CN116440060A - 一种负载干细胞外泌体促进区域快速上皮化的微针的制备方法 - Google Patents
一种负载干细胞外泌体促进区域快速上皮化的微针的制备方法 Download PDFInfo
- Publication number
- CN116440060A CN116440060A CN202310424373.0A CN202310424373A CN116440060A CN 116440060 A CN116440060 A CN 116440060A CN 202310424373 A CN202310424373 A CN 202310424373A CN 116440060 A CN116440060 A CN 116440060A
- Authority
- CN
- China
- Prior art keywords
- stem cell
- exosome
- supernatant
- needle
- micro
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000001808 exosome Anatomy 0.000 title claims abstract description 72
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 32
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 239000006228 supernatant Substances 0.000 claims abstract description 30
- 239000000017 hydrogel Substances 0.000 claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 16
- 238000010438 heat treatment Methods 0.000 claims abstract description 13
- 210000002901 mesenchymal stem cell Anatomy 0.000 claims abstract description 12
- 238000000432 density-gradient centrifugation Methods 0.000 claims abstract description 11
- 238000000016 photochemical curing Methods 0.000 claims abstract description 10
- 230000005540 biological transmission Effects 0.000 claims abstract description 9
- 238000013508 migration Methods 0.000 claims abstract description 6
- 230000000877 morphologic effect Effects 0.000 claims abstract description 5
- 230000008556 epithelial cell proliferation Effects 0.000 claims abstract description 4
- 239000004017 serum-free culture medium Substances 0.000 claims abstract description 4
- 230000010393 epithelial cell migration Effects 0.000 claims abstract description 3
- 238000002156 mixing Methods 0.000 claims abstract description 3
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 20
- 229910052802 copper Inorganic materials 0.000 claims description 20
- 239000010949 copper Substances 0.000 claims description 20
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 14
- 229930006000 Sucrose Natural products 0.000 claims description 14
- 239000005720 sucrose Substances 0.000 claims description 14
- 238000001723 curing Methods 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 6
- OOIBFPKQHULHSQ-UHFFFAOYSA-N (3-hydroxy-1-adamantyl) 2-methylprop-2-enoate Chemical compound C1C(C2)CC3CC2(O)CC1(OC(=O)C(=C)C)C3 OOIBFPKQHULHSQ-UHFFFAOYSA-N 0.000 claims description 5
