CN116790492A - Method for producing umbilical cord blood exosomes - Google Patents
Method for producing umbilical cord blood exosomes Download PDFInfo
- Publication number
- CN116790492A CN116790492A CN202310763216.2A CN202310763216A CN116790492A CN 116790492 A CN116790492 A CN 116790492A CN 202310763216 A CN202310763216 A CN 202310763216A CN 116790492 A CN116790492 A CN 116790492A
- Authority
- CN
- China
- Prior art keywords
- cord blood
- umbilical cord
- exosomes
- mesenchymal stem
- microcarrier
- 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 110
- 210000004700 fetal blood Anatomy 0.000 title claims abstract description 62
- 238000004519 manufacturing process Methods 0.000 title abstract description 11
- 210000002901 mesenchymal stem cell Anatomy 0.000 claims abstract description 45
- 238000000034 method Methods 0.000 claims abstract description 30
- 210000002381 plasma Anatomy 0.000 claims abstract description 21
- 239000001963 growth medium Substances 0.000 claims abstract description 13
- 238000002360 preparation method Methods 0.000 claims abstract description 10
- 239000006285 cell suspension Substances 0.000 claims abstract description 5
- 210000004027 cell Anatomy 0.000 claims description 31
- 239000002609 medium Substances 0.000 claims description 12
- 238000012258 culturing Methods 0.000 claims description 10
- 201000010099 disease Diseases 0.000 claims description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 7
- 238000011081 inoculation Methods 0.000 claims description 6
- 229910002091 carbon monoxide Inorganic materials 0.000 claims description 5
- 229910052799 carbon Inorganic materials 0.000 claims description 4
- 230000001737 promoting effect Effects 0.000 claims description 3
- 230000008929 regeneration Effects 0.000 claims description 3
- 238000011069 regeneration method Methods 0.000 claims description 3
- 230000008439 repair process Effects 0.000 claims description 3
- 208000024172 Cardiovascular disease Diseases 0.000 claims description 2
- 208000012902 Nervous system disease Diseases 0.000 claims description 2
- 208000025966 Neurological disease Diseases 0.000 claims description 2
- 239000003814 drug Substances 0.000 claims description 2
- 208000026278 immune system disease Diseases 0.000 claims description 2
- 230000004071 biological effect Effects 0.000 abstract description 7
- 238000005516 engineering process Methods 0.000 abstract description 7
- 108010019160 Pancreatin Proteins 0.000 abstract description 5
- 229940055695 pancreatin Drugs 0.000 abstract description 5
- 239000000126 substance Substances 0.000 abstract description 5
- 230000009286 beneficial effect Effects 0.000 abstract description 4
- 238000000926 separation method Methods 0.000 abstract description 4
- 238000005406 washing Methods 0.000 description 22
- 102000004169 proteins and genes Human genes 0.000 description 19
- 108090000623 proteins and genes Proteins 0.000 description 19
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 14
- 229910052802 copper Inorganic materials 0.000 description 14
- 239000010949 copper Substances 0.000 description 14
- 239000006228 supernatant Substances 0.000 description 12
- 238000001816 cooling Methods 0.000 description 11
- 239000007788 liquid Substances 0.000 description 11
- 239000012528 membrane Substances 0.000 description 10
- 230000001954 sterilising effect Effects 0.000 description 10
- 239000002244 precipitate Substances 0.000 description 9
- 102100025222 CD63 antigen Human genes 0.000 description 8
- 101000934368 Homo sapiens CD63 antigen Proteins 0.000 description 8
- 102100027221 CD81 antigen Human genes 0.000 description 7
- 101000914479 Homo sapiens CD81 antigen Proteins 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 238000011534 incubation Methods 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 238000012546 transfer Methods 0.000 description 7
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 6
- 238000001514 detection method Methods 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 4
- 230000010261 cell growth Effects 0.000 description 4
- 239000012228 culture supernatant Substances 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 238000011068 loading method Methods 0.000 description 4
- 239000002105 nanoparticle Substances 0.000 description 4
- 230000003287 optical effect Effects 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 229910001220 stainless steel Inorganic materials 0.000 description 4
- 239000010935 stainless steel Substances 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 3
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 3
- 229930182816 L-glutamine Natural products 0.000 description 3
- 229930182555 Penicillin Natural products 0.000 description 3
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 3
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 3
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- QVYARBLCAHCSFJ-UHFFFAOYSA-N butane-1,1-diamine Chemical compound CCCC(N)N QVYARBLCAHCSFJ-UHFFFAOYSA-N 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 230000001678 irradiating effect Effects 0.000 description 3
- 229920000609 methyl cellulose Polymers 0.000 description 3
- 239000001923 methylcellulose Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229940049954 penicillin Drugs 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000002791 soaking Methods 0.000 description 3
- 229910052708 sodium Inorganic materials 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 238000010561 standard procedure Methods 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- 229960005322 streptomycin Drugs 0.000 description 3
- 239000011782 vitamin Substances 0.000 description 3
- 229940088594 vitamin Drugs 0.000 description 3
- 229930003231 vitamin Natural products 0.000 description 3
- 235000013343 vitamin Nutrition 0.000 description 3
- 150000003722 vitamin derivatives Chemical class 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- XRASPMIURGNCCH-UHFFFAOYSA-N zoledronic acid Chemical compound OP(=O)(O)C(P(O)(O)=O)(O)CN1C=CN=C1 XRASPMIURGNCCH-UHFFFAOYSA-N 0.000 description 3
- 229960004276 zoledronic acid Drugs 0.000 description 3
- 102100037904 CD9 antigen Human genes 0.000 description 2
- 101000836492 Dictyostelium discoideum ALG-2 interacting protein X Proteins 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 2
- 101000738354 Homo sapiens CD9 antigen Proteins 0.000 description 2
- 101001134621 Homo sapiens Programmed cell death 6-interacting protein Proteins 0.000 description 2
- 102100033344 Programmed cell death 6-interacting protein Human genes 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 230000006835 compression Effects 0.000 description 2
- 238000007906 compression Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 238000002331 protein detection Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000003892 spreading Methods 0.000 description 2
- 230000007480 spreading Effects 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 208000030507 AIDS Diseases 0.000 description 1
