CN106957818A - A kind of method for efficiently promoting fat stem cell to breed and its kit - Google Patents
A kind of method for efficiently promoting fat stem cell to breed and its kit Download PDFInfo
- Publication number
- CN106957818A CN106957818A CN201710303383.3A CN201710303383A CN106957818A CN 106957818 A CN106957818 A CN 106957818A CN 201710303383 A CN201710303383 A CN 201710303383A CN 106957818 A CN106957818 A CN 106957818A
- Authority
- CN
- China
- Prior art keywords
- stem cell
- fat stem
- cell
- culture
- growth factor
- 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
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 128
- 238000000034 method Methods 0.000 title claims abstract description 25
- 230000001737 promoting effect Effects 0.000 title claims abstract description 11
- 108010010803 Gelatin Proteins 0.000 claims abstract description 52
- 239000008273 gelatin Substances 0.000 claims abstract description 52
- 229920000159 gelatin Polymers 0.000 claims abstract description 52
- 235000019322 gelatine Nutrition 0.000 claims abstract description 52
- 235000011852 gelatine desserts Nutrition 0.000 claims abstract description 52
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims abstract description 44
- 210000004027 cell Anatomy 0.000 claims abstract description 42
- 239000003102 growth factor Substances 0.000 claims abstract description 29
- 102000004877 Insulin Human genes 0.000 claims abstract description 22
- 108090001061 Insulin Proteins 0.000 claims abstract description 22
- 102000002070 Transferrins Human genes 0.000 claims abstract description 22
- 108010015865 Transferrins Proteins 0.000 claims abstract description 22
- 229940125396 insulin Drugs 0.000 claims abstract description 22
- 210000001772 blood platelet Anatomy 0.000 claims abstract description 19
- 230000006798 recombination Effects 0.000 claims abstract description 17
- 238000005215 recombination Methods 0.000 claims abstract description 17
- 230000012010 growth Effects 0.000 claims abstract description 11
- 101500025419 Homo sapiens Epidermal growth factor Proteins 0.000 claims abstract description 7
- 229940116978 human epidermal growth factor Drugs 0.000 claims abstract description 7
- GVUGOAYIVIDWIO-UFWWTJHBSA-N nepidermin Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CS)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CS)NC(=O)[C@H](C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C(C)C)C(C)C)C1=CC=C(O)C=C1 GVUGOAYIVIDWIO-UFWWTJHBSA-N 0.000 claims abstract description 7
- 101001052035 Homo sapiens Fibroblast growth factor 2 Proteins 0.000 claims abstract description 5
- 210000001519 tissue Anatomy 0.000 claims description 34
- 238000004113 cell culture Methods 0.000 claims description 21
- 239000000243 solution Substances 0.000 claims description 18
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 16
- 210000002950 fibroblast Anatomy 0.000 claims description 15
- 239000002504 physiological saline solution Substances 0.000 claims description 15
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims description 14
- 108010019160 Pancreatin Proteins 0.000 claims description 14
- 229940055695 pancreatin Drugs 0.000 claims description 14
- 102100022464 5'-nucleotidase Human genes 0.000 claims description 13
- 102100037241 Endoglin Human genes 0.000 claims description 13
- 102000009024 Epidermal Growth Factor Human genes 0.000 claims description 13
- 101000678236 Homo sapiens 5'-nucleotidase Proteins 0.000 claims description 13
- 101000881679 Homo sapiens Endoglin Proteins 0.000 claims description 13
- 101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 claims description 13
- 102100033523 Thy-1 membrane glycoprotein Human genes 0.000 claims description 13
- 230000029087 digestion Effects 0.000 claims description 13
- 102000006354 HLA-DR Antigens Human genes 0.000 claims description 12
- 108010058597 HLA-DR Antigens Proteins 0.000 claims description 12
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 claims description 12
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 claims description 12
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 claims description 10
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 claims description 9
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 claims description 9
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 claims description 9
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 claims description 9
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 claims description 9
- 239000012930 cell culture fluid Substances 0.000 claims description 9
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
- 239000012228 culture supernatant Substances 0.000 claims description 8
- 238000004043 dyeing Methods 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 8
- 102000018386 EGF Family of Proteins Human genes 0.000 claims description 7
- 108010066486 EGF Family of Proteins Proteins 0.000 claims description 7
- 102000004218 Insulin-Like Growth Factor I Human genes 0.000 claims description 7
- 238000001035 drying Methods 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- 241000233855 Orchidaceae Species 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 6
- 230000004927 fusion Effects 0.000 claims description 6
- 235000015097 nutrients Nutrition 0.000 claims description 6
- 239000000047 product Substances 0.000 claims description 6
- 238000012549 training Methods 0.000 claims description 6
- 239000011248 coating agent Substances 0.000 claims description 5