- 239000000287 crude extract Substances 0.000 claims description 5
- 230000001737 promoting effect Effects 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 5
- 239000000853 adhesive Substances 0.000 claims description 3
- IYDGMDWEHDFVQI-UHFFFAOYSA-N phosphoric acid;trioxotungsten Chemical compound O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.OP(O)(O)=O IYDGMDWEHDFVQI-UHFFFAOYSA-N 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 claims description 2
- 229920001287 Chondroitin sulfate Polymers 0.000 claims description 2
- 108010010803 Gelatin Proteins 0.000 claims description 2
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 claims description 2
- 229940059329 chondroitin sulfate Drugs 0.000 claims description 2
- 238000004043 dyeing Methods 0.000 claims description 2
- 239000008273 gelatin Substances 0.000 claims description 2
- 229920000159 gelatin Polymers 0.000 claims description 2
- 235000019322 gelatine Nutrition 0.000 claims description 2
- 235000011852 gelatine desserts Nutrition 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 239000006143 cell culture medium Substances 0.000 claims 1
- 238000004627 transmission electron microscopy Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 9
- 210000001519 tissue Anatomy 0.000 abstract description 5
- 239000000463 material Substances 0.000 abstract description 3
- 230000008439 repair process Effects 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 13
- 239000000243 solution Substances 0.000 description 7
- 238000010586 diagram Methods 0.000 description 4
- 210000002919 epithelial cell Anatomy 0.000 description 3
- 230000005012 migration Effects 0.000 description 3
- XLYOFNOQVPJJNP-ZSJDYOACSA-N Heavy water Chemical compound [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 230000008508 epithelial proliferation Effects 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 241001122767 Theaceae Species 0.000 description 1
- 239000000980 acid dye Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 238000001085 differential centrifugation Methods 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 230000003076 paracrine Effects 0.000 description 1
- 230000037368 penetrate the skin Effects 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 238000013271 transdermal drug delivery Methods 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