- 230000005653 Brownian motion process Effects 0.000 description 1
- 102000010831 Cytoskeletal Proteins Human genes 0.000 description 1
- 108010037414 Cytoskeletal Proteins Proteins 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 208000025499 G6PD deficiency Diseases 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 208000005176 Hepatitis C Diseases 0.000 description 1
- 102000005755 Intercellular Signaling Peptides and Proteins Human genes 0.000 description 1
- 108010070716 Intercellular Signaling Peptides and Proteins Proteins 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 108020005198 Long Noncoding RNA Proteins 0.000 description 1
- 108700011259 MicroRNAs Proteins 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 208000002903 Thalassemia Diseases 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- COQLPRJCUIATTQ-UHFFFAOYSA-N Uranyl acetate Chemical compound O.O.O=[U]=O.CC(O)=O.CC(O)=O COQLPRJCUIATTQ-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- MHMCOSLCKPWGQG-UHFFFAOYSA-H [U+6].[O-]C(=O)C([O-])=O.[O-]C(=O)C([O-])=O.[O-]C(=O)C([O-])=O Chemical compound [U+6].[O-]C(=O)C([O-])=O.[O-]C(=O)C([O-])=O.[O-]C(=O)C([O-])=O MHMCOSLCKPWGQG-UHFFFAOYSA-H 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 230000004900 autophagic degradation Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 238000005537 brownian motion Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000003085 diluting agent Substances 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
- 238000000635 electron micrograph Methods 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 208000008605 glucosephosphate dehydrogenase deficiency Diseases 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- QNILTEGFHQSKFF-UHFFFAOYSA-N n-propan-2-ylprop-2-enamide Chemical compound CC(C)NC(=O)C=C QNILTEGFHQSKFF-UHFFFAOYSA-N 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000003921 particle size analysis Methods 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 210000003370 receptor cell Anatomy 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 208000006379 syphilis Diseases 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 239000000107 tumor biomarker Substances 0.000 description 1
- 210000003954 umbilical cord Anatomy 0.000 description 1
- -1 uranyl oxalate Chemical compound 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
- C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- 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
-
- 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
- C12N2531/00—Microcarriers
-
- 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
- C12N2539/00—Supports and/or coatings for cell culture characterised by properties
Abstract
The invention discloses a method for producing umbilical cord blood exosomes. The method comprises the following steps: and inoculating the mesenchymal stem cells and the temperature-sensitive microcarrier into a culture medium containing umbilical cord blood plasma for culture, collecting mesenchymal stem cell suspension after culture, and centrifuging to obtain exosomes. According to the invention, the temperature-sensitive microcarrier is utilized for the first time to culture the mesenchymal stem cells in the culture container containing the umbilical cord blood plasma, so that the yield of exosomes can be greatly improved, the mesenchymal stem cells and the exosomes are safer by utilizing the umbilical cord blood plasma and are suitable for clinical application, and the application of the temperature-sensitive technology prevents the mesenchymal stem cells from being damaged by chemical substances such as pancreatin during separation, thereby being beneficial to improving the yield and uniformity of the exosomes, being capable of obtaining more exosomes with biological activity and realizing large-scale exosome acquisition and large-scale exosome preparation.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a method for producing umbilical cord blood exosomes.
Background
Exosomes (exosomes) are small vesicles secreted by cells with lipid bilayer membranes, approximately 30-150nm in diameter. The exosomes are capable of carrying and transmitting important signaling molecules, such as DNA, mRNA, microRNA, cytoskeletal proteins, transcription factors, adhesion factors, growth factors, and the like. The exosomes have various biological effects, perform the functions of protein and nucleic acid transportation, specific targeting of receptor cells and the like, participate in immune response and autophagy interaction, and the lncRNA can also be used as a tumor biomarker, so that the exosomes are widely paid attention to disease diagnosis and treatment.
Exosomes can be secreted by a variety of cells, different cells determining the function and effect of exogenesis. The mesenchymal stem cells (mesenchymal stem cell, MSC) have the functions of anti-inflammatory, immunoregulation, angiogenesis, pathological protein removal, matrix reconstruction and the like. Whereas the mesenchymal stem cell exosomes (mesenchymal stem cellderived exosome, MSC-EXO) have not only similar functions as mesenchymal stem cells, but also better safety. Human umbilical blood mesenchymal stem cells are obtained from umbilical cord blood of a pregnant woman with healthy term delivery, have strong self-renewal capacity and multidirectional differentiation potential, and the derived exosomes have therapeutic effects on various diseases.
At present, the MSC for producing exosomes is cultivated in a manner of adherent cultivation in a common culture bottle, so that the quantity of the cultivated MSC is limited, pancreatin digestion and separation are needed after cultivation, certain damage is caused to cells, and the obtained exosomes have low yield and poor uniformity. With the development of technology, a method for preparing mesenchymal stem cell exosomes starts to use microcarriers, for example CN110669729a discloses a method for preparing mesenchymal stem cell exosomes, which uses a culture solution containing gelatin microcarriers to culture mesenchymal stem cells, but the culture medium of the method uses various growth factors, has high cost, and is easy to cause damage to cells when separating mesenchymal stem cells, thus being unfavorable for improving the yield of exosomes.
In summary, the existing method for producing exosomes is complex in operation process, and the mesenchymal stem cells are easy to damage cells when separated, so that the acquisition of the subsequent exosomes is affected, the used culture medium has more components, the price is high, the obtained exosomes have low yield and low purity, and the requirements of the exosomes in research and clinical application are greatly limited. How to provide a method for producing exosomes conveniently, efficiently and at low cost has become one of the problems to be solved in the biotechnology field at present.
