CN111304164A - Preparation method and application of adipose-derived stem cell exosomes - Google Patents
Preparation method and application of adipose-derived stem cell exosomes Download PDFInfo
- Publication number
- CN111304164A CN111304164A CN201911208966.3A CN201911208966A CN111304164A CN 111304164 A CN111304164 A CN 111304164A CN 201911208966 A CN201911208966 A CN 201911208966A CN 111304164 A CN111304164 A CN 111304164A
- Authority
- CN
- China
- Prior art keywords
- exosome
- stem cell
- adipose
- preparation
- derived
- 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 65
- 238000002360 preparation method Methods 0.000 title claims abstract description 35
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 25
- 238000004519 manufacturing process Methods 0.000 claims abstract description 9
- 239000002537 cosmetic Substances 0.000 claims abstract description 8
- 210000004027 cell Anatomy 0.000 claims description 35
- 210000002901 mesenchymal stem cell Anatomy 0.000 claims description 32
- 238000000034 method Methods 0.000 claims description 21
- 239000006228 supernatant Substances 0.000 claims description 21
- 238000001914 filtration Methods 0.000 claims description 12
- 238000012258 culturing Methods 0.000 claims description 11
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 9
- 239000000843 powder Substances 0.000 claims description 8
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 6
- 210000003743 erythrocyte Anatomy 0.000 claims description 6
- 239000012894 fetal calf serum Substances 0.000 claims description 6
- 239000001963 growth medium Substances 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 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 3
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 3
- 102000029816 Collagenase Human genes 0.000 claims description 3
- 108060005980 Collagenase Proteins 0.000 claims description 3
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 3
- 229920001503 Glucan Polymers 0.000 claims description 3
- 229930195725 Mannitol Natural products 0.000 claims description 3
- 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 3
- 102000004142 Trypsin Human genes 0.000 claims description 3
- 108090000631 Trypsin Proteins 0.000 claims description 3
- 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 3
- 230000001640 apoptogenic effect Effects 0.000 claims description 3
- 239000006143 cell culture medium Substances 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 229960002424 collagenase Drugs 0.000 claims description 3
- 239000000287 crude extract Substances 0.000 claims description 3
- 230000029087 digestion Effects 0.000 claims description 3
- 210000003195 fascia Anatomy 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 125000002566 glucosaminyl group Chemical group 0.000 claims description 3
- 229920002674 hyaluronan Polymers 0.000 claims description 3
- 229960003160 hyaluronic acid Drugs 0.000 claims description 3
- 239000000411 inducer Substances 0.000 claims description 3
- 238000007443 liposuction Methods 0.000 claims description 3
- 239000006166 lysate Substances 0.000 claims description 3
- 239000000594 mannitol Substances 0.000 claims description 3
- 235000010355 mannitol Nutrition 0.000 claims description 3
- 239000002609 medium Substances 0.000 claims description 3
- 239000008188 pellet Substances 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 239000004017 serum-free culture medium Substances 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 229960005322 streptomycin Drugs 0.000 claims description 3
- 239000012588 trypsin Substances 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 238000005303 weighing Methods 0.000 claims description 3
- 230000004069 differentiation Effects 0.000 claims description 2
- 238000012136 culture method Methods 0.000 claims 1
- 102100032912 CD44 antigen Human genes 0.000 abstract description 7
- 102100037241 Endoglin Human genes 0.000 abstract description 7
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 abstract description 7
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 abstract description 7
- 101000881679 Homo sapiens Endoglin Proteins 0.000 abstract description 7
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 abstract description 7
- 101000935043 Homo sapiens Integrin beta-1 Proteins 0.000 abstract description 7
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 abstract description 7
- 101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 abstract description 7
- 102100025304 Integrin beta-1 Human genes 0.000 abstract description 7
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 abstract description 7
- 102100033523 Thy-1 membrane glycoprotein Human genes 0.000 abstract description 7
- 238000007796 conventional method Methods 0.000 abstract description 5
- 230000003712 anti-aging effect Effects 0.000 abstract description 4
- 238000000684 flow cytometry Methods 0.000 abstract description 2
- 230000014509 gene expression Effects 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 230000032683 aging Effects 0.000 description 4
- 239000011324 bead Substances 0.000 description 4
- 230000003053 immunization Effects 0.000 description 4