- A61K9/0021—Intradermal administration, e.g. through microneedle arrays, needleless injectors
-
- 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
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
-
- 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
- A61L27/14—Macromolecular materials
- A61L27/20—Polysaccharides
-
- 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
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/222—Gelatin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
- A61M37/0015—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin by using microneedles
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
- A61M37/0015—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin by using microneedles
- A61M2037/0046—Solid microneedles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
- A61M37/0015—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin by using microneedles
- A61M2037/0053—Methods for producing microneedles
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
-
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Dermatology (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Transplantation (AREA)
- Pharmacology & Pharmacy (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Zoology (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Inorganic Chemistry (AREA)
- Rheumatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Anesthesiology (AREA)
- Virology (AREA)
- Medical Informatics (AREA)
- Urology & Nephrology (AREA)
- Botany (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Sampling And Sample Adjustment (AREA)
Abstract
本发明属于组织工程修复材料技术领域，涉及一种负载干细胞外泌体促进区域快速上皮化的微针的制备方法；收集无血清培养的间充质干细胞培养基上清液，采用密度梯度离心法提取干细胞外泌体，PBS溶解后，取新鲜样本进行透射电子显微镜，鉴定外泌体形态；研究干细胞外泌体对上皮细胞增殖和迁移性能的影响；在模具中加入提取的干细胞外泌体共混后的光固化水凝胶，除泡3‑5次，烘箱加热浓缩3‑5次，UV405nm光固化后脱模，并通过扫描电镜观察其形貌特征。
Description
技术领域
本发明属于组织工程修复材料技术领域，本发明涉及一种负载干细胞外泌体促进区域快速上皮化的微针的制备方法。
背景技术
微针材料是一种通常以阵列的形式放置的微型针头，可以穿透皮肤等屏障，为经皮给药提供一种微创的途径。水凝胶微针于2012年首次报道，是微针的最新形式。其由可膨胀的聚合物(交联水凝胶)组成，与之前提到的其他微针的形式相比，水凝胶微针具有不同的工作机制。当插入皮肤时，水凝胶微针会因为水凝胶的亲水性而膨胀，这意味着它们很容易吸收水分，也更适合于生物医学应用。
间充质干细胞作是一种合适的组织工程中种子细胞，由于其储存方面的限制和细胞在体外扩增时发生容易发生衰老等不足，在临床应用中存在着一定局限性。由间充质干细胞通过旁分泌信号分泌的外泌体，不仅具有与间充质干细胞相同的效果，而且还具有定向输送、低免疫原性和高修复性等优点，一般认为外泌体的直径约为30至150纳米(nm)，外泌体含有其来源细胞的各种分子成分，外泌体也可以作为细胞信号传导的有效媒介而广泛用于医学再生领域，它们能够将RNA和蛋白质的信息从来源细胞转移到周围环境中的其他细胞。
发明内容