Disclosure of Invention
Aiming at the defects and actual demands of the prior art, the invention provides the method for producing the umbilical cord blood exosomes, solves the problems of low yield, low purity and the like of the exosomes obtained by the method for producing the exosomes, realizes the convenient, efficient and low-cost production of the exosomes, and has important significance for promoting cell regeneration and repair and disease diagnosis.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method of producing an umbilical cord blood exosome, the method comprising:
and inoculating the mesenchymal stem cells and the temperature-sensitive microcarrier into a culture medium containing umbilical cord blood plasma for culture, collecting mesenchymal stem cell suspension after culture, and centrifuging to obtain exosomes.
According to the invention, the temperature-sensitive microcarrier is utilized for the first time to culture the mesenchymal stem cells in the culture container containing the umbilical cord blood plasma, so that the yield of exosomes can be greatly improved, the mesenchymal stem cells and the exosomes are safer by utilizing the umbilical cord blood plasma and are suitable for clinical application, and the application of the temperature-sensitive technology prevents the mesenchymal stem cells from being damaged by chemical substances such as pancreatin during separation, thereby being beneficial to improving the yield and uniformity of the exosomes, being capable of obtaining more exosomes with biological activity and realizing large-scale exosome acquisition and large-scale exosome preparation.
The temperature-sensitive microcarrier is a microcarrier with temperature-sensitive property, cells can grow normally on the microcarrier at 37 ℃, when the temperature is reduced to a certain value, the cells can not adhere to the microcarrier and fall off, for example, the microcarrier grafted by poly-N-isopropyl acrylamide (PNIPAAm) can be selected, and the surface of the microcarrier contains hydrophobic isopropyl and hydrophilic amide groups, so that the microcarrier becomes a typical temperature-sensitive microcarrier, and when the temperature is lower than 32 ℃, the microcarrier presents hydrophilicity in a water-soluble culture medium and swells, so that the cells are difficult to adhere.
300mL of cell culture supernatant was also prepared to extract exosomes, and microcarriers were cultured 1 time in only 1 culture tank; in the traditional adherence method, a plurality of culture bottles are needed, for example, 30 culture bottles (10 mL of culture medium is placed in each T75 culture bottle) are needed when a T150 culture bottle is used, the workload of microcarrier is small, the obtained exosomes are good in uniformity, stable in batch and low in pollution risk.
Preferably, the mesenchymal stem cells comprise: umbilical cord blood mesenchymal stem cells.
Preferably, the inoculation density of the temperature-sensitive microcarrier is 1-5g/L.
Specific point values in the above 1-5g/L may be selected from 1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, etc.
Preferably, the volume percentage of the cord blood plasma in the culture medium is 5-10%.
Specific point values in the above 5-10% may be selected from 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 6%, 7%, 8%, 9%, 9.6%, 9.8%, 9.9%, 10%, etc.
Preferably, the mesenchymal stem cells have an seeding density of 2×10 5 -3×10 5 Individual cells/mL.
The above 2×10 5 -3×10 5 The specific point value in (2X 10) can be selected 5 、2.1×10 5 、2.2×10 5 、2.3×10 5 、2.4×10 5 、2.5×10 5 、2.6×10 5 、2.7×10 5 、2.8×10 5 、2.9×10 5 、3×10 5 Etc.
Preferably, the time of the cultivation is 4-12 hours.
Specific point values in the above 4-12h may be selected from 4h, 4.1h, 4.2h, 4.3h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 11.7h, 11.8h, 11.9h, 12h, etc.
Preferably, the conditions of the culture are: the temperature is 35-38deg.C, CO 2 The concentration is 3-8%.
The specific values of 35-38deg.C can be 35 deg.C, 35.1 deg.C, 35.2 deg.C, 35.3 deg.C, 35.4 deg.C, 35.5 deg.C, 36 deg.C, 37.3 deg.C, 37.5 deg.C, 37.7 deg.C, 37.8 deg.C, 37.9 deg.C, 38 deg.C, etc.
Specific point values in the above 3-8% may be selected from 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 4%, 5%, 6%, 7%, 7.7%, 7.8%, 7.9%, 8%, etc.
As a preferred embodiment, the method for producing umbilical cord blood exosomes comprises the steps of:
(1) Ultraviolet autoclaving the temperature sensitive microcarrier;
(2) Incubating the inoculated and sterilized temperature-sensitive microcarrier and umbilical cord blood mesenchymal stem cells in a medium containing umbilical cord blood plasma, wherein the inoculation density of the temperature-sensitive microcarrier is 1-5g/L, the volume percentage of the umbilical cord blood plasma in the medium is 5-10%, and the inoculation density of the umbilical cord blood mesenchymal stem cells is 2 multiplied by 10 5 -3×10 5 Individual cells/mL;
(3) And after culturing, collecting mesenchymal stem cell suspension of umbilical cord blood, and centrifuging to obtain exosomes.
In a second aspect, the present invention provides an umbilical cord blood exosome produced by the method for producing an umbilical cord blood exosome of the first aspect.
In a third aspect, the present invention provides the use of the umbilical cord blood exosomes of the second aspect for promoting cell regeneration and repair and for the preparation of a medicament for treating a disease.
Preferably, the disease comprises any one or a combination of at least two of cardiovascular disease, neurological disease or immune system disease.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, the temperature-sensitive microcarrier is utilized for the first time to culture the mesenchymal stem cells in the culture container containing the umbilical cord blood plasma, so that the yield of exosomes can be greatly improved, the mesenchymal stem cells and the exosomes are safer by utilizing the umbilical cord blood plasma and are suitable for clinical application, and the application of the temperature-sensitive technology prevents the mesenchymal stem cells from being damaged by chemical substances such as pancreatin during separation, thereby being beneficial to improving the yield and uniformity of the exosomes, being capable of obtaining more exosomes with biological activity and realizing large-scale exosome acquisition and large-scale exosome preparation.
Drawings
FIG. 1 is an electron microscope image after cells are grown on microcarriers;
FIG. 2 is a view showing the microcarrier after autoclaving and cooling;
FIG. 3 is a morphology of MSC exosomes observed by transmission electron microscopy;
FIG. 4 is a phenotype flow chart of MSC exosomes CD81 and CD 63;
FIG. 5 is a graph of exosome vesicle particle size analysis;
FIG. 6A is a graph showing the detection results of CD9 protein;
FIG. 6B is a graph showing the detection results of CD63 protein;
FIG. 6C is a graph showing the results of Alix protein detection.
Detailed Description
The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
Example 1
1. Obtaining umbilical cord blood
The pregnant and lying-in women need to sign an 'umbilical cord blood collection informed consent' and an 'donation item informed acknowledgement list' before umbilical cord blood collection, and are required to provide prenatal assay detection results, including detection results of hepatitis B surface antigen, hepatitis C antibody, AIDS antibody, syphilis antibody, macrophage virus antibody, thalassemia, G-6-PD deficiency and the like, so as to ensure safety and reliability of the umbilical cord blood. The collection of the umbilical cord blood is completed in time after delivery of the puerpera by a professional nurse, so that the probability of pollution in the process of collecting the umbilical cord blood is reduced to be extremely low. Then, the umbilical cord blood hematopoietic stem cell bank is transported back at low temperature within 24 hours for timely treatment.
2. Culture of umbilical cord blood MSC
(1) Selecting temperature-sensitive Cytodex-3 microcarrier (brand Cytiva product number 17048503), performing ultraviolet treatment on an ultra-clean bench according to a standard procedure, spreading the temperature-sensitive microcarrier on a filter membrane as much as possible, irradiating ultraviolet in the ultra-clean bench for 30min each time, turning the microcarrier after one irradiation, performing ultraviolet irradiation again, soaking the microcarrier in PBS overnight after 4 times of ultraviolet treatment, discarding the PBS, adding fresh PBS for rinsing 3 times, finally leaving PBS capable of immersing the volume of the microcarrier, sterilizing at 121 ℃ for 30min under high pressure, cooling for 4 ℃ for later use, and using medium (glucose 0.6%, L-glutamine 2mM (added in use), VEGF 20ng/mL, bFGF-210ng/mL and NaHCO) containing 8% of umbilical cord blood plasma by volume percent just before use 3 3 mM, hepes5mM, vitamin C0.4 mM, butanediamine 60mM, zoledronate sodium 3. Mu.M, penicillin 100U/mL, streptomycin 100U/mL, DMEM/F12 (1:1) medium to 1L) for replacement, and after incubation at 37℃for 8 h;
(2) Inoculating the microcarrier of step (1) to a culture flask at a density of 3g/L, and sterilizing the umbilical cord blood MSC suspension 3×10 before sterilization 5 Density of individual cells/mL of culture medium inoculated into the above flask at 37℃with 5% CO 2 Culturing in incubator, observing cell growth state and adherence effect, photographing with common inverted optical microscope to obtain microcarrier with cell growth state shown in figure 1, autoclaving, cooling to 27deg.C, and microcarrier state shown in figure 2.
3. Preparation of exosomes
A high-speed refrigerating table type centrifuge and a miniature freeze dryer are adopted.
(1) Collecting 300mL of umbilical cord blood mesenchymal stem cell culture supernatant;
(2) Centrifuging at 4deg.C for 2000 Xg for 10min to obtain supernatant (dead cells removed);
(3) Centrifuging at 4deg.C for 10,000Xg for 30min to obtain supernatant (removing cell debris);
(4) Centrifuging at 4deg.C for 100,000Xg for 70min to retain precipitate, and discarding supernatant (mainly exosomes and heteroproteins;
(5) Pre-cooling the precipitate with sterile PBS, centrifuging for 70min to retain the precipitate, and discarding supernatant (the precipitate is mainly exosomes);
(6) The precipitate was blown off with 1mL pre-chilled sterile PBS and lyophilized using a lyophilizer, frozen at-20 ℃ for future use.
4. Identification of exosomes
4.1. Electron microscope identification
(1) The exosomes were suspended with buffer PBS;
(2) Fixing exosomes on sample-carrying copper net
(1) The centrifuged exosomes were resuspended at 100,000×g to 100 μl of 2% PFA;
(2) 5. Mu.L of exosome suspension was added to the Formvar-carbon-loaded copper mesh, 3 copper meshes were prepared for each exosome sample, covered, and the Formvar membrane was allowed to absorb for 20min in a dry environment;
(3) 100 μLPBS was added to the sealing film. The copper mesh was washed with tweezers on PBS drop
In all steps, the Formvar film side should be kept wet, while the other side is dried;
(4) the copper mesh was placed on 50 μl of 1% glutaraldehyde droplets for 5min;
(5) the copper mesh was placed at 100. Mu.LddH 2 O2 min (washing 8 times)
(6) The copper mesh was placed on 50. Mu.L uranium oxalate drops at pH7 for 5min
(7) Placing the copper mesh on 50 methyl cellulose-UA liquid drops for 10min, and operating on ice;
(8) the copper mesh was removed with a stainless steel ring, excess liquid was gently sucked off the filter paper, leaving a thin layer of methylcellulose film, the thickness of which was controlled to a proper range, which affected the contrast of the image. The stainless steel ring was fixed on a 1mL blue gun head;
(9) the copper net is still on the stainless steel ring, and is dried in the air for 10min;
after drying, the blue-gold interference fringes indicate a suitable and uniform thickness of the methylcellulose film.
The copper mesh was placed in a box and an electron micrograph taken at 80kV, and under electron microscopy, the negative dye exosome was shown to be 50-90nm cup-shaped bubbles, and 10-20nm lipid particles were also observed.
The reagent is used: 4% (w/v) PFA, PBS, 1% glutaraldehyde, uranyl oxalate, pH7, methylcellulose-UA, pH4, 9 parts of 2% methylcellulose +1 part of 4% uranyl acetate, pre-use mixing, formvar-carbon sample-carrying copper mesh, sealing film, no. 5 tweezers, glass culture dish, stainless steel ring, no. 1 filter paper slightly larger than copper mesh, copper mesh storage box.
The instrument is used: single particle freeze transmission electron microscope model: talosarc G2.
Results: as shown in FIG. 3, the MSC cell exosomes are observed by a transmission electron microscope to be in an oval cup-shaped vesicle-like structure, have the diameter of about 30-150nm, have a complete membrane structure, contain low-density substances and have good integrity.
MSC cell exosomes CD81 and CD63 phenotypes assays
The expression of two surface markers, CD63 and CD81, in MSC exosomes was analyzed using a BD accuriC6 flow cytometer, and the results were shown in FIG. 4 to have positive signals for both surface markers, with positive rates of 84.6% and 84.5%, respectively.
4.3. Nanoparticle Tracking Analysis (NTA) identification
Nanoparticle tracking analysis (Nanoparticle TrackingAnalysis, NTA) is based on the principle of tracking and analyzing the brownian motion of each particle, and calculating the hydrodynamic diameter and concentration of the nanoparticles in combination with the stock-Einstein equation. NTA technology has been accepted by the exosome research field as one of the exosome characterization means; compared with other characterization modes, the NTA technology has the advantages that the sample processing is simpler, the original state of the exosome can be ensured, and the detection speed is higher.
The experimental steps are as follows:
(1) Washing the sample cell with deionized water;
(2) The instrument was calibrated with polystyrene microspheres (100 nm);
(3) Wash the cuvette with 1×pbs buffer (Biological Industries, israel);
(4) Samples were taken and diluted with 1 XPBS buffer (Biological Industries, israel) and assayed by sampling.
Results: as shown in FIG. 5, the exosome vesicles have a narrow particle size distribution, indicating that the extracted exosome has high purity, which can reach 97%.
Wb identification
(1) Collecting protein samples
Adherent cells, suspension cells or tissue samples are lysed using an appropriate lysate RIPA lysate or the like. After collection of the protein samples, it is necessary to determine the protein concentration of each protein sample in order to ensure consistent loading of each protein sample.
(2) Electrophoresis
(1) SDS-PAGE gel preparation
(2) Sample processing
An appropriate amount of concentrated SDS-PAGE protein loading buffer was added to the collected protein samples. Heating in boiling water bath for 5min to thoroughly denature protein.
(3) Loading and electrophoresis
And (3) cooling to room temperature, and directly loading the protein sample into an SDS-PAGE gel loading hole. To facilitate observation of the electrophoresis effect and the transfer effect, and judgment of the molecular weight of the protein, a pre-dye protein molecular weight standard was used. The voltage is usually set at 100V by adopting a constant voltage mode in the whole SDS-PAGE process, and then the timing time is set to be 120min. Setting the timing can avoid frequent electrophoresis overhead. And when the bromophenol blue reaches the position near the bottom end of the gel, the electrophoresis can be stopped.
(3) Transfer film
The film transfer current was set to 300mA and the film transfer time was set to 60min using a standard wet film transfer apparatus of Bio-Rad.
(4) Closure
After the transfer of the membrane, the protein membrane is immediately placed into a prepared Western washing liquid, and rinsed for 2min to wash the membrane transfer liquid on the membrane. From all steps after finishing transferring the film, care must be taken to keep the film moist, avoid drying the film, otherwise, a higher background is very easy to generate, the film is slowly shaken on a shaking table, and the film is sealed for 60 minutes at room temperature.
(5) Incubation with primary antibody
Referring to the instructions for primary antibodies, western primary antibody dilutions were used at a 1:1000 ratio. Anti-CD9Anti [ EPR2949] abcam company cat#: ab92726, anti-CD63 Anti-ibody [ EPR5702]
abcam company cat No.: ab134045, anti-ALIX antibody abcam company cat No.: ab88388.
The blocking solution is sucked up by a miniature bench vacuum pump or a dropper, diluted primary antibody is added immediately, and the mixture is slowly shaken on a side swing table at 4 ℃ for incubation for 1h. If the primary antibody is not effective for 1h, it can be incubated overnight with slow shaking at 4 ℃. Recovering the primary antibody. Western washing liquid is added, and the mixture is slowly shaken on a side swinging table for washing for 10min. After the washing liquid is absorbed, the washing liquid is added for washing for 10min. The washing was performed 3 times. If the result background is higher, the washing time can be prolonged and the washing times can be increased appropriately.
(6) Second antibody incubation
Reference is made to the product number of the second anti-Goatti-Rabbit IgG H & L (HRP) abcam: ab205718, horseradish peroxidase (HRP) -labeled secondary antibodies were diluted with a Western secondary antibody diluent at a ratio of 1:8000. The secondary antibody is selected according to the primary antibody, the washing liquid is sucked by a miniature desk vacuum pump or a dropper, and the diluted secondary antibody is immediately added, and the secondary antibody is slowly shaken on a side swing table at 4 ℃ for incubation for 1h. Recovering the secondary antibody. Western washing liquid is added, and the mixture is slowly shaken on a side swinging table for washing for 10min. After the washing liquid is absorbed, the washing liquid is added for washing for 10min. The washing was performed 3 times. If the result background is higher, the washing time can be prolonged and the washing times can be increased appropriately.
(7) Protein detection
Proteins were detected using BeyoECL Plus ECL-like reagents, and compression can be performed using a dedicated compression cassette. An X-ray film automatic film developing machine can be used for developing films. The X-ray film is kodak X-OMAT BT film specially used for the biological experiment of the kodak original package.
(8) Gel image analysis
The film is scanned or photographed and the molecular weight and net optical density values of the target band, such as the quality One of bio-rad, are analyzed using a gel image processing system.
Results: the surface markers CD9, CD63 and Alix protein (figures 6A-C) of the MSC cell exosomes can be successfully detected by the method, which shows that more exosomes with biological activity can be obtained by the method, and the yield of the exosomes can be improved.
Example 2
1. Obtaining umbilical cord blood
Reference is made to example 1.
2. Culture of umbilical cord blood MSC
(1) Selecting a temperature-sensitive Cytodex-3 microcarrier (brand Cytiva product number 17048503), carrying out ultraviolet treatment on an ultra-clean bench according to a standard procedure, paving the temperature-sensitive microcarrier on a filter membrane as much as possible, irradiating ultraviolet in the ultra-clean bench for 30min each time, turning the microcarrier after one irradiation, carrying out ultraviolet irradiation again, soaking the microcarrier in PBS overnight after 4 times of ultraviolet treatment, discarding the PBS, adding fresh PBS for rinsing 3 times, finally leaving PBS capable of immersing the volume of the microcarrier, sterilizing at 121 ℃ for 30min under high pressure, cooling for 4 ℃ for later use, and using a medium (glucose 0.6%, L-glutamine 2mM (added in use), VEGF 20ng/mL, bFGF-210ng/mL and NaHCO) containing 5% of umbilical cord blood plasma by volume percentage 3 3 mM, hepes5mM, vitamin C0.4 mM, butanediamine 60mM, zoledronate sodium 3. Mu.M, penicillin 100U/mL, streptomycin 100U/mL, DMEM/F12 (1:1) medium to 1L) substitution, and after incubation at 37℃for 4 h;
(2) Inoculating the microcarrier of step (1) to a culture flask at a density of 1g/L, and sterilizing the umbilical cord blood MSC suspension 3×10 before sterilization 5 Density of individual cells/mL of culture medium inoculated into the above flask at 35℃with 3% CO 2 Culturing in incubator, observing cell growth state and adherence effect, photographing with common inverted optical microscope to obtain the result shown in figure 1, culturing cells to obtain microcarrier, autoclaving, cooling to 20deg.C, and cooling to obtain microcarrier state shown in figure 2.
3. Preparation of exosomes
A high-speed refrigerating table type centrifuge and a miniature freeze dryer are adopted.
(1) Collecting 500mL of umbilical cord blood mesenchymal stem cell culture supernatant;
(2) Centrifuging at 4deg.C for 2000 Xg for 10min to obtain supernatant (dead cells removed);
(3) Centrifuging at 4deg.C for 10,000Xg for 30min to obtain supernatant (removing cell debris);
(4) Centrifuging at 4deg.C for 100,000Xg for 70min to retain precipitate, and discarding supernatant (mainly exosomes and heteroproteins;
(5) Washing the precipitate with sterile PBS, centrifuging for 70min at 100,000Xg, and discarding supernatant to obtain the exosome.
4. Identification of exosomes
Results: the purity of extracted exosome protein is 97%, and the positive signals of the two surface markers of CD63 and CD81 in MSC cell exosome are 83.1% and 82.1% respectively.
Example 3
1. Obtaining umbilical cord blood
Reference is made to example 1.
2. Culture of umbilical cord blood MSC
(1) Selecting a temperature-sensitive Cytodex-3 microcarrier (brand Cytiva product number 17048503), performing ultraviolet treatment on an ultra-clean bench according to a standard procedure, spreading the temperature-sensitive microcarrier on a filter membrane as much as possible, irradiating ultraviolet in the ultra-clean bench for 30min each time, turning the microcarrier after one irradiation, performing ultraviolet irradiation again, soaking the microcarrier in PBS overnight after 4 times of ultraviolet treatment, discarding the PBS, adding fresh PBS for rinsing 3 times, finally leaving PBS capable of immersing the volume of the microcarrier, sterilizing at 121 ℃ for 30min under high pressure, cooling for 4 ℃ for later use, and using a culture medium (glucose 0.6%, L-glutamine 2mM (added in use), VEGF 20ng/mL, bFGF-210ng/mL and NaHCO) containing 10% of umbilical cord blood plasma by volume percent just before use 3 3 mM, hepes5mM, vitamin C0.4 mM, butanediamine 60mM, zoledronate sodium 3 μM, penicillin 100U/mL, streptomycin 100U/mL, medium added to 1L), and after incubation at 37℃for 12 h;
(2) Inoculating the microcarrier of step (1) to a culture flask at a density of 5g/L, and sterilizing the umbilical cord blood MSC suspension at a concentration of 2×10 before sterilization 5 Density of individual cells/mL of culture medium inoculated into the above flask at 38deg.C, 8% CO 2 Culturing in incubator, observing cell growth state and adherence effect, photographing with common inverted optical microscope to obtain the result shown in figure 1, culturing cells to obtain microcarrier, sterilizing under high pressure, cooling, and cooling to obtain microcarrier shown in figure 2.
3. Preparation of exosomes
A high-speed refrigerating table type centrifuge and a miniature freeze dryer are adopted.
(1) Collecting 500mL of umbilical cord blood mesenchymal stem cell culture supernatant;
(2) Centrifuging at 4deg.C for 2000 Xg for 10min to obtain supernatant (dead cells removed);
(3) Centrifuging at 4deg.C for 10,000Xg for 30min to obtain supernatant (removing cell debris);
(4) Centrifuging at 4deg.C for 100,000Xg for 70min to retain precipitate, and discarding supernatant (mainly exosomes and heteroproteins;
(5) Washing the precipitate with sterile PBS, centrifuging for 70min at 100,000Xg, and discarding supernatant to obtain the exosome.
4. Identification of exosomes
Results: the purity of extracted exosome protein is 96%, and the positive signals of the two surface markers of CD63 and CD81 in MSC cell exosome are 81.6% and 81.5% respectively.
Comparative example 1
The only difference from example 1 is that the medium was a medium specific for purchased human umbilical cord mesenchymal stem cells (cat# CM-CL 11). Results: the purity of extracted exosome protein is 95%, and the positive signals of the two surface markers of CD63 and CD81 in MSC cell exosome are 63.6% and 62.5% respectively.
The data obtained in examples 1-3 show that the purity of the exosome protein obtained by the method can reach 92%, the positive signals of the CD63 and CD81 surface markers in the MSC exosome can reach 84.6% and 84.5% at the highest positive rate, and the exosome obtained by the method has high purity, high yield, good uniformity and good integrity, can obtain more exosomes with biological activity, and compared with the traditional adherence method for culturing mesenchymal stem cells, no cord blood plasma is added in the culture medium, so that the purity of the exosomes is obviously reduced, and the positive rate of the surface markers is obviously reduced.
In conclusion, the mesenchymal stem cells are cultured in the culture container containing the umbilical cord blood plasma by utilizing the temperature-sensitive microcarrier for the first time, so that the yield of exosomes can be greatly improved, the mesenchymal stem cells and exosomes are safer and suitable for clinical application by utilizing the umbilical cord blood plasma, the mesenchymal stem cells are prevented from being damaged by chemical substances such as pancreatin when being separated by utilizing the temperature-sensitive technology, the yield and uniformity of the exosomes are improved, more exosomes with biological activity can be obtained, and large-scale exosomes acquisition and large-scale exosomes preparation can be realized.
The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
Claims (10)
1. A method of producing an umbilical cord blood exosome, the method comprising:
and inoculating the mesenchymal stem cells and the temperature-sensitive microcarrier into a culture medium containing umbilical cord blood plasma for culture, collecting mesenchymal stem cell suspension after culture, and centrifuging to obtain exosomes.
2. The method of claim 1, wherein the mesenchymal stem cells comprise: umbilical cord blood mesenchymal stem cells.
3. The method according to claim 1 or 2, wherein the temperature sensitive microcarrier has an inoculation density of 1-5g/L.
4. A method according to any one of claims 1-3, wherein the volume percentage of cord blood plasma in the medium is 5-10%.
5. The method according to any one of claims 1-4, wherein the mesenchymal stem cells are seeded at a density of 2 x 10 5 -3×10 5 Individual cells/mL.
6. The method according to any one of claims 1 to 5, wherein the time of the culturing is 4 to 12 hours.
7. The method according to any one of claims 1 to 6, wherein the culturing conditions are: the temperature is 35-38deg.C, CO 2 The concentration is 3-8%.
8. The method according to any one of claims 1-7, characterized in that the method comprises the steps of:
(1) Ultraviolet autoclaving the temperature sensitive microcarrier;
(2) Incubating the inoculated and sterilized temperature-sensitive microcarrier and umbilical cord blood mesenchymal stem cells in a medium containing umbilical cord blood plasma, wherein the inoculation density of the temperature-sensitive microcarrier is 1-5g/L, the volume percentage of the umbilical cord blood plasma in the medium is 5-10%, and the inoculation density of the umbilical cord blood mesenchymal stem cells is 2 multiplied by 10 5 -3×10 5 Individual cells/mL;
(3) And after culturing, collecting mesenchymal stem cell suspension of umbilical cord blood, and centrifuging to obtain exosomes.
9. An umbilical cord blood exosome produced by the method of producing an umbilical cord blood exosome of any one of claims 1-8.
10. Use of the umbilical cord blood exosomes of claim 9 for promoting cell regeneration repair and for the preparation of a medicament for treating a disease;
preferably, the disease comprises any one or a combination of at least two of cardiovascular disease, neurological disease or immune system disease.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310763216.2A CN116790492A (en) | 2023-06-27 | 2023-06-27 | Method for producing umbilical cord blood exosomes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310763216.2A CN116790492A (en) | 2023-06-27 | 2023-06-27 | Method for producing umbilical cord blood exosomes |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116790492A true CN116790492A (en) | 2023-09-22 |
Family
ID=88034320
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310763216.2A Pending CN116790492A (en) | 2023-06-27 | 2023-06-27 | Method for producing umbilical cord blood exosomes |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116790492A (en) |
Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103409361A (en) * | 2013-06-24 | 2013-11-27 | 上海瀚正生物技术服务有限公司 | Thermosensitive microcarrier as well as preparation technology and application method thereof |
| CN107988153A (en) * | 2017-12-15 | 2018-05-04 | 英科博雅生命科技有限公司 | The method of mesenchymal stem cells derived from human umbilical blood source separation excretion body and the reagent used |
| CN108103014A (en) * | 2017-12-28 | 2018-06-01 | 重庆斯德姆生物技术有限公司 | A kind of method for preserving of umbilical cord mesenchymal stem cells culture supernatant |
| CN111154648A (en) * | 2019-12-27 | 2020-05-15 | 新乡医学院三全学院 | Stem cell source exosome culture device |
| CN111450117A (en) * | 2020-03-30 | 2020-07-28 | 苏州大学 | Temperature-sensitive exosome system with maternal impression and preparation method and application thereof |
| CN111808809A (en) * | 2020-06-05 | 2020-10-23 | 广州医科大学附属第二医院 | Exosome derived from human umbilical cord blood and preparation method and application thereof |
| CN112442484A (en) * | 2020-11-25 | 2021-03-05 | 大连普瑞康生物技术有限公司 | Method for large-scale cell culture based on porous nanoscale temperature-sensitive soft colloid |
| CN112553155A (en) * | 2020-12-28 | 2021-03-26 | 山东省齐鲁干细胞工程有限公司 | Umbilical cord blood mesenchymal stem cell culture method |
| CN114712302A (en) * | 2022-03-25 | 2022-07-08 | 浙江大学 | Exosome hydrogel and application thereof |
| CN114748510A (en) * | 2020-12-29 | 2022-07-15 | 南京优智源医药科技有限公司 | Application of human umbilical cord blood plasma exosome in preparation of medicine for treating degenerative diseases caused by aging |
| CN115025034A (en) * | 2022-06-24 | 2022-09-09 | 广州美蔻生物科技有限公司 | Mesenchymal stem cell exosome composition and preparation method and application thereof |
| US20230144187A1 (en) * | 2021-11-05 | 2023-05-11 | The First Hospital of Lanzhou University | Preparation method of thermosensitive gel for treating androgenetic alopecia |
| CN219239646U (en) * | 2022-10-16 | 2023-06-23 | 北京三一造血技术有限公司 | High-purity exosome separator |
| CN116286624A (en) * | 2023-03-14 | 2023-06-23 | 青岛安融生物健康科技有限公司 | Application of water-soluble chitosan in stem cell in-vitro culture |
-
2023
- 2023-06-27 CN CN202310763216.2A patent/CN116790492A/en active Pending
Patent Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103409361A (en) * | 2013-06-24 | 2013-11-27 | 上海瀚正生物技术服务有限公司 | Thermosensitive microcarrier as well as preparation technology and application method thereof |
| CN107988153A (en) * | 2017-12-15 | 2018-05-04 | 英科博雅生命科技有限公司 | The method of mesenchymal stem cells derived from human umbilical blood source separation excretion body and the reagent used |
| CN108103014A (en) * | 2017-12-28 | 2018-06-01 | 重庆斯德姆生物技术有限公司 | A kind of method for preserving of umbilical cord mesenchymal stem cells culture supernatant |
| CN111154648A (en) * | 2019-12-27 | 2020-05-15 | 新乡医学院三全学院 | Stem cell source exosome culture device |
| CN111450117A (en) * | 2020-03-30 | 2020-07-28 | 苏州大学 | Temperature-sensitive exosome system with maternal impression and preparation method and application thereof |
| CN111808809A (en) * | 2020-06-05 | 2020-10-23 | 广州医科大学附属第二医院 | Exosome derived from human umbilical cord blood and preparation method and application thereof |
| CN112442484A (en) * | 2020-11-25 | 2021-03-05 | 大连普瑞康生物技术有限公司 | Method for large-scale cell culture based on porous nanoscale temperature-sensitive soft colloid |
| CN112553155A (en) * | 2020-12-28 | 2021-03-26 | 山东省齐鲁干细胞工程有限公司 | Umbilical cord blood mesenchymal stem cell culture method |
| CN114748510A (en) * | 2020-12-29 | 2022-07-15 | 南京优智源医药科技有限公司 | Application of human umbilical cord blood plasma exosome in preparation of medicine for treating degenerative diseases caused by aging |
| US20230144187A1 (en) * | 2021-11-05 | 2023-05-11 | The First Hospital of Lanzhou University | Preparation method of thermosensitive gel for treating androgenetic alopecia |
| CN114712302A (en) * | 2022-03-25 | 2022-07-08 | 浙江大学 | Exosome hydrogel and application thereof |
| CN115025034A (en) * | 2022-06-24 | 2022-09-09 | 广州美蔻生物科技有限公司 | Mesenchymal stem cell exosome composition and preparation method and application thereof |
| CN219239646U (en) * | 2022-10-16 | 2023-06-23 | 北京三一造血技术有限公司 | High-purity exosome separator |
| CN116286624A (en) * | 2023-03-14 | 2023-06-23 | 青岛安融生物健康科技有限公司 | Application of water-soluble chitosan in stem cell in-vitro culture |
Non-Patent Citations (1)
| Title |
|---|
| INAARA MAWJI ET AL.,: "Challenges and opportunities in downstream separation processes for mesenchymal stromal cells cultured in microcarrier‐based stirred suspension bioreactors", BIOTECHNOLOGY AND BIOENGINEERING, vol. 119, pages 3062 - 3078 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ryan et al. | Methods for microcarrier culture of bovine pulmonary artery endothelial cells avoiding the use of enzymes | |
| CN114591905B (en) | Method for preparing apoptotic vesicles from human erythrocytes and application of apoptotic vesicles | |
| CN110669729B (en) | Method for preparing mesenchymal stem cell exosome | |
| CN112899218A (en) | Dual tangential flow filtration system for exosome extraction, and preparation method and application of exosome | |
| CN112553142B (en) | 3D organ of nasal mucosa epithelial cells and culture method and application thereof | |
| CN106980018B (en) | A kind of kit and its application using CD45 immunofluorescences joint CEP17 probe identification circulating tumor cells | |
| CN106970225B (en) | A kind of kit and its application for combining 8 probe identification circulating tumor cells of CEP using CD45 immunofluorescences | |
| CN111334469A (en) | PBMC (peripheral blood mononuclear cell) in-vitro 3D (three-dimensional) methylcellulose agarose hydrogel culture medium and preparation method thereof | |
| CN111759862A (en) | Application of stem cell exosome in preparation of anti-colitis-exacerbation medicine | |
| CN108384745A (en) | A kind of method that improved two steps enzyme is separately cultured sustentacular cell of testis | |
| CN112852725B (en) | Preparation method and application for extracting and purifying stem cell exosome by using protein cross-linked nano affinity microspheres | |
| CN110592001B (en) | Purification culture system for oviduct epithelial cells | |
| CN105647869B (en) | Human lung adenocarcinoma cell strain HA1221 and establishment method thereof | |
| CN116790492A (en) | Method for producing umbilical cord blood exosomes | |
| CN108410796B (en) | Method for inducing differentiation of human mesenchymal stem cells into vascular endothelial cells | |
| CN111172104A (en) | Separation culture method of umbilical blood mesenchymal stem cells | |
| CN112458055B (en) | Method for preparing cell microvesicles under stimulation effect based on shear stress | |
| CN111394301B (en) | Application of piceatannol in increasing number of secreted exosomes of pluripotent stem cells and bioactivity | |
| CN113018501A (en) | Endothelial progenitor cell exosome medical dressing, preparation method and application thereof | |
| CN112980006A (en) | Protein cross-linked nano affinity microsphere, preparation method and application | |
| CN111893088A (en) | Method for in vitro isolated culture of Mongolian horse testis spermatogonium stem cells | |
| CN115006350B (en) | Construction of milk fat plastid and application thereof in macrophage immunocompetence regulation | |
| CN109439614A (en) | A kind of excretion body preparation of maintenance and reply hair papilla cell stemness | |
| CN117660325B (en) | Culture medium for preparing umbilical cord blood MSC and method thereof | |
| CN111979176B (en) | Preparation method of human corneal epithelial cells, conditioned medium thereof and preparation method thereof |
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 |