- 238000002649 immunization Methods 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 238000000108 ultra-filtration Methods 0.000 description 4
- 230000003796 beauty Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 210000001163 endosome Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 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
- 230000037361 pathway Effects 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000030968 tissue homeostasis Effects 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000037314 wound repair 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
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/84—Products or compounds obtained by lyophilisation, freeze-drying
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Developmental Biology & Embryology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Cell Biology (AREA)
- Dermatology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Rheumatology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Cosmetics (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of preparation methods of stem cell exosomes, and discloses a preparation method and application of an adipose-derived stem cell exosome, wherein the prepared exosome is subjected to Flow cytometry analysis by adopting a conventional method, and data are analyzed by Flow software7.6.1 software, and results show that CD29, CD44, CD90 and CD105 in the exosome are positively expressed, the positive rates are 97.7%, 89.3%, 97.1% and 84.6%, CD34 and CD45 are negatively expressed, and the negative rates are 97.7% and 92.7%, so that the later-stage use is improved, the problems of poor repairing and anti-aging effects, poor general stability and high production cost of a cosmetic preparation are solved, and the aims of improving the repairing and anti-aging effects, improving the stability and reducing the production cost are fulfilled.
Description
Technical Field
The invention relates to the technical field of preparation methods of stem cell exosomes, in particular to a preparation method and application of an adipose-derived stem cell exosome.
Background
The mesenchymal stem cell is one of important seed cells, has self-renewal and multidirectional differentiation potential, has stable genetic information, and can be extracted in large quantity by a simpler method. Compared with other mesenchymal stem cell sources, adipose tissues are tissues which are rich in sources, can be consumed and are easily obtained, so that the adipose mesenchymal stem cells gradually become a new favorite for stem cell research, and the research on the adipose mesenchymal stem cells is more and more. However, the storage condition of the adipose-derived mesenchymal stem cells is harsh, the transportation is inconvenient, and the clinical application is difficult.
The exosome is a cystic vesicle which is generated through an endosome pathway and released outside cells, is coated by a phospholipid double-layer membrane, and is internally coated with a plurality of bioactive substances such as lipid, protein, nucleic acid and the like, is cupped under the observation of an electron microscope, and plays an important role in the transfer of the substances among the cells and the information transmission. Recent studies have shown that exosomes play an important role in intercellular signal communication, have the functions of immunoregulation, promotion of angiogenesis, mediation of cell proliferation, differentiation, migration, apoptosis and the like, maintain the normal physiological state of the body and participate in disease processes. The mesenchymal stem cell-derived exosome has the characteristics of derived cells, and can promote self-repair and tissue regeneration of cells in a damaged area, restore tissue homeostasis, accelerate wound repair and the like.
The exosome secreted by the human adipose-derived mesenchymal stem cell has a stable structure, is not easy to decompose, and provides a new possibility for the adipose-derived mesenchymal stem cell in clinical application.
The traditional exosome preparation technology comprises ultra-separation, ultra-filtration, magnetic bead immunization, a PEG precipitation method and the like, CD29, CD44, CD90 and CD105 in the prepared exosome are positively expressed, the positive rate is respectively lower than 80%, CD34 and CD45 are negatively expressed, the negative rate is respectively lower than 90%, the later-stage use is influenced, and the repairing damage and anti-aging effects of the existing cosmetic preparations are not ideal.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a preparation method and application of an adipose-derived stem cell exosome, and solves the problems that CD29, CD44, CD90 and CD105 in the exosome prepared by the existing equipment are all positively expressed, the positive rates are respectively lower than 70.1, CD34 and CD45 are negatively expressed, the negative rates are respectively lower than 90%, the repairing and anti-aging effects of a cosmetic preparation are not ideal, the stability is not good, the production cost is high and the like.