本发明的目的在于解决现有技术中的不足，提供一种负载干细胞外泌体促进区域快速上皮化的微针的制备方法，发挥干细胞外泌体促进上皮再生的作用，并构建合适的负载干细胞外泌体的水凝胶微针。
为了达到上述目的，本发明是通过以下技术方案实现的：一种负载干细胞外泌体促进区域快速上皮化的微针的制备方法，所述方法如下：
步骤(1)密度梯度离心法提取间充质干细胞外泌体及鉴定
收集无血清培养的间充质干细胞培养基上清液，采用密度梯度离心法提取干细胞外泌体，将干细胞外泌体使用PBS溶解后，取新鲜样本进行透射电子显微镜，鉴定外泌体形态；
步骤(2)检测干细胞外泌体对上皮细胞增殖和迁移性能的影响；
步骤(3)光固化水凝胶微针支架的制备
在模具中加入与步骤(1)提取的干细胞外泌体共混后的光固化水凝胶；除泡3-5次，烘箱加热浓缩3-5次，UV405nm光固化后微针脱模，并通过扫描电镜观察其形貌特征。
优选的，步骤(1)中收集无血清培养的间充质干细胞培养基上清液，采用密度梯度离心法提取干细胞外泌体，将干细胞外泌体使用PBS溶解的具体步骤如下：
步骤(1-1)：于超净台中取实验样本100mL加入离心管，配平后300g，4℃离心10min，取上清液转移至新的离心管中；
步骤(1-2)：将收集的上清液配平后2000g，4℃离心10min，取上清液转移至高速离心机配套的离心管中；
步骤(1-3)：配平所收集的超速离心机配套离心管中的上清液后10000g，4℃离心30min，取上清液转移至超速离心机配套的离心管中；
步骤(1-4)：配平所收集的超速离心机配套离心管中的上清液后120000g，4℃离心70min，取透明沉淀，并用1000μL的PBS重悬；所得即为外泌体溶液粗提液；
步骤(1-5)：准备新的超离管，每管加入6mL质量体积比为30％蔗糖垫，并于蔗糖垫上轻柔加入步骤(1-4)中外泌体溶液粗提液，配平后120000g，4℃离心70min，弃去上清，用1mL无菌PBS重悬外泌体沉淀。
优选的，步骤(1-5)中所述蔗糖垫的组分为1MTris-HCI，MTris-HCI的pH＝7.4、蔗糖、D2O。
优选的，步骤(1)中取新鲜样本进行透射电子显微镜，鉴定外泌体形态的具体步骤如下：取一枚铜网置于不干胶底纸上，吸取30ul外泌体样品滴于铜网上，静置10min，使用滤纸去除铜网上多余的样品，吸取30ul磷钨酸染液滴于铜网上，染色2min；使用滤纸去除铜网上多余的染液，将铜网放置于红外烘灯下烘干10min后透射电镜观察形貌。
优选的，步骤(3)光固化水凝胶微针支架的制备的具体步骤如下：将10％(w/v)HAMA水凝胶溶液与10-50ug/mL浓度的外泌体共混，并滴加在模具中，室温真空除泡法3-5次，30℃烘箱加热浓缩3-5次，UV405nm光固化10min后微针脱模，并通过扫描电镜观察其形貌特征。
优选的，步骤(3)光固化水凝胶微针支架的制备的具体步骤如下：将10％(w/v)GelMA水凝胶溶液与10-50ug/mL浓度的外泌体共混，并滴加在模具中，45-50℃水浴加热除泡3-5次，30-35℃烘箱加热浓缩3-5次，UV405nm光固化5-10min后微针脱模，并通过扫描电镜观察其形貌特征。
优选的，步骤(3)中所述光固化水凝胶采用光固化甲基丙烯酸酰化明胶、光固化硫酸软骨素代替。
本发明具有以下有益效果：(1)本发明制备的负载外泌体的微针可以有效促进上皮细胞的增殖迁移，有望用于组织工程修复领域。
(2)本发明制备一种负载干细胞外泌体促进区域快速上皮化的微针，使得负载成分易于进入目标组织的深部，而不是停留在表面，其在目标区域可以有效稳定的达到缓释效果。
(3)本发明一种负载干细胞外泌体促进区域快速上皮化的微针的制备方法，采用密度梯度离心，将差速离心与蔗糖密度相结合，可将外泌体从其他颗粒物和污染物种分离，可分离处低丰度的珍贵外泌体，其分离纯度高于单纯超速离心法，使得制备的微针效果更优。
附图说明
图1为蔗糖垫密度梯度离心示意图及提取后的外泌体透射电镜下的形态图；
图2为外泌体对上皮细胞增殖、迁移的影响；
图3为负载间充质干细胞外泌体的微针的设计概念图与实物图；
图4为负载间充质干细胞外泌体的微针的扫描电镜图。
具体实施方式
下面结合实施例对本发明作进一步的说明，但并不作为对本发明限制的依据。
实施例1、样本外泌体分离(密度梯度离心法)
1)于超净台中取实验样本100mL分装加入到50mL的离心管，配平后300g，4℃离心10min，取上清液转移至新的50mL离心管中，此步骤目的为去除上清中未贴壁的细胞；
2)将收集的上清液配平后2000g，4℃离心10min，取上清液转移至高速离心机配套的50mL离心管中，此步骤目的在于去除死细胞；
3)配平所收集的超速离心机配套离心管中的上清液后10000g，4℃离心30min，取上清液转移至超速离心机配套的离心管中，此步骤目的为去除细胞碎片；
4)配平所收集的超速离心机配套离心管中的上清液后120000g，4℃离心70min，取透明沉淀，并用1000μL的PBS重悬；所得即为外泌体溶液粗提液；
5)提前配置好30％(质量体积比)的蔗糖垫，组分为1MTris-HCI(pH7.4)、蔗糖、D2O(重水)；
6)准备新的超离管，每管加入6mL 30％蔗糖垫，并于蔗糖垫上轻柔加入外泌体，配平后120000g,4℃离心70min，可见管中分为4层，弃去上清，1mL无菌PBS重悬外泌体沉淀；
7)取一枚铜网置于不干胶底纸上，吸取30ul外泌体样品滴于铜网上，静置10min，使用滤纸去除铜网上多余的样品，吸取30ul磷钨酸染液滴于铜网上，染色2min。使用滤纸去除铜网上多余的染液，将铜网放置于红外烘灯下烘干10min后透射电镜观察形貌。
