can be cultured by the method of the present invention.
  • Cell culture of the present invention can be made in conventionally used cell culture medium.
  • the cell culture medium may be appropriately selected and used depending on the type of cultured cell and the purpose of the culture.
  • the basal medium constituting the medium of the present invention is a conventional medium in the art, such as DMEM (Dulbecco's modification of Eagle's medium, Dulbecco, R. et al., Virology 8: 396 (1959)), Eagle's MEM [Eagle's minimum essensial medium, Eagle, H. Science 130: 142 (1959)], ⁇ -MEM [Stanner, CP et al., NAT. New Biol . 230: 52 (1971)], Iscove's MEM [Iscove, N.
  • DMEM Dulbecco's modification of Eagle's medium, Dulbecco, R. et al., Virology 8: 396 (1959)
  • Eagle's MEM Eagle's minimum essensial
  • the medium of the present invention may additionally include antibiotics, and may include, but are not limited to, for example, penicillin, streptomycin, gentamycin, neomycin, polymyxin or amphotericin B.
  • the present invention provides a composition for inducing stem cell differentiation comprising the above-described cell support.
  • the "stem cells” of the present invention are not particularly limited, and all of the cells having the characteristics of stem cells, that is, undifferentiated, indefinite proliferation and differentiation into specific cells are cells that can be applied to the present invention.
  • Stem cells to which the present invention is applied are largely divided into pluripotent stem cells and multipotent stem cells, including embryonic stem cells (ES) and embryonic germ cells (EG).
  • Embryonic stem cells are derived from the internal cell mass (ICM) of the blastocyst, and embryonic germ cells are derived from primordial germ cells of the 5-10 week old gonadal ridge.
  • Pluripotent stem cells are found in embryonic, fetal and adult tissues, including adult stem cells. Pluripotent stem cells proliferate indefinitely in vitro and have the ability to differentiate into a variety of cells derived from all three embryonic layers (ectoderm, mesoderm and endoderm). Pluripotent stem cells, on the other hand, have the ability to differentiate into the specific tissue from which they originate, and the ability to autoreproduce is typically limited to the life of the organism. Sources of pluripotent stem cells are all tissue types and are primarily isolated from bone marrow, blood, liver, skin, intestine, pancreas, brain, skeletal muscle and pulp.
  • the present invention is characterized by inducing colonization regardless of the concentration of cultured cells using the above-described cell support, and promoting cell differentiation by cell-cell binding formed at this time, and other culture conditions are greatly limited. Instead, the cells can be appropriately selected and adjusted according to the cultured cells and the culture purpose.
  • the composition of the present invention may include a conventionally known stem cell differentiation condition medium, the differentiation condition medium can be used by appropriately selecting a conventionally known medium according to the type of cells to be obtained as a result of differentiation.
  • the present invention comprises the step of contacting the stem cell differentiation induction composition and the stem cells described above and culturing in chondrocyte differentiation conditions medium, stem cell culture method for differentiation into chondrocytes To provide.
  • chondrocyte differentiation condition medium refers to a medium under conditions that induce differentiation of stem cells into chondrocytes, and as a representative example, insulin, transferrin, selenium acid, woo supplemented with growth factors Serum-deficient media (Johnstone et al., 1998) comprising serum albumin, linoleic acid, pyruvate, ascorbate and / or dexamethasone, and DMEM / F12, dexamethasone, L used in one embodiment of the present invention.
  • a medium containing ascorbic acid, insulin and / or TGF- ⁇ 1 may be used, but is not limited thereto.
  • stem cell culture method for differentiation into chondrocytes of the present invention in particular, in the case of using a cell support further comprising an integrin-attached oligopeptide, as well as a cadrerin-attached oligopeptide, a cadherin- or integrin-attached Gene expression of chondrocyte differentiation factor was significantly increased as compared with the case of using a cell support including sex oligoptide alone. Therefore, preferably, stem cells may be cultured using a cell support including both a caherin-adhering oligopeptide and an integrin-adhering oligopeptide, and more effectively differentiated into chondrocytes.