(II) technical scheme
The invention provides the following technical scheme: a preparation method and application of adipose-derived stem cell exosomes are disclosed, wherein the preparation method at least comprises the following steps:
(1) culturing human adipose-derived mesenchymal stem cells, and adding an inducer into a cell culture medium in a culturing stage;
(2) pretreating the cultured stem cells;
(3) extracting the human adipose-derived mesenchymal stem cell exosomes from the pretreated culture medium supernatant;
(4) and (3) preparing an exosome preparation.
Preferably, the exosome is differentiated from a mesenchymal stem cell.
Preferably, the mesenchymal stem cell source is human adipose mesenchymal stem cells.
Preferably, the method also comprises a method for culturing the human adipose-derived mesenchymal stem cells:
(1) taking 30mL of fat after liposuction, washing with PBS containing penicillin-streptomycin double antibody for 3 times, removing fascia and erythrocytes, adding 30mL of 0.075% type I collagenase [7-11], digesting at 37 deg.C and 150r/min for 40-45 min;
(2) adding DMEM/F12 culture medium containing 10% fetal calf serum in volume fraction to stop digestion, centrifuging at 15000 r/min for 10min, removing upper layer oil and middle layer clear liquid, re-suspending with 5mL erythrocyte lysate, standing at room temperature for 5min, and centrifuging at 1000r/min for 5 min;
(3) removing supernatant, resuspending with low-sugar DMEM medium containing fetal calf serum with volume fraction of 15%, filtering with 70 μm cell sieve, transferring into culture dish for primary culture, changing liquid every three days, digesting with trypsin with volume fraction of 0.25% when cell concentration is 70% -80%, and subculturing at 1: 2;
(4) taking 3-6 generation cells, culturing for 24-48h with serum-free culture medium when the cells are fused and grown to 70-80%, collecting cell supernatant, and storing in-80 deg.C refrigerator.
Preferably, the method also comprises the following steps:
(1) centrifuging the collected supernatant at 4 deg.C under aseptic condition at 300 × g for 10min to remove dead cells and large cell debris, and centrifuging at 2000 × g for 10min to remove dead cells and cell debris; centrifuging at 10000 Xg/min for 30min to remove vesicles with larger cell debris; filtering with 0.22 μm needle filter to remove microbubbles and possible apoptotic bodies;
(2) transferring the supernatant into an ultracentrifuge tube by using a 20mL empty needle, centrifuging for 70min at 106 Xg, removing the supernatant, and collecting the precipitate to obtain a crude extract exosome;
(3) centrifugation at 106 Xg for 70min removed the supernatant and pellet solubilized with 100. mu.LPBS to give relatively pure exosomes.
Preferably, the preparation method comprises the following steps:
(1) taking exosome, adding 10% mannitol, mixing uniformly, filtering with a 0.22 μm needle filter, subpackaging, and placing in a refrigerator at minus 80 ℃ for 10 hours. Then freeze-drying under vacuum (-50 ℃, 24 h) to obtain the human mesenchymal stem cell exosome freeze-dried powder;
(2) weighing exosome freeze-dried powder 10mg, glucosaminyl glucan 15mg, hyaluronic acid 15mg and trehalose 10mg, dissolving in 100ml of glycerol, mixing and stirring to be transparent, and filtering by using a 0.22 mu m needle filter to obtain the cosmetic preparation.
(III) advantageous effects
Compared with the prior art, the invention provides a preparation method and application of an adipose-derived stem cell exosome, and the preparation method has the following beneficial effects:
(1) according to the preparation method of the adipose-derived stem cell exosome, the prepared exosome is analyzed by a Flow cytometer by adopting a conventional method, data are analyzed by Flow software7.6.1 software, and the result shows that CD29, CD44, CD90 and CD105 in the exosome are all positive expressions, the positive rates are respectively 97.7%, 89.3%, 97.1% and 84.6%, CD34 and CD45 are negative expressions, the negative rates are respectively 97.7% and 92.7%, and the later-stage use is improved.