如图1所示，图1右侧可见经过密度梯度离心法提取的外泌体在透射镜下呈现电信的茶托样结构；不同细胞(A)来源外泌体应用于不同细胞(B)的起的作用效果是不一样的，如图2所示，外泌体有促进上皮细胞增殖、迁移的效果。对照组细胞数为522.7778±38.5382，外泌体组细胞数为737.5556±21.2374，具有统计学差异，这里验证了其对我目标细胞(上皮细胞)的具体效果。
实施例2、水凝胶微针支架的制备，以HAMA水凝胶为例
1)滴加10％(w/v)HAMA水凝胶溶液与10-50ug/mL浓度的外泌体混合液；
2)真空机除泡(重复3次)；
3)30℃烘箱加热12h，3次重复加热并添加10％HAMA水凝胶后浓缩；
4)UV405nm光固化5-10min；
5)微针脱模。
如表1所示，不同浓度Exosome对上皮细胞增殖的影响，数据形式为均数±标准差；
表1、不同浓度Exosome对上皮细胞增殖的影响
实施例3、微针的制备，以GelMA为例
(1)滴加10％(w/v)GelMA水凝胶和10-50ug/mL浓度的外泌体混合液，
(2)45-50℃水浴加热除泡(重复3次)，
(3)30-35℃烘箱加热，3-5次加热浓缩，每次10-12h，
(4)UV405nm光固化5-10min后微针脱模，扫描电镜下观察其形貌特征。
以上显示和描述了本发明的基本原理、主要特征及优点。但是以上所述仅为本发明的具体实施例，本发明的技术特征并不局限于此，任何本领域的技术人员在不脱离本发明的技术方案下得出的其他实施方式均应涵盖在本发明的专利范围之中。
Claims (7)
1.一种负载干细胞外泌体促进区域快速上皮化的微针的制备方法，其特征在于，所述方法如下：
步骤(1)密度梯度离心法提取间充质干细胞外泌体及鉴定
收集无血清培养的间充质干细胞培养基上清液，采用密度梯度离心法提取干细胞外泌体，将干细胞外泌体使用PBS溶解后，取新鲜样本进行透射电子显微镜，鉴定外泌体形态；
步骤(2)检测干细胞外泌体对上皮细胞增殖和迁移性能的影响；
步骤(3)光固化水凝胶微针支架的制备
在模具中加入与步骤(1)提取的干细胞外泌体共混后的光固化水凝胶；除泡3-5次，烘箱加热浓缩3-5次，UV405nm光固化后微针脱模，并通过扫描电镜观察其形貌特征。
2.根据权利要求1所述的负载干细胞外泌体促进区域快速上皮化的微针的制备方法，其特征在于，步骤(1)中收集无血清培养的间充质干细胞培养基上清液，采用密度梯度离心法提取干细胞外泌体，将干细胞外泌体使用PBS溶解的具体步骤如下：
步骤(1-1)：于超净台中取实验样本100mL加入离心管，配平后300g，4℃离心10min，取上清液转移至新的离心管中；
步骤(1-2)：将收集的上清液配平后2000g，4℃离心10min，取上清液转移至高速离心机配套的离心管中；
步骤(1-3)：配平所收集的超速离心机配套离心管中的上清液后10000g，4℃离心30min，取上清液转移至超速离心机配套的离心管中；
步骤(1-4)：配平所收集的超速离心机配套离心管中的上清液后120000g，4℃离心70min，取透明沉淀，并用1000μL的PBS重悬；所得即为外泌体溶液粗提液；
步骤(1-5)：准备新的超离管，每管加入6mL质量体积比为30％蔗糖垫，并于蔗糖垫上轻柔加入步骤(1-4)中外泌体溶液粗提液，配平后120000g，4℃离心70min，弃去上清，用1mL无菌PBS重悬外泌体沉淀。
3.根据权利要求2所述的负载干细胞外泌体促进区域快速上皮化的微针的制备方法，其特征在于，步骤(1-5)中所述蔗糖垫的组分为1MTris-HCI，MTris-HCI的pH＝7.4、蔗糖、D2O。
4.根据权利要求1所述的负载干细胞外泌体促进区域快速上皮化的微针的制备方法，其特征在于，步骤(1)中取新鲜样本进行透射电子显微镜，鉴定外泌体形态的具体步骤如下：取一枚铜网置于不干胶底纸上，吸取30ul外泌体样品滴于铜网上，静置10min，使用滤纸去除铜网上多余的样品，吸取30ul磷钨酸染液滴于铜网上，染色2min；使用滤纸去除铜网上多余的染液，将铜网放置于红外烘灯下烘干10min后透射电镜观察形貌。
5.根据权利要求1所述的负载干细胞外泌体促进区域快速上皮化的微针的制备方法，其特征在于，步骤(3)光固化水凝胶微针支架的制备的具体步骤如下：将10％(w/v)HAMA水凝胶溶液与10-50ug/mL浓度的外泌体共混，并滴加在模具中，室温真空除泡法3-5次，30℃烘箱加热浓缩3-5次，UV405nm光固化10min后微针脱模，并通过扫描电镜观察其形貌特征。
6.根据权利要求1所述的负载干细胞外泌体促进区域快速上皮化的微针的制备方法，其特征在于，步骤(3)光固化水凝胶微针支架的制备的具体步骤如下：将10％(w/v)GelMA水凝胶溶液与10-50ug/mL浓度的外泌体共混，并滴加在模具中，45-50℃水浴加热除泡3-5次，30-35℃烘箱加热浓缩3-5次，每次10-12h，UV405nm光固化5-10min后微针脱模，并通过扫描电镜观察其形貌特征。
7.根据权利要求1所述的负载干细胞外泌体促进区域快速上皮化的微针的制备方法，其特征在于，步骤(3)中所述光固化水凝胶采用光固化甲基丙烯酸酰化明胶、光固化硫酸软骨素代替。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310424373.0A CN116440060A (zh) | 2023-04-20 | 2023-04-20 | 一种负载干细胞外泌体促进区域快速上皮化的微针的制备方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310424373.0A CN116440060A (zh) | 2023-04-20 | 2023-04-20 | 一种负载干细胞外泌体促进区域快速上皮化的微针的制备方法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116440060A true CN116440060A (zh) | 2023-07-18 |