  • the present invention provides a composition for transplantation in vivo cells comprising (a) the cell support described above, (b) cells for transplantation in vivo bound to the cell support.
  • Cells for transplantation of the present invention are specifically, for example, chondrocytes, myoblasts, hepatocytes, osteoblasts, embryonic stem cells, embryonic germ cells (embryonic) germ cells, adult stem cells, mesenchymal stem cells, neural stem cells, vascular endothelial stem cells, hematopoietic stem cells, liver stem cells, heart stem cells, pancreatic stem cells, endothelial progenitors, growth Outgrowth endothelial cells, mesenchymal stem cells, hematopoietic stem cells, neural stem cells, satellite cells, intestinal epithelial cells, smooth muscle cells and fibers There are blasts, but not limited thereto.
  • the cell composition for implantation of the body of the present invention can be injected at a desired location using, for example, injection or surgical methods.
  • the cells for transplantation in the body of the invention are stem cells.
  • the stem cells are as described above in the present specification, and may be used in a conventionally known use mode for stem cell therapy for treating diseases by introducing stem cells into the body.
  • the cells for transplantation in the body of the present invention are chondrocytes differentiated from stem cells.
  • composition of the present invention may additionally include a pharmaceutically acceptable carrier, the pharmaceutically acceptable carrier included in the therapeutic composition of the present invention is commonly used in the formulation, lactose, dextrose, sucrose, Sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzo 8, talc, magnesium stearate, mineral oil, and the like.
  • a pharmaceutically acceptable carrier included in the therapeutic composition of the present invention is commonly used in the formulation, lactose, dextrose, sucrose, Sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxy
  • compositions of the present invention may further comprise lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like in addition to the above components.
  • lubricants wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like in addition to the above components.
  • Pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
  • compositions of the present invention can be administered parenterally, and local injection is the most preferred method of administration.
  • Suitable dosages of the compositions of the present invention may be prescribed in various ways depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of the patient, food, time of administration, route of administration, rate of excretion and response to reaction. have.
  • the dosage of the composition is preferably 1 times 1 x 10 5 - 5 x 10 8 cells / ml and can be adjusted by the amount required.
  • compositions of the present invention allow for excellent formation of intercellular networking to fully exert the function of the cells to be transplanted, thereby actually exhibiting improved therapeutic efficacy in diseased animals.
  • Intracellular graft cell compositions of the present invention can be used for the treatment of cartilage-injury diseases, specifically, for example, osteoarthritis, rheumatoid arthritis, meniscus cartilage injury (Meniscus Injury), It can be used for the treatment of Costoschondritis, Relapsing polychondritis, and Chondrosacoma.
  • the present invention provides a polymer cell support for forming cell populations.
  • the present invention provides a cell colonization culture method.
  • the present invention provides a composition for inducing stem cell differentiation.
  • the present invention provides a stem cell culture method for differentiation into chondrocytes.
  • the present invention provides a cell composition for implantation in the body.
  • the polymer cell support of the present invention can induce cell-cell conjugation to form cell populations even when small concentrations of cells are used in cell culture.
  • composition for inducing stem cell differentiation of the present invention and the stem cell culture method, it is possible to effectively differentiate the stem cells into desired cells.
  • Figure 1 schematically shows the attachment process of the cells using the support to which the cell adhesion peptide of the present invention is immobilized.
  • Figure 2 shows the process and predicted structure of the cell-adhesive peptide of the present invention binds to the polymer structure of the polymer support.
  • Figure 3 shows the results of confirming that the cell-adhesive peptide is bound to the polymer support as a result of the IR analysis of the polymer support to which the cell-adhesive peptide is bound.
  • Figure 4 shows the results of measuring the physical properties of the polymer support bound to the cell-adhesive peptide of the present invention.
  • Figure 5 shows the results of observing the phenotype of each cell by attaching a variety of cells to the polymer support to which the cell adhesion peptide of the present invention is bound.
  • Figure 6 shows the results of observing the binding force between cells of the cells attached to the polymer support to which the cell-adhesive peptide of the present invention is bound.
  • Figure 7 shows the effect of the cell-adhesive peptides of the present invention on the phenotype of stem cells, and shows the results confirmed that they are attached by a specific binding force between cells.
  • Figure 8 shows the results of observing the phenotype of the stem cells and the binding force between the cells when differentiating stem cells into chondrocytes using a low concentration of the cell number of the cell adhesion peptide conjugated polymer support of the present invention.
  • Figure 9 shows the results of analyzing the differentiation capacity when differentiating stem cells to chondrocytes using low concentration of the cell-adhesive peptide conjugated polymer support of the present invention.
  • Figure 10 shows the results of observing the phenotype and the intercellular binding force of the stem cells when the high concentration of stem cells to the chondrocytes using the polymer support conjugated to the cell-adhesive peptide of the present invention.
  • Figure 11 shows the results of analyzing the differentiation power when differentiation of high concentration of stem cells into chondrocytes using the polymer support to which the cell-adhesive peptide of the present invention is bound.
  • FIG. 12 shows a method for forming a polymer microsphere and a process of binding a cell-adhesive peptide to the polymer microsphere.
  • Figure 13 shows the process of forming aggregates using the polymer microspheres and stem cells of the present invention.
  • Figure 14 shows the results of measuring the physical properties of the aggregates formed of the polymer microspheres and stem cells of the present invention.
  • Figure 15 shows the results of observing the phenotype of the stem cells in the aggregate formed of the polymer microspheres and stem cells of the present invention.
  • Figure 16 shows the results of observing the survival rate of stem cells in the aggregate formed of the polymer microspheres and stem cells of the present invention.
  • Figure 17 shows the results of histological evaluation (Alcian blue, serious red) to analyze the differentiation ability of stem cells into cartilage cells in aggregates formed of the polymer microspheres and stem cells of the present invention.
  • Figure 18 shows the results of analyzing the differentiation power when differentiating stem cells into chondrocytes in the aggregate formed of the polymer microspheres and stem cells of the present invention.
  • Figure 19 shows the results of analyzing the growth ability of the stem cells according to the concentration of the card herin peptide of the present invention.
  • Figure 20 shows the results of analyzing the phenotype and adhesion of stem cells according to the concentration of the cardinin peptide of the present invention.
  • Synthesis was performed using alginic acid, a natural polymer, and the EDC / NHS reaction was used to bind the carboxyl group of alginic acid to the amine group of the cell-adhesive peptide.
  • Alginic acid was dissolved in MES buffer at pH 6.5, and peptides were bound using EDC and NHS, and then the peptide-bound alginic acid solution was dialyzed with distilled water and NaCl for 3 days.
  • Activated carbon was added to the aqueous solution of alginic acid to complete dialysis to remove impurities for 30 minutes, filtered through a 0.22 ⁇ l filter, and lyophilized (see FIG. 2).
  • the amino acid analysis was performed on the polymer support to which the LRP5 peptide was attached to determine whether amino acids were efficiently bound (see Table 1).
  • HSC hematopoietic cells
  • M3T3 osteoblasts
  • D1 stem cells adult stem cells
  • NIH3T3 fibroblasts
  • Observation of cell adhesion and cell phenotype in the RGD peptide-bound polymer scaffold confirmed the binding to hematopoietic cells (HSC), osteoblasts (MC3T3), adult stem cells (D1 stem cells) and fibroblasts (NIH3T3). And evenly binding to single cells (see FIG. 5A).
  • HSC hematopoietic cells
  • M3T3 osteoblasts
  • D1 stem cells adult stem cells
  • NIH3T3T3 fibroblasts
  • each cell was combined to form a cell population in the polymer support to which the LRP5 peptides provided intercellular binding (see FIG. 5B).
  • HSC Hepatoblasts
  • M3T3 osteoblasts
  • D1 stem cells adult stem cells
  • fibroblasts NIH3T3
  • Example 2 Evaluation of Chondrocyte Differentiation Ability of Stem Cells Using Polymer Supports Providing Cell-to-Cell Binding
  • the adult stem cells (D1 stem cell, CRL-12424, ATCC) used are adult stem cells derived from mouse bone marrow, and are very suitable for the study of differentiation into bone, cartilage and adipocytes.
  • the peptide was not bound as a result of binding to the support after inhibiting cell-cell binding by treating EGTA solution. Cell adhesion similar to that of the polymeric support could be observed (see FIG. 7). This is a result showing that the polymer support into which the catherine-adherent oligopeptide of the present invention is introduced provides cell-cell binding.
  • chondrocyte differentiation medium conditions DMEM / F12, 10 nM dexamethasone, 50 ⁇ g / ml L-ascorbic acid, 5 ⁇ l / ml Insulin, 10 ng / ml TGF-beta1
  • concentration of the stem cells used was incubated with the number of cells (1.0 x 10 6 cells / ml) of a concentration lower than one tenth of the cell concentration used in the general three-dimensional cell culture method.
  • the cell phenotype was observed.
  • stem cells existed as single cells and immunostained beta-catenin, a protein that can confirm cell-to-cell binding. In contrast, it was confirmed that the binding was not performed, but in contrast, the polymer support in which the LRP5 peptide was introduced showed that the stem cells were clustered, and the beta-catenin protein expression was also markedly increased. (See FIG. 8).
  • Stem cells (D1 stem cell, CRL-) were cultured in chondrocyte differentiation medium (DMEM / F12, 10 nM dexamethasone, 50 ⁇ g / ml L-ascorbic acid, 5 ⁇ l / ml Insulin, 10 ng / ml TGF-beta1) for 2 weeks. 12424, ATCC) induced the differentiation of chondrocytes, and it was observed that RGD peptides were also forming a cell population in the polymer scaffold to which the RGD peptides provided the binding between the cell and the support. In the polymer scaffold to which the LRP5 peptide was bound, more cells were observed to form colonies (see FIG. 10).
  • chondrocyte differentiation medium DMEM / F12, 10 nM dexamethasone, 50 ⁇ g / ml L-ascorbic acid, 5 ⁇ l / ml Insulin, 10 ng / ml TGF-beta
  • Example 3 cell Intercellular Polymeric microspheres that provide a bond ( microsphere Cartilage cells of stem cells differentiation Ability evaluation
  • GGGGSHAVSS specific site peptide
  • Microspheres were prepared using alginic acid, a natural polymer, and a water-in-oil emulsion was used to prepare alginic acid microspheres.
  • Alginate polymer was dissolved in an organic solvent containing isooctane, SPAN 80, and Tween 80, and reacted in an aqueous solution of 20 wt% CaCl 2 for one hour to prepare microspheres. Thereafter, an EDC / NHS reaction was used to bind the carboxyl group of the alginic acid microspheres to the amine group of the caherin-adhesive oligopeptide (HAV peptide).
  • HAV peptide caherin-adhesive oligopeptide
  • Alginic acid microspheres were dissolved in MES buffer at pH 6.5, peptides were bound using EDC and NHS, and the peptide-alginated aqueous alginic acid solution was dialyzed with distilled water and NaCl for 3 days. Alginate aqueous solution completed dialysis was added to activated carbon to remove impurities for 30 minutes, filtered through a 0.22 ⁇ m filter and lyophilized (see Fig. 12).
  • a hydrogel was prepared by introducing peptides of various concentrations (7, 14, 28, 70 ⁇ g / mg) into the alginic acid, to each hydrogel Stem cells were attached at a concentration of 2.0 ⁇ 10 6 cells / ml and the experiment was performed for a total of 5 days.
  • the hydrogel was removed with a solution containing EDTA, and after the polymer support was removed, the number of cells was measured by a hemocytometer to evaluate the growth ability of the stem cells for 5 days. .
  • Agglomerates composed of stem cells and polymer microspheres were prepared by mixing the polymer microspheres and the stem cells having the binding force with the cells.
  • the polymer microspheres were used at a concentration of 1.0 x 10 8 / ml at 10 wt%, and the cells were 1.0 x 10.
  • Aggregates were formed using a concentration of 7 / ml. Aggregates thus formed were also incubated using round bottom well plates (see FIG. 13).
  • RGD-alg represents the RGD peptide introduced polymer microspheres
  • Cadherin-alg represents the HAV peptide introduced polymer microspheres
  • Alginate represents a polymer microsphere control group without the peptide introduced
  • solid line is the elastic modulus ( G ') and the dotted line represent the loss modulus (G ").
  • Polymeric scaffolds with RGD peptides alone which provide the binding between cells and scaffolds, to assess the binding capacity of stem cells and to identify cell phenotype and cell-cell binding using polymer microspheres that provide cell-to-cell binding. Experiment was carried out using as a comparison group.
  • RGD-alginate represents the RGD peptide introduced polymer microspheres
  • CAD-alginate represents the HAV peptide introduced polymer microspheres
  • R / C-alginate refers to the polymer microspheres to which the RGD peptide and HAV peptide is simultaneously bonded
  • Alginate indicates a polymer microsphere control group without peptide.
  • the survival rate of stem cells in the aggregates formed from the polymer microspheres and the stem cells was evaluated.
  • the aggregates using the polymer microspheres to which the RGD peptide and / or the HAV peptide were not bound can be observed that the aggregates did not form because there was no binding force between the cells and the microspheres, and the cell survival rate was lower than that of the other groups.
  • the aggregates using the polymer microspheres in which the RGD peptide or the HAV peptide were respectively bound it was confirmed that a specific volume of aggregates were formed by the binding force between the cells and the microspheres, and the cell survival rate was also high.
  • stem cells were mixed to form aggregates and cultured under chondrocyte differentiation medium conditions for 2 weeks (DMEM / F12, 10 nM dexamethasone, 50 ⁇ g / ml L-ascorbic acid, 5 ⁇ l / ml Insulin, 10 ng / ml TGF). -beta1).
  • RGD-Alginate represents RGD peptide-introduced polymer microspheres
  • Cadherin-Alginate represents HAV peptide-introduced polymer microspheres
  • Alginate represents a polymer microsphere control in which no peptide is introduced.

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Abstract

La présente invention permettant d'induire la liaison entre les cellules. Le support cellulaire polymère de la présente invention permet l'induction de l'adhérence intercellulaire en culture, ce qui permet d'obtenir une composition permettant d'induire la différenciation cellulaire, une composition permettant la transplantation cellulaire, ou analogues.
PCT/KR2016/003739 2015-04-09 2016-04-08 Support polymère permettant d'augmenter la liaison entre les cellules et procédé de culture de cellules l'utilisant WO2016163828A1 (fr)

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KR20150050380 2015-04-09
KR10-2015-0050380 2015-04-09
KR1020160043529A KR20160121780A (ko) 2015-04-09 2016-04-08 세포와 세포간의 결합 증진을 위한 고분자 지지체 및 이를 이용한 세포 배양 방법
KR10-2016-0043529 2016-04-08

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7446120B2 (en) * 2000-01-24 2008-11-04 Adherex Technologies, Inc. Peptidomimetic modulators of cell adhesion

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7446120B2 (en) * 2000-01-24 2008-11-04 Adherex Technologies, Inc. Peptidomimetic modulators of cell adhesion

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LEE, JAE - WON ET AL.: "(108-16) A Control of Stem Cell Phenotype Using Cell-interactive Hydrogels", THE POLYMER SOCIETY OF KOREA, vol. 39, no. 2, 2014, pages 43 *
LEE, JAE - WON ET AL.: "Controlling Chondrogenic Differentiation of Stem Cell-instructive Hydrogels", THE POLYMER SOCIETY OF KOREA, vol. 39, no. 1, 2014, pages 143 *
SCHENSE, JASON C. ET AL.: "Enzymatic Incorporation of Bioactive Peptides into Fibrin Matrices Enhances Neurite Extension", NATURE BIOTECHNOLOGY, vol. 18, no. 4, 2000, pages 415 - 419, XP001134964 *
ZHANG, JIANJUN ET AL.: "Physically Associated Synthetic Hydrogels with Long-term Covalent Stabilization for Cell Culture and Stem Cell Transplantation", ADVANCED MATERIALS, vol. 23, no. 43, 2011, pages 5098 - 5103, XP055320633 *

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