(2) The application of the adipose-derived stem cell exosome solves the problems that the effect of repairing damage and resisting aging of a beauty preparation is not ideal, the stability is poor and the production cost is high generally, so that the aims of improving the effect of repairing damage and resisting aging, improving the stability and reducing the production cost are fulfilled.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example (b): a preparation method and application of adipose-derived stem cell exosomes are disclosed, wherein the preparation method at least comprises the following steps:
(1) culturing human adipose-derived mesenchymal stem cells, and adding an inducer into a cell culture medium in a culturing stage;
(2) pretreating the cultured stem cells;
(3) extracting the human adipose-derived mesenchymal stem cell exosomes from the pretreated culture medium supernatant;
(4) and (3) preparing an exosome preparation.
Further, exosomes are differentiated from mesenchymal stem cells.
Further, the mesenchymal stem cell source is human adipose mesenchymal stem cells.
Further, the method also comprises a method for culturing the human adipose-derived mesenchymal stem cells:
(1) taking 30mL of fat after liposuction, washing with PBS containing penicillin-streptomycin double antibody for 3 times, removing fascia and erythrocytes, adding 30mL of 0.075% type I collagenase [7-11], digesting at 37 deg.C and 150r/min for 40-45 min;
(2) adding DMEM/F12 culture medium containing 10% fetal calf serum in volume fraction to stop digestion, centrifuging at 15000 r/min for 10min, removing upper layer oil and middle layer clear liquid, re-suspending with 5mL erythrocyte lysate, standing at room temperature for 5min, and centrifuging at 1000r/min for 5 min;
(3) removing supernatant, resuspending with low-sugar DMEM medium containing fetal calf serum with volume fraction of 15%, filtering with 70 μm cell sieve, transferring into culture dish for primary culture, changing liquid every three days, digesting with trypsin with volume fraction of 0.25% when cell concentration is 70% -80%, and subculturing at 1: 2;
(4) taking 3-6 generation cells, culturing for 24-48h with serum-free culture medium when the cells are fused and grown to 70-80%, collecting cell supernatant, and storing in-80 deg.C refrigerator.
Further, the method for extracting the human adipose-derived mesenchymal stem cell exosomes comprises the following steps:
(1) centrifuging the collected supernatant at 4 deg.C under aseptic condition at 300 × g for 10min to remove dead cells and large cell debris, and centrifuging at 2000 × g for 10min to remove dead cells and cell debris; centrifuging at 10000 Xg/min for 30min to remove vesicles with larger cell debris; filtering with 0.22 μm needle filter to remove microbubbles and possible apoptotic bodies;
(2) transferring the supernatant into an ultracentrifuge tube by using a 20mL empty needle, centrifuging for 70min at 106 Xg, removing the supernatant, and collecting the precipitate to obtain a crude extract exosome;
(3) centrifugation at 106 Xg for 70min removed the supernatant and pellet solubilized with 100. mu.LPBS to give relatively pure exosomes.
A preparation method of a freeze-dried powder cosmetic preparation of human adipose-derived mesenchymal stem cell exosomes comprises the following steps:
(1) taking exosome, adding 10% mannitol, mixing uniformly, filtering with a 0.22 μm needle filter, subpackaging, and placing in a refrigerator at minus 80 ℃ for 10 hours. Then freeze-drying under vacuum (-50 ℃, 24 h) to obtain the human mesenchymal stem cell exosome freeze-dried powder;
(2) weighing exosome freeze-dried powder 10mg, glucosaminyl glucan 15mg, hyaluronic acid 15mg and trehalose 10mg, dissolving in 100ml of glycerol, mixing and stirring to be transparent, and filtering by using a 0.22 mu m needle filter to obtain the cosmetic preparation.