Family
ID=87127215
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310424373.0A Pending CN116440060A (zh) | 2023-04-20 | 2023-04-20 | 一种负载干细胞外泌体促进区域快速上皮化的微针的制备方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116440060A (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117143812A (zh) * | 2023-10-31 | 2023-12-01 | 中国人民解放军军事科学院军事医学研究院 | 一种富含活性线粒体的细胞外微囊泡的微针贴片的制备及其应用 |
-
2023
- 2023-04-20 CN CN202310424373.0A patent/CN116440060A/zh active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117143812A (zh) * | 2023-10-31 | 2023-12-01 | 中国人民解放军军事科学院军事医学研究院 | 一种富含活性线粒体的细胞外微囊泡的微针贴片的制备及其应用 |
| CN117143812B (zh) * | 2023-10-31 | 2024-01-26 | 中国人民解放军军事科学院军事医学研究院 | 一种富含活性线粒体的细胞外微囊泡的微针贴片的制备及其应用 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112280734A (zh) | 一种外泌体的制备方法及含有外泌体的干细胞增殖试剂 | |
| CN116440060A (zh) | 一种负载干细胞外泌体促进区域快速上皮化的微针的制备方法 | |
| CN112522191A (zh) | 一种间充质干细胞的培养方法 | |
| US20220325247A1 (en) | METHOD FOR PROMOTING GROWTH OF PARIETAL DECIDUAL BASALIS-MESENCHYMAL STEM CELLS (PDB-MSCs) | |
| CN110564682A (zh) | 一种大规模生产人脂肪间充质干细胞外泌体的方法 | |
| CN113940949B (zh) | 一种负载外泌体的GelMA水凝胶微针及其制备方法和应用 | |
| CN105085938B (zh) | 白芨多糖水凝胶、培养基质及其应用与诱导脐带间充质干细胞向角膜上皮细胞分化的方法 | |
| WO2016082882A1 (en) | Secretomes and method for producing secretomes | |
| CN115305233B (zh) | 大豆苷元与芹菜素复合琼脂糖-胶原蛋白水凝胶三维培养干细胞及细胞外囊泡的制备与用途 | |
| CN113957036B (zh) | 一种子宫内膜类器官培养基及培养方法 | |
| CN114807015A (zh) | 一种促进胰岛α细胞转化为β细胞的诱导方法及其应用 | |
| CN110846280B (zh) | 人肠癌原代细胞及其培养方法与应用 | |
| CN112029717A (zh) | 无血清体外驯化培养人间充质干细胞 | |
| CN116590231A (zh) | 一种用于肠道肿瘤细胞培养的水凝胶支架及其制备方法 | |
| CN113444679B (zh) | 一种人泪腺干细胞及其分化培养方法与应用 | |
| CN113712897B (zh) | 一种可溶性微针介导的载紫草素的透皮递药系统及其制备 | |
| CN116121174A (zh) | 一种鸡胚胎成纤维细胞体外三维培养和分离的方法 | |
| CN114736855A (zh) | 一种干细胞外泌体高纯度提取方法 | |
| CN111925985B (zh) | 间充质干细胞的驯化培养方法 | |
| CN111004782B (zh) | 人肠癌原代细胞及其培养方法与应用 | |
| TW202302840A (zh) | 間質幹細胞培養物及其製備方法 | |
| CN114231491A (zh) | 一种利用肿瘤患者自体胸、腹水制备水凝胶个性化培养肿瘤类器官的方法 | |
| CN113018501A (zh) | 一种内皮祖细胞外泌体医用敷料、制备方法及其应用 | |
| CN116790493A (zh) | 一种经血源干细胞外泌体递送体系及其制备方法和应用 | |
| CN114703125B (zh) | 外泌体的获得方法、制剂及其在制备抗衰老、皮肤修复制品上的应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |