CN116790490A - Collagen-combined exosome, exosome freeze-dried powder and preparation method - Google Patents
Collagen-combined exosome, exosome freeze-dried powder and preparation method Download PDFInfo
- Publication number
- CN116790490A CN116790490A CN202310688610.4A CN202310688610A CN116790490A CN 116790490 A CN116790490 A CN 116790490A CN 202310688610 A CN202310688610 A CN 202310688610A CN 116790490 A CN116790490 A CN 116790490A
- Authority
- CN
- China
- Prior art keywords
- exosome
- freeze
- stem cells
- exosomes
- collagen
- 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 151
- 239000000843 powder Substances 0.000 title claims abstract description 33
- 238000002360 preparation method Methods 0.000 title abstract description 16
- 210000000130 stem cell Anatomy 0.000 claims abstract description 43
- 102000008186 Collagen Human genes 0.000 claims abstract description 21
- 108010035532 Collagen Proteins 0.000 claims abstract description 21
- 229920001436 collagen Polymers 0.000 claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 14
- 239000013604 expression vector Substances 0.000 claims abstract description 13
- 238000003259 recombinant expression Methods 0.000 claims abstract description 13
- 239000002773 nucleotide Substances 0.000 claims abstract description 3
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 3
- 238000004108 freeze drying Methods 0.000 claims description 25
- 210000004027 cell Anatomy 0.000 claims description 18
- 239000003223 protective agent Substances 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 16
- 241000700605 Viruses Species 0.000 claims description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- 239000008176 lyophilized powder Substances 0.000 claims description 8
- 229940099596 manganese sulfate Drugs 0.000 claims description 7
- 235000007079 manganese sulphate Nutrition 0.000 claims description 7
- 239000011702 manganese sulphate Substances 0.000 claims description 7
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 7
- 238000001890 transfection Methods 0.000 claims description 7
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 6
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 6
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 4
- 239000008055 phosphate buffer solution Substances 0.000 claims description 4
- 210000001185 bone marrow Anatomy 0.000 claims description 3
- 239000002158 endotoxin Substances 0.000 claims description 3
- 210000003954 umbilical cord Anatomy 0.000 claims description 3
- 239000012141 concentrate Substances 0.000 claims description 2
- 210000004698 lymphocyte Anatomy 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 239000008363 phosphate buffer Substances 0.000 claims description 2
- 239000002994 raw material Substances 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 18
- 230000006378 damage Effects 0.000 abstract description 11
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 21
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 14
- 230000008569 process Effects 0.000 description 11
- 239000000243 solution Substances 0.000 description 10
- 230000008685 targeting Effects 0.000 description 9
- 239000006228 supernatant Substances 0.000 description 8
- 208000027418 Wounds and injury Diseases 0.000 description 7
- 238000007710 freezing Methods 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 208000014674 injury Diseases 0.000 description 7
- 230000035755 proliferation Effects 0.000 description 7
- 230000004071 biological effect Effects 0.000 description 6
- 239000012091 fetal bovine serum Substances 0.000 description 6
- 239000013598 vector Substances 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 5
- 230000004663 cell proliferation Effects 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 238000001514 detection method Methods 0.000 description 4
- 210000002889 endothelial cell Anatomy 0.000 description 4
- 210000003606 umbilical vein Anatomy 0.000 description 4
- 210000003050 axon Anatomy 0.000 description 3
- 210000004204 blood vessel Anatomy 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 230000008929 regeneration Effects 0.000 description 3
- 238000011069 regeneration method Methods 0.000 description 3
- 239000013049 sediment Substances 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 238000010257 thawing Methods 0.000 description 3
- 238000005199 ultracentrifugation Methods 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 210000003169 central nervous system Anatomy 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 238000010362 genome editing Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 102000015427 Angiotensins Human genes 0.000 description 1
- 108010064733 Angiotensins Proteins 0.000 description 1
- 102000004452 Arginase Human genes 0.000 description 1
- 108700024123 Arginases Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241001112695 Clostridiales Species 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 241000906682 Hemsleya Species 0.000 description 1
- 101000934368 Homo sapiens CD63 antigen Proteins 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 description 1
- 108010039918 Polylysine Proteins 0.000 description 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 102000019197 Superoxide Dismutase Human genes 0.000 description 1
- 108010012715 Superoxide dismutase Proteins 0.000 description 1