To sum up, according to the preparation method of the adipose-derived stem cell exosome, the prepared exosome is subjected to Flow cytometry analysis by using a conventional method, and the data is analyzed by using Flow software7.6.1 software, and the result shows that CD29, CD44, CD90 and CD105 in the exosome are positive expressions, the positive rates are 97.7%, 89.3%, 97.1% and 84.6% respectively, the CD34 and CD45 are negative expressions, and the negative rates are 97.7% and 92.7% respectively.
The application of the adipose-derived stem cell exosome solves the problems that the effect of repairing damage and resisting aging of a beauty preparation is not ideal, the stability is poor and the production cost is high generally, so that the aims of improving the effect of repairing damage and resisting aging, improving the stability and reducing the production cost are fulfilled.
Experimental example 1: exosomes were prepared by the example, ultra-separation, ultra-filtration, magnetic bead immunization and PEG precipitation methods, two each prepared, 10g each.
| Positive/% | CD29 | CD44 | CD90 | CD105 |
| Super separation | 85 | 80 | 84 | 76 |
| Ultrafiltration | 80 | 73 | 81 | 76 |
| Magnetic bead immunization | 84 | 75 | 83 | 72 |
| PEG precipitation method | 79 | 77 | 76 | 78 |
| Examples | 97.7 | 89.3 | 97.1 | 84.6 |
| Negative/% | CD34 | CD45 |
| Super separation | 85 | 87 |
| Ultrafiltration | 90 | 83 |
| Magnetic bead immunization | 94 | 79 |
| PEG precipitation method | 89 | 80 |
| Examples | 97.7 | 92.7 |
The judging method comprises the following steps: the prepared exosomes are analyzed by a flow cytometer by adopting a conventional method, and data are analyzed by FlowSoftware7.6.1 software.
The structure shows that: the prepared exosome is analyzed by a Flow cytometer by adopting a conventional method, and data is analyzed by Flow software7.6.1 software, and the result shows that CD29, CD44, CD90 and CD105 in the exosome are all positive expressions, the positive rates are 97.7%, 89.3%, 97.1% and 84.6% respectively, the CD34 and CD45 are negative expressions, the negative rates are 97.7% and 92.7% respectively, and the later-period use is improved.
It is to be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. A preparation method of an adipose-derived stem cell exosome is characterized by comprising the following steps: the preparation method at least comprises the following steps:
(1) culturing human adipose-derived mesenchymal stem cells, and adding an inducer into a cell culture medium in a culturing stage;
(2) pretreating the cultured stem cells;
(3) extracting the human adipose-derived mesenchymal stem cell exosomes from the pretreated culture medium supernatant;
(4) and (3) preparing an exosome preparation.
2. The method for producing an adipose-derived stem cell exosome according to claim 1, wherein: the exosome is derived from mesenchymal stem cells by differentiation.
3. The method for producing an adipose-derived stem cell exosome according to claim 1, wherein: the mesenchymal stem cell source is human adipose mesenchymal stem cells.
4. The preparation method and application of the adipose-derived stem cell exosome according to claim 1, further comprising a culture method of human adipose-derived mesenchymal stem cells:
(1) taking 30mL of fat after liposuction, washing with PBS containing penicillin-streptomycin double antibody for 3 times, removing fascia and erythrocytes, adding 30mL of 0.075% type I collagenase [7-11], digesting at 37 deg.C and 150r/min for 40-45 min;
(2) adding DMEM/F12 culture medium containing 10% fetal calf serum in volume fraction to stop digestion, centrifuging at 15000 r/min for 10min, removing upper layer oil and middle layer clear liquid, re-suspending with 5mL erythrocyte lysate, standing at room temperature for 5min, and centrifuging at 1000r/min for 5 min;
(3) removing supernatant, resuspending with low-sugar DMEM medium containing fetal calf serum with volume fraction of 15%, filtering with 70 μm cell sieve, transferring into culture dish for primary culture, changing liquid every three days, digesting with trypsin with volume fraction of 0.25% when cell concentration is 70% -80%, and subculturing at 1: 2;
(4) taking 3-6 generation cells, culturing for 24-48h with serum-free culture medium when the cells are fused and grown to 70-80%, collecting cell supernatant, and storing in-80 deg.C refrigerator.
5. The method for preparing an adipose-derived stem cell exosome according to claim 1, further comprising a method for extracting a human adipose-derived mesenchymal stem cell exosome:
(1) centrifuging the collected supernatant at 4 deg.C under aseptic condition at 300 × g for 10min to remove dead cells and large cell debris, and centrifuging at 2000 × g for 10min to remove dead cells and cell debris; centrifuging at 10000 Xg/min for 30min to remove vesicles with larger cell debris; filtering with 0.22 μm needle filter to remove microbubbles and possible apoptotic bodies;
(2) transferring the supernatant into an ultracentrifuge tube by using a 20mL empty needle, centrifuging for 70min at 106 Xg, removing the supernatant, and collecting the precipitate to obtain a crude extract exosome;
(3) centrifugation at 106 Xg for 70min removed the supernatant and pellet solubilized with 100. mu.LPBS to give relatively pure exosomes.
6. The application of the adipose-derived stem cell exosome according to claim 1, which is characterized in that the preparation method of the freeze-dried powder cosmetic preparation of the human adipose-derived stem cell exosome comprises the following steps:
(1) taking exosome, adding 10% mannitol, mixing uniformly, filtering with a 0.22 μm needle filter, subpackaging, and placing in a refrigerator at minus 80 ℃ for 10 hours.
7. Then freeze-drying under vacuum (-50 ℃, 24 h) to obtain the human mesenchymal stem cell exosome freeze-dried powder;
(2) weighing exosome freeze-dried powder 10mg, glucosaminyl glucan 15mg, hyaluronic acid 15mg and trehalose 10mg, dissolving in 100ml of glycerol, mixing and stirring to be transparent, and filtering by using a 0.22 mu m needle filter to obtain the cosmetic preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911208966.3A CN111304164A (en) | 2019-11-30 | 2019-11-30 | Preparation method and application of adipose-derived stem cell exosomes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911208966.3A CN111304164A (en) | 2019-11-30 | 2019-11-30 | Preparation method and application of adipose-derived stem cell exosomes |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111304164A true CN111304164A (en) | 2020-06-19 |
Family
ID=71148570
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911208966.3A Pending CN111304164A (en) | 2019-11-30 | 2019-11-30 | Preparation method and application of adipose-derived stem cell exosomes |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111304164A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111944748A (en) * | 2020-08-14 | 2020-11-17 | 福建医科大学附属协和医院 | high-IL-10-expression human adipose-derived mesenchymal stem cell exosome for treating myocardial infarction and application thereof |
| CN111944747A (en) * | 2020-08-14 | 2020-11-17 | 福建医科大学附属协和医院 | Human adipose-derived mesenchymal stem cell exosome for treating myocardial infarction and application thereof |
| CN111944749A (en) * | 2020-08-14 | 2020-11-17 | 福建医科大学附属协和医院 | Preparation method of human adipose-derived mesenchymal stem cell exosome for treating myocardial infarction |
| CN113171411A (en) * | 2021-04-28 | 2021-07-27 | 奥启(深圳)创投科技有限公司 | Composition containing adipose-derived stem cell exosomes with whitening function |
| CN113662965A (en) * | 2021-06-11 | 2021-11-19 | 盛小伍 | Application of adipose-derived stem cells and cathepsin F inhibitor |
| WO2023169594A1 (en) * | 2022-03-08 | 2023-09-14 | 中山大学 | Application of blood-derived sample in preparation of vesicles |