- 241000202898 Ureaplasma Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000008499 blood brain barrier function Effects 0.000 description 1
- 210000001218 blood-brain barrier Anatomy 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 239000002360 explosive Substances 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000005090 green fluorescent protein Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 102000057640 human CD63 Human genes 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 210000002510 keratinocyte Anatomy 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 229910052748 manganese Inorganic materials 0.000 description 1
- 239000011572 manganese Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 238000002715 modification method Methods 0.000 description 1
- 229920000656 polylysine Polymers 0.000 description 1
- 239000003761 preservation solution Substances 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 238000003146 transient transfection Methods 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Classifications
-
- 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
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
-
- 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
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- 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
- C12N2510/00—Genetically modified cells
-
- 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
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Pharmacology & Pharmacy (AREA)
- Rheumatology (AREA)
- Virology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Developmental Biology & Embryology (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of exosome preparation, and in particular discloses a collagen-combined exosome, and a preparation method of the exosome comprises the following steps: constructing a recombinant expression vector pHBLV-CMV-CD63-EF1-ZsGreen-T2A-puro, wherein the nucleotide sequence of the recombinant expression vector is shown as SEQ ID NO. 1; the stem cells are transfected by the recombinant expression vector, so that the stem cells secrete exosomes with collagen binding properties. The exosomes prepared by the method can well target the collagen at the damaged part, so that the exosomes can play a role more stably. The invention also discloses exosome freeze-dried powder which can be stored and transported for a long time at normal temperature, can effectively retain activity, can be widely and conveniently used for repairing various injuries, and has convenient use and wide application.
Description
Technical Field
The invention relates to the technical field of exosome preparation, in particular to collagen-combined exosome, exosome freeze-dried powder and a preparation method thereof.
Background
The exosomes are lipid bilayer structure vesicles actively secreted by cells, and the exosomes contain a large number of factors, RNA, DNA and other substances, participate in information communication among cells, play an important bridge role in interaction among cells, so that the exosomes are widely applied. However, the existing exosomes still have the problem of poor targeting in application, so how to make the exosomes secreted by the stem cells play a role in local targeting stabilization is important, in recent years, along with the development of biotechnology, the stem cells are modified in a gene editing mode, and the secreted exosomes contain target molecules which are hot spots for research, for example, chinese patent publication No. CN115093459A discloses a delivery complex capable of carrying out axon targeting modification on the exosomes and a modification method for the exosomes, wherein the exosomes targeted by the axons have good blood stability, stealth and targeting property, and can effectively identify the axon tissues of the central nervous system. The Chinese patent with publication number of CN115177742A discloses a preparation method and application of a drug-loaded brain-targeted exosome, wherein the exosome is derived from cell supernatant of mouse mononuclear macrophage leukemia cells, and the brain-targeted exosome drug-loaded system can load drugs into exosome without obvious biotoxic effect, and small amount of tail vein drug administration can enter the central nervous system through the blood brain barrier to inhibit the expression of angiotensin (RAS).
Exosomes have great potential in promoting wound healing, applicable to lesions, and have been shown in animal models to promote collagen synthesis and proliferation and migration of fibroblasts and keratinocytes. In the prior art, the application of exosomes to injury is continuously researched and improved, such as the exosomes are applied to injury after being mixed into gel, and the exosomes are more beneficial than single direct administration of exosomes due to slow and stable delivery rate of the exosomes. However, in the application of the exosomes to the injury, the problem of reduced curative effect caused by lack of targeting and local dissipation of the exosomes exists, so that development of the exosomes capable of targeting the injury part has good research value and application prospect in improving the curative effect of the exosomes and enabling the exosomes to be better applied to injury treatment.
In addition, in the existing exosomes preservation and use process, the common mode is to preserve the flowing liquid exosomes at the temperature of-80 ℃, and the cryopreservation liquid is protected by dimethyl sulfoxide/glycerol, so that the storage cost is high and inconvenient. And the mixture prepared by using the dimethyl sulfoxide/glycerol and the exosome is easy to cause stimulation and damage to the body cells, so that the biological curative effect of the exosome is reduced. Therefore, the invention aims to develop a collagen-binding exosome and a preparation method of the collagen-binding exosome freeze-dried powder so as to prepare the collagen-binding exosome freeze-dried powder which can be stored and transported at normal temperature and effectively retain activity.
Disclosure of Invention
The technical problem solved by the invention is to provide an exosome to solve the problems of lack of targeting property, easy dissipation in the process of local injury application and poor curative effect of the exosome derived from stem cells in the prior art.
The second technical problem to be solved by the invention is to provide the exosome freeze-dried powder, which solves the problems of lack of targeting property, poor curative effect, easy damage and degradation and inactivation in the storage process of exosome in the prior art.
The third technical problem to be solved by the invention is to provide a preparation method of the exosome freeze-dried powder.
The technical problems solved by the invention are realized by adopting the following technical scheme:
a collagen-binding exosome, the preparation method of which comprises:
constructing a recombinant expression vector pHBLV-CMV-CD63-EF1-ZsGreen-T2A-puro, wherein the nucleotide sequence of the recombinant expression vector is shown as SEQ ID NO. 1;
the stem cells are transfected by the recombinant expression vector, so that the stem cells secrete exosomes with collagen binding properties.
Further, the stem cells are preferably human adipose-derived stem cells or human bone marrow-derived stem cells or human umbilical cord-derived stem cells or human ureagenic stem cells. In the technical scheme of the invention, the preferred stem cells include but are not limited to the above, and other human stem cells can also be adopted.
Further, the recombinant expression vector is transfected by a slow virus transfection method, and the implementation steps of slow virus transfection comprise:
transfecting the recombinant expression vector without endotoxin into lymphocytes, and collecting cell sap after culture to obtain virus liquid;
mixing the virus liquid with the stem cells after culture, and culturing the stem cells which are successfully infected to 60-70% for extracting exosomes.
The exosome freeze-dried powder is prepared by adopting the exosome as a raw material and a freeze-drying protective agent as an auxiliary material, wherein the freeze-drying protective agent comprises 30-40g/L trehalose, 2.0-3.0mM manganese sulfate and 0.01-0.015mol/L phosphate buffer solution, and the pH value of the freeze-drying protective agent is 7.35-7.45.
Further, the mass ratio of the exosome to the lyoprotectant is 1:1.
Further, the exosomes are exosome concentrated solution, and the concentration of the exosome concentrated solution is 400-600 ug/ml.
Further, the lyoprotectant comprises 35g/L trehalose, 2.5mM manganese sulfate and 0.012mol/L phosphate buffer solution, and the pH value of the lyoprotectant is 7.4.
The preparation method of the exosome freeze-dried powder comprises the steps of mixing exosome and stem cells, filtering, quick-freezing in liquid nitrogen for 5-10 min, transferring to a vacuum freeze dryer, and freeze-drying to obtain the exosome freeze-dried powder.
The beneficial effects are that: the exosomes of the invention construct a unique lentiviral vector by adopting a gene editing technology and are transfected into stem cells, so that the stem cells secrete exosomes with collagen binding property, and the exosomes can be bound with collagen at a damaged part in a targeted manner, thereby playing a role more stably.
The exosome freeze-dried powder disclosed by the invention has the advantages of effectively retaining activity, being capable of being targeted and combined to a damaged part, effectively promoting local cell proliferation and angiogenesis, being capable of being stored and transported for a long time at normal temperature, being widely and conveniently used for repairing various injuries, being convenient to use and being wide in application.
According to the preparation method of the exosome freeze-dried powder, the adopted freeze-dried protective agent can provide stable microenvironment for the exosome, can assist the exosome function in the use process, effectively promote the regeneration of locally damaged blood vessels, and meanwhile, the exosome freeze-dried protective agent presents weak alkalinity, can neutralize the slightly acidic environment of the locally damaged part, so that the local PH of the damaged part tends to be attached to the PH value of proper growth and proliferation of body cells; the unique freeze-drying protective agent is combined with the liquid nitrogen quick-freezing pretreatment technology, so that the conventional glycerol and dimethyl sulfoxide anti-icing crystal effect in the exosome preservation solution is replaced, the protein protection effect of the fetal bovine serum albumin on the exosome is avoided, the immune response of the exosome freeze-drying powder to the stimulation of organisms and the foreign protein in the use process is avoided, a unique protective film can be formed through the freeze-drying protective agent, the damage of the exosome in the low temperature and drying process is reduced under the synergistic effect of all components of the freeze-drying protective agent, and the exosome biological film is kept complete, so that the activity of biomolecules in the exosome is well maintained.
Drawings
FIG. 1 is a schematic diagram of pHBLV-CMV-MCS-EF1-ZsGreen-T2A-puro vector structure;
FIG. 2 is a schematic diagram of pHBLV-CMV-CD63-EF1-ZsGreen-T2A-puro vector structure;
FIG. 3 is a graph showing the in vitro rat tail collagen binding sustained release effect of CD63-BSP exosomes versus untransfected Exosomes (EXO);
FIG. 4 is a contrast image of the exosomes of the CD63-BSP before lyophilization (CD 63-BSP EXO) and after lyophilization (F-CD 63-BSP EXO);
FIG. 5 is a graph of electron microscopy particle size contrast analysis of CD63-BSP exosomes before lyophilization (CD 63-BSP EXO) and after lyophilization (F-CD 63-BSP EXO);
FIG. 6 shows the effect of CD63-BSP exosome lyophilized powder on umbilical vein endothelial cell proliferation after two months of storage at ambient temperature and-80 ℃;
FIG. 7 is a graph showing the comparative effect of the exosome lyophilized powder of the present invention and the exosome lyophilized powder obtained by conventional methods.
Detailed Description
In order that the manner in which the invention is attained, as well as the features and advantages thereof, will be readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof.
Example 1
The exosome of this embodiment, the preparation method thereof includes:
construction of pHBLV-CMV-CD63-EF1-ZsGreen-T2A-puro vector
1) Based on the gene information of CDS sequence and BSP sequence on mRNA of human CD63 in GeneBank, xbaI-CD63-EF1-ZsGreen-T2A-puro-NotI gene DNA fragment (Hemsleya Biotechnology (Shanghai) Co., ltd.) was designed and synthesized. Double-stranded DNA molecules of the synthetic XbaI-CD63-EF1-ZsGreen-T2A-puro-NotI gene were digested with restriction enzymes XbaI and NotI, respectively, and the digestion system was: xba I1. Mu.L, not I1. Mu.L, buffer 3. Mu.L, synthetic DNA 1ug, make up water to 30ul, and digested at 37℃for 4h, and the digested product was recovered.
2) The restriction enzyme pair XbaI and NotI is used as shown in FIG. 1
pHBLV-CMV-MCS-EF1-ZsGreen-T2A-puro (Hayota Biotechnology (Shanghai) Co., ltd.) cleavage, cleavage System: xba I1. Mu.L, not I1. Mu.L, buffer 3. Mu.L, pHBLV-CMV-MCS-EF1-ZsGreen-T2A-puro plasmid 1. Mu.g, make up water to 30. Mu.L. And (3) enzyme cutting at 37 ℃ for 4 hours, and recovering the carrier framework.
3) Connecting the enzyme digestion product in the step 1) with the carrier framework in the step 2), wherein a connecting system is as follows: t4DNA ligase 1. Mu.L, buffer 1. Mu.L, recovered synthetic DNA 20ng, recovered plasmid 10ng. After overnight ligation at 16 ℃, E.coli was transformed, positive bacteria were selected and plasmids thereof were extracted to obtain recombinant plasmids pHBLV-CMV-CD63-EF1-ZsGreen-T2A-puro as shown in FIG. 2. The sequencing result of the recombinant plasmid is shown as SEQ ID NO. 1.
(II) transient transfection
293T cells are taken out of liquid nitrogen, placed in a water bath kettle at 37 ℃ for thawing, centrifuged, diluted by a culture medium and cultured in a T25 culture dish coated by polylysine, and when the density reaches 60% -70%, the culture dish can be used for subsequent transfection experiments.
The pHBLV-CMV-CD63-EF1-ZsGreen-T2A-puro vector without endotoxin is transfected into 293T cells with 15 mug, pLP-110 mug, pLP-210 mug and pLP-VSVG with 5 mug, a DNA-calcium phosphate mixture is added, the culture medium is replaced for about 10 hours, the culture medium is continuously cultured for 60 hours, the cell culture solution is collected, the supernatant is collected after centrifugation, the filtration is carried out by a filter membrane with 0.45-micro, the centrifugation is continuously carried out, the supernatant is discarded, and the sediment is resuspended by PBS; the virus solution was stored at-80 ℃.
Virus titer detection: HEK 293T cells were inoculated into 24-well plates, and at the time of transfection, the virus solution stored in a refrigerator at-80℃was thawed in a 37℃water bath, and 10-fold gradient dilution was performed with a cell culture medium containing 5% fetal bovine serum FBS by volume fraction, from 10 -1 Diluted to 10 -10 . Sucking culture solution from corresponding culture hole, adding multiple diluted virus solution, adding into CO 2 Incubator incubate for 48 hours, add complete medium. After 4 days, RNA extraction was performed according to TRIZOL protocol from Invitrogen company, cDNA was obtained after reverse transcription of the RNA, and finally Real-time quantitative PCR was performed on iQ5 of Bio-Rad to obtain a higher titer of 1X10 virus 8 TU/ml。
Transfection of Stem cells with pHBLV-CMV-CD63-EF1-ZsGreen-T2A-puro vector
Human bone marrow mesenchymal stem cells hBMSC stored at-80 ℃ are thawed in a water bath at 37 ℃ and are used for subsequent transfection when cultured to 60-70% fusion degree. In addition to hbmscs used in this example, stem cells (human adipose mesenchymal stem cells (hADSC), human umbilical cord mesenchymal stem cells (hcscs), human ureaplasma stem cells (hscs), and the like may be used.
Thawing virus solution stored at-80deg.C in 37 deg.C water bath. 2X 10 of the Stem cells per well 5 cells/m L were inoculated into 24-well plates, and 500. Mu.L of the corresponding stem cell-dedicated medium containing 10% FBS was added to each well for overnight culture. And adding 5 mu L of virus liquid into 500 mu L of culture liquid special for the corresponding stem cells containing 2% FBS, uniformly mixing, then changing the original culture liquid, culturing overnight, then changing back to the culture liquid special for the corresponding stem cells containing 10% FBS, and observing the expression condition of the green fluorescent protein for 72 hours. Successfully transfected stem cells were stored at-80 ℃.
(IV) exosome extraction
The transfected stem cells (T-hBMSC) were cultured to 60-70% for exosome extraction, and the same batch of corresponding untransfected stem cells was used as a control group. The stem cells were treated with serum-free medium for 24 hours, and the separated supernatant was centrifuged at 3500 Xg for 30 minutes at 4℃and filtered through a 0.22 μm filter. The filtered supernatant was placed in a 100kd ultrafiltration concentration centrifuge tube and centrifuged at 3500 Xg for 30min at 4℃to obtain a primary exosome concentrate, which was packed with PBS and subjected to ultracentrifugation at 200000 Xg for 1 hour. The obtained pellet was resuspended in a large amount of PBS, filtered through a 0.22 μm filter, and subjected to ultracentrifugation at 200000 Xg for 2 hours, and the obtained pellet was resuspended in PBS as an exosome and frozen at-80 ℃.
(V) detection of collagen binding of exosomes
Thawing the extracted exosomes, mixing with rat tail collagen, placing in a 24-well plate, adding PBS after solidification, and incubating at 37deg.C. The detection results of the protein content detection by absorbing PBS every 24 hours are shown in fig. 3, the protein content in the PBS of the transfected stem cell exosomes shows a slow increasing trend, and the untransfected stem cell exosomes of the control group shows explosive increase, which indicates that the transfected stem cell exosomes have good collagen binding characteristics, can be bound to collagen in a targeting way and achieve the effect of slow release.
Example 2
The preparation method of the exosome freeze-dried powder comprises the following steps: the supernatant of the successfully transfected stem cells from example 1 was collected, centrifuged and the supernatant after centrifugation was filtered through a 0.22 μm filter. And (3) placing the filtered supernatant in an ultrafiltration concentration centrifuge tube with the volume of 100kd, continuing to centrifuge to obtain a primary exosome concentrated solution, filling the primary exosome concentrated solution with PBS, then performing ultracentrifugation, taking the sediment to use PBS for resuspension, filtering, centrifuging, and continuing to resuspension the obtained sediment with PBS to obtain a concentrated exosome. The concentrated exosomes and the freeze-drying protective agent are uniformly mixed in a ratio of 1:1, then a 0.22 mu m filter is filtered in a quick-heating cup, the quick-freezing is immediately carried out in liquid nitrogen in a sterile operation cabinet, the freezing is carried out for 5-10 min, the quick-freezing is carried out, the quick-freezing is rapidly transferred into a vacuum freeze dryer, the freeze drying is carried out for 24h, the collagen-binding angiogenesis-promoting exosome freeze-dried powder is obtained, and the collagen-binding angiogenesis-promoting exosomes freeze-dried powder is stored in a dark place at room temperature.
The lyoprotectant preferably comprises 35g/L trehalose, 2.5mM manganese sulfate and 0.012mol/L phosphate buffer, and the pH of the lyoprotectant is 7.4. The added trehalose can form a layer of special protective film in the processes of freezing and drying the exosomes to protect and maintain the activity of biomolecules in the exosomes, phosphate buffer solution is added, the pH of the mixed system is regulated to be maintained at 7.37-7.45, the pH is fit with the living environment of the most suitable cells of the human body, particularly immune cells, and the cells can play the maximum functional effect under the pH. And the weakly alkaline freeze-dried powder can neutralize weak acidic microenvironment at the damaged part in the use process, is regulated to be the weakly alkaline environment which is most suitable for the growth of human cells, and the exosomes exert biological effects of the exosomes more quickly and effectively in the use process, so that the bioremediation function of the cells at the damaged part is started quickly and the exosomes react with signal molecules of the exosomes. Manganese is used as one of key microelements required by human body, is a key element containing manganese superoxide dismutase, arginase, pyruvate clostridial enzyme and the like, participates in three metabolic processes of the organism, effectively promotes processes of cell proliferation, blood vessel regeneration and the like, and manganese sulfate with a given content is added in the preparation process of freeze-dried powder to serve as an ion balance protective agent in the freeze-drying process, and simultaneously, the manganese sulfate is also effective to cooperate with biological effects of exosomes, improve biological activity, promote cell proliferation, blood vessel regeneration and the like.
Structural changes of exosomes before and after lyophilization
The microstructure of the exosome before and after freeze-drying is shown in figure 4, and the particle size of the exosome freeze-dried powder before and after freeze-drying is shown in figure 5. The microstructure and the particle size distribution are not greatly different before and after freeze-drying, which indicates that the structure of the exosome is not greatly changed before and after freeze-drying and the exosome is less damaged.
Biological Effect of (II) exosomes on umbilical vein endothelial cells (HUVECs) before and after lyophilization
The exosomes obtained in example 1 are preserved for two months at-80 ℃, the exosomes freeze-dried powder obtained in the preparation method of example 2 is preserved for two months at normal temperature, the exosomes freeze-dried powder is added into a culture medium and HUVECs for culture according to the concentration of 10ug/ml, proliferation experiments are carried out, a control group is a culture medium without exosomes, and the result is shown in figure 6, which shows that compared with the control group, the exosomes preserved at-80 ℃ and the exosomes freeze-dried powder preserved at room temperature both obviously promote the proliferation of HUVECs, and the CD63-BSP exosomes freeze-dried powder can more effectively promote the proliferation of umbilical vein endothelial cells compared with the exosomes preserved at-80 ℃, so that the exosomes freeze-dried powder obtained in the invention can be preserved at normal temperature, has lower preservation cost and is more convenient, and meanwhile, the biological activity of the exosomes freeze-dried powder is better preserved, and has better curative effect.
And (III) biological effects of the exosome freeze-dried powder prepared by the freeze-dried protective agent and the exosome freeze-dried powder which is directly dried in a vacuum freeze dryer without adopting liquid nitrogen speed by adopting glycerol and dimethyl sulfoxide as protective agents on umbilical vein endothelial cells (HUVECs).
The exosome freeze-dried powder and exosome freeze-dried powder obtained by adopting glycerol and dimethyl sulfoxide as protective agents are respectively taken out and added into a culture medium and HUVECs for culture according to the concentration of 10ug/ml, proliferation experiments are carried out, and a blank control group is a culture medium without exosome. As shown in FIG. 7, the HUVECs proliferation of the exosome lyophilized powder group directly lyophilized by using glycerol and dimethyl sulfoxide as protective agents is even lower than that of the control group, and the cell proliferation is obviously inhibited, which indicates that the conventional lyophilized powder prepared by using glycerol and dimethyl sulfoxide as protective agents has a certain toxic effect on cells in the process of directly culturing the cells.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (8)
1. A collagen-bound exosome, characterized in that the exosome is prepared by a method comprising:
constructing a recombinant expression vector pHBLV-CMV-CD63-EF1-ZsGreen-T2A-puro, wherein the nucleotide sequence of the recombinant expression vector is shown as SEQ ID NO. 1;
the stem cells are transfected by the recombinant expression vector, so that the stem cells secrete exosomes with collagen binding properties.
2. The collagen-bound exosome according to claim 1, wherein the stem cells comprise human adipose-derived stem cells or human bone marrow-derived stem cells or human umbilical cord-derived stem cells or human ureagenic stem cells.
3. The collagen-bound exosome of claim 1, wherein the recombinant expression vector is transfected by lentiviral transfection, the step of performing lentiviral transfection comprising:
transfecting the recombinant expression vector without endotoxin into lymphocytes, and collecting cell sap after culture to obtain virus liquid;
mixing the virus liquid with the stem cells after culture, and culturing the stem cells which are successfully infected to 60-70% for extracting exosomes.
4. The exosome freeze-dried powder is characterized by being prepared by taking the exosome as a raw material and taking a freeze-drying protective agent as an auxiliary material according to any one of claims 1-3, wherein the freeze-drying protective agent comprises 30-40g/L trehalose, 2.0-3.0mM manganese sulfate and 0.01-0.015mol/L phosphate buffer solution, and the pH value of the freeze-drying protective agent is 7.35-7.45.
5. The exosome lyophilized powder of claim 4, wherein the mass ratio of exosome to lyoprotectant is 1:1.
6. The exosome lyophilized powder according to claim 4, wherein the exosome is an exosome concentrate having a concentration of 400-600 ug/ml.
7. The exosome lyophilized powder according to claim 4, wherein the lyoprotectant comprises 35g/L trehalose, 2.5mM manganese sulfate, 0.012mol/L phosphate buffer, and the pH of the lyoprotectant is 7.4.