| CN117771260A (en) * | 2024-02-07 | 2024-03-29 | 中国中医科学院中医基础理论研究所 | Fat mesenchymal stem cell exosome-icariin drug loading system, preparation method and application |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103184191A (en) * | 2012-12-04 | 2013-07-03 | 东南大学 | Extracting method and special culture medium for rat omentum majus adipose source mesenchymal stem cells |
| CN104762260A (en) * | 2015-04-23 | 2015-07-08 | 广州赛莱拉干细胞科技股份有限公司 | Preparation method and application of adipose-derived mesenchymal stem cells and preparation thereof |
| CN105219707A (en) * | 2015-11-17 | 2016-01-06 | 广州赛莱拉干细胞科技股份有限公司 | A kind of method of recovery fat mesenchymal stem cell |
| CN105267240A (en) * | 2014-12-16 | 2016-01-27 | 天津医科大学眼科医院 | Applications of exosome from mesenchymal stem cells |
| WO2018182356A1 (en) * | 2017-03-31 | 2018-10-04 | (주)안트로젠 | Culture containing high concentration of mesenchymal stem cell-derived high-purity exosome, and preparation method therefor |
| CN108721200A (en) * | 2018-06-07 | 2018-11-02 | 武汉赛云博生物科技有限公司 | A kind of preparation method and application of the excretion body cosmetic formulation in human mesenchymal stem cell source |
| CN109536440A (en) * | 2018-11-19 | 2019-03-29 | 深圳市第二人民医院 | The extracting method of excretion body |
| CN109880797A (en) * | 2019-04-08 | 2019-06-14 | 济南磐升生物技术有限公司 | A method of preparing human umbilical cord mesenchymal stem cells excretion body |
| CN110151679A (en) * | 2019-06-24 | 2019-08-23 | 广州远想生物科技有限公司 | A kind of excretion body freeze-dried powder and the preparation method and application thereof |
-
2019
- 2019-11-30 CN CN201911208966.3A patent/CN111304164A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103184191A (en) * | 2012-12-04 | 2013-07-03 | 东南大学 | Extracting method and special culture medium for rat omentum majus adipose source mesenchymal stem cells |
| CN105267240A (en) * | 2014-12-16 | 2016-01-27 | 天津医科大学眼科医院 | Applications of exosome from mesenchymal stem cells |
| CN104762260A (en) * | 2015-04-23 | 2015-07-08 | 广州赛莱拉干细胞科技股份有限公司 | Preparation method and application of adipose-derived mesenchymal stem cells and preparation thereof |
| CN105219707A (en) * | 2015-11-17 | 2016-01-06 | 广州赛莱拉干细胞科技股份有限公司 | A kind of method of recovery fat mesenchymal stem cell |
| WO2018182356A1 (en) * | 2017-03-31 | 2018-10-04 | (주)안트로젠 | Culture containing high concentration of mesenchymal stem cell-derived high-purity exosome, and preparation method therefor |
| CN108721200A (en) * | 2018-06-07 | 2018-11-02 | 武汉赛云博生物科技有限公司 | A kind of preparation method and application of the excretion body cosmetic formulation in human mesenchymal stem cell source |
| CN109536440A (en) * | 2018-11-19 | 2019-03-29 | 深圳市第二人民医院 | The extracting method of excretion body |
| CN109880797A (en) * | 2019-04-08 | 2019-06-14 | 济南磐升生物技术有限公司 | A method of preparing human umbilical cord mesenchymal stem cells excretion body |
| CN110151679A (en) * | 2019-06-24 | 2019-08-23 | 广州远想生物科技有限公司 | A kind of excretion body freeze-dried powder and the preparation method and application thereof |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111944748A (en) * | 2020-08-14 | 2020-11-17 | 福建医科大学附属协和医院 | high-IL-10-expression human adipose-derived mesenchymal stem cell exosome for treating myocardial infarction and application thereof |
| CN111944747A (en) * | 2020-08-14 | 2020-11-17 | 福建医科大学附属协和医院 | Human adipose-derived mesenchymal stem cell exosome for treating myocardial infarction and application thereof |
| CN111944749A (en) * | 2020-08-14 | 2020-11-17 | 福建医科大学附属协和医院 | Preparation method of human adipose-derived mesenchymal stem cell exosome for treating myocardial infarction |
| CN113171411A (en) * | 2021-04-28 | 2021-07-27 | 奥启(深圳)创投科技有限公司 | Composition containing adipose-derived stem cell exosomes with whitening function |
| CN113662965A (en) * | 2021-06-11 | 2021-11-19 | 盛小伍 | Application of adipose-derived stem cells and cathepsin F inhibitor |
| WO2023169594A1 (en) * | 2022-03-08 | 2023-09-14 | 中山大学 | Application of blood-derived sample in preparation of vesicles |
| CN117771260A (en) * | 2024-02-07 | 2024-03-29 | 中国中医科学院中医基础理论研究所 | Fat mesenchymal stem cell exosome-icariin drug loading system, preparation method and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111304164A (en) | Preparation method and application of adipose-derived stem cell exosomes | |
| Wang et al. | Functional engineered human cardiac patches prepared from nature's platform improve heart function after acute myocardial infarction | |
| JP5371445B2 (en) | Method for culturing adipose tissue-derived cells and use thereof | |
| CN110540956B (en) | Method for simply preparing cell factor from placenta mesenchymal stem cells | |
| CN102876630B (en) | Method for efficiently separating and expanding mesenchymal stem cells in human umbilical cord blood | |
| Saleh et al. | Turning round: multipotent stromal cells, a three-dimensional revolution? | |
| US20170304366A1 (en) | Composition containing mesenchymal stem cell-hydrogel and method for producing the composition | |
| US20110217385A1 (en) | Method for extracting mesenchymal stem cell from human or animal embryo and for extracting the secretion product thereof | |
| CN107254443B (en) | Induction medium and induction method for promoting differentiation of mesenchymal stem cells to neurons | |
| Xiao et al. | Extracellular matrix from human umbilical cord-derived mesenchymal stem cells as a scaffold for peripheral nerve regeneration | |
| US20220325247A1 (en) | METHOD FOR PROMOTING GROWTH OF PARIETAL DECIDUAL BASALIS-MESENCHYMAL STEM CELLS (PDB-MSCs) | |
| WO2012068710A1 (en) | Methods for extracting mesenchymal stem cell from slight amount human adipose tissue and mass cultivation thereof | |
| CN111840646B (en) | Stem cell composition for filling chest fat and application thereof | |
| Baghalishahi et al. | Cardiac extracellular matrix hydrogel together with or without inducer cocktail improves human adipose tissue-derived stem cells differentiation into cardiomyocyte–like cells | |
| CN111826345A (en) | Human umbilical cord mesenchymal stem cell source exosome preparation | |
| Zhang et al. | Comparison of beneficial factors for corneal wound-healing of rat mesenchymal stem cells and corneal limbal stem cells on the xenogeneic acellular corneal matrix in vitro | |
| Lu et al. | The in vitro differentiation of GDNF gene-engineered amniotic fluid-derived stem cells into renal tubular epithelial-like cells | |
| CN114540298A (en) | Stem cell serum-free medium and preparation method thereof | |
| CN1778905B (en) | Separating culture and use for fatty mesenchymal dry cell | |
| Xie et al. | Construction of engineered corpus cavernosum with primary mesenchymal stem cells in vitro | |
| CN109897815B (en) | Efficient separation and culture method of adipose endothelial progenitor cells without coating | |
| CN106606512B (en) | Mixed cell preparation for treating myocardial infarction and preparation method and application thereof | |
| WO2006134951A1 (en) | Long-term culture proliferation method for fat-derived stem cell | |
| CN101760445B (en) | Method for amplifying autologous bone marrow mesenchymal stem cells | |
| Esposito et al. | Culture conditions allow selection of different mesenchymal progenitors from adult mouse bone marrow |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200619 |