8. The method for preparing the exosome freeze-dried powder according to claim 4, wherein the exosome and the stem cells are mixed, filtered, quickly frozen in liquid nitrogen for 5-10 min, and then transferred to a vacuum freeze dryer for freeze-drying to obtain the exosome freeze-dried powder.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310688610.4A CN116790490A (en) | 2023-06-12 | 2023-06-12 | Collagen-combined exosome, exosome freeze-dried powder and preparation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310688610.4A CN116790490A (en) | 2023-06-12 | 2023-06-12 | Collagen-combined exosome, exosome freeze-dried powder and preparation method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116790490A true CN116790490A (en) | 2023-09-22 |
Family
ID=88043266
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310688610.4A Pending CN116790490A (en) | 2023-06-12 | 2023-06-12 | Collagen-combined exosome, exosome freeze-dried powder and preparation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116790490A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117427202A (en) * | 2023-10-27 | 2024-01-23 | 东莞胶原生物科技有限公司 | Exosome and collagen sponge biological scaffold and preparation method and application thereof |
-
2023
- 2023-06-12 CN CN202310688610.4A patent/CN116790490A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117427202A (en) * | 2023-10-27 | 2024-01-23 | 东莞胶原生物科技有限公司 | Exosome and collagen sponge biological scaffold and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Toma et al. | Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart | |
| CN112760293B (en) | Method for preparing high-activity exosomes by 3D culture of MSC stem cells by using xeno-free serum | |
| US20210032598A1 (en) | Activation-induced tissue-effector cells suitable for cell therapy and extracelluar vesicles derived therefrom | |
| Leblond et al. | Developing cell therapy techniques for respiratory disease: intratracheal delivery of genetically engineered stem cells in a murine model of airway injury | |
| CN116790490A (en) | Collagen-combined exosome, exosome freeze-dried powder and preparation method | |
| US20230157966A1 (en) | Compositions and methods related to megakaryocyte-derived extracellular vesicles | |
| CN107937442A (en) | A kind of immortal human fat mesenchymal stem cell system and its method for building up | |
| CN112410304A (en) | Gene-modified exosome and preparation method and application thereof | |
| Sato et al. | Effects of B-cell lymphoma 2 gene transfer to myoblast cells on skeletal muscle tissue formation using magnetic force-based tissue engineering | |
| CN113144221B (en) | Exosome preparation and preparation method and application thereof | |
| Song et al. | Multiplexed targeting of microRNA in stem cell-derived extracellular vesicles for regenerative medicine | |
| Gálvez-Montón et al. | Preclinical safety evaluation of allogeneic induced pluripotent stem cell-based therapy in a swine model of myocardial infarction | |
| CN110075122B (en) | Liver cancer therapeutic exosome medicine | |
| Kealy et al. | Comparison of viral and nonviral vectors for gene transfer to human endothelial progenitor cells | |
| Jin et al. | VP22 and cytosine deaminase fusion gene modified tissue-engineered neural stem cells for glioma therapy | |
| US20210130784A1 (en) | De-differentiated fibroblast-conditioned media for stimulation of disc regeneration | |
| Nie et al. | Preparing adipogenic hydrogel with neo-mechanical isolated adipose-derived extracellular vesicles for adipose tissue engineering | |
| US20240148937A1 (en) | Adipose-derived hydrogel compositions and methods of use | |
| Yamada et al. | Development of direct cardiac reprogramming for clinical applications | |
| CN115029320A (en) | Engineered exosome for tumor radiotherapy sensitization, preparation method and application | |
| Wang et al. | Engineered stem cells by emerging biomedical stratagems | |
| CN110982802A (en) | Recombinant human SGK3 protein kinase hydrogel, preparation method thereof and application thereof in promoting myocardial regeneration | |
| CN111187761A (en) | Fusion protein of MNK2 protein kinase and cell-penetrating peptide, hydrogel thereof and application of fusion protein to promotion of myocardial regeneration | |
| CN110904070A (en) | Recombinant human CHK1 protein kinase hydrogel for promoting myocardial regeneration and preparation method and application thereof | |
| CN114470002B (en) | Application of placenta mesenchymal stem cell freeze-dried powder in preparation of medicine |
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 |