- 238000000576 coating method Methods 0.000 claims description 5
- 239000012737 fresh medium Substances 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 5
- 108010035532 Collagen Proteins 0.000 claims description 4
- 102000008186 Collagen Human genes 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 4
- 210000000577 adipose tissue Anatomy 0.000 claims description 4
- 230000010261 cell growth Effects 0.000 claims description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 229920001436 collagen Polymers 0.000 claims description 4
- 229940088598 enzyme Drugs 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- 102000012422 Collagen Type I Human genes 0.000 claims description 3
- 108010022452 Collagen Type I Proteins 0.000 claims description 3
- 239000006285 cell suspension Substances 0.000 claims description 3
- 239000004519 grease Substances 0.000 claims description 3
- 230000010355 oscillation Effects 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- 239000011550 stock solution Substances 0.000 claims description 3
- 239000012267 brine Substances 0.000 claims description 2
- 238000011084 recovery Methods 0.000 claims description 2
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 claims description 2
- 239000012531 culture fluid Substances 0.000 claims 1
- 239000000835 fiber Substances 0.000 claims 1
- 235000015110 jellies Nutrition 0.000 claims 1
- 239000008274 jelly Substances 0.000 claims 1
- 239000008055 phosphate buffer solution Substances 0.000 claims 1
- 238000007443 liposuction Methods 0.000 abstract description 9
- 208000002193 Pain Diseases 0.000 abstract description 5
- 230000036407 pain Effects 0.000 abstract description 5
- 230000009467 reduction Effects 0.000 abstract description 2
- 201000010099 disease Diseases 0.000 abstract 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 17
- 230000003796 beauty Effects 0.000 description 14
- 210000000038 chest Anatomy 0.000 description 13
- 210000003491 skin Anatomy 0.000 description 11
- 231100000241 scar Toxicity 0.000 description 10
- 238000002360 preparation method Methods 0.000 description 8
- 230000037303 wrinkles Effects 0.000 description 8
- 108090000695 Cytokines Proteins 0.000 description 6
- 102000004127 Cytokines Human genes 0.000 description 6
- 230000004663 cell proliferation Effects 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 4
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 4
- 210000001015 abdomen Anatomy 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 230000004069 differentiation Effects 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 238000011282 treatment Methods 0.000 description 4
- 210000000689 upper leg Anatomy 0.000 description 4
- 208000003351 Melanosis Diseases 0.000 description 3
- 108010025020 Nerve Growth Factor Proteins 0.000 description 3
- 102000015336 Nerve Growth Factor Human genes 0.000 description 3
- 210000001339 epidermal cell Anatomy 0.000 description 3
- 238000011049 filling Methods 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 230000008439 repair process Effects 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 description 2
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 description 2
- 102000013275 Somatomedins Human genes 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 235000006708 antioxidants Nutrition 0.000 description 2
- 239000002537 cosmetic Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 229940053128 nerve growth factor Drugs 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 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 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 102000016942 Elastin Human genes 0.000 description 1
- 108010014258 Elastin Proteins 0.000 description 1
- 102000008857 Ferritin Human genes 0.000 description 1
- 108050000784 Ferritin Proteins 0.000 description 1
- 238000008416 Ferritin Methods 0.000 description 1
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 1
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 1
- 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 1
- 241000282412 Homo Species 0.000 description 1
- 108010085895 Laminin Proteins 0.000 description 1
- 101710098940 Pro-epidermal growth factor Proteins 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 210000001789 adipocyte Anatomy 0.000 description 1
- 210000004504 adult stem cell Anatomy 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000001949 anaesthesia Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 210000003321 cartilage cell Anatomy 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 238000002737 cell proliferation kit Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000034373 developmental growth involved in morphogenesis Effects 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229920002549 elastin Polymers 0.000 description 1
- 230000008472 epithelial growth Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229940126864 fibroblast growth factor Drugs 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000009499 grossing Methods 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000000492 lymphangiogenic effect Effects 0.000 description 1
- 210000001365 lymphatic vessel Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 210000003739 neck Anatomy 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 210000004927 skin cell Anatomy 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 239000012588 trypsin Substances 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/20—Transition metals
- C12N2500/24—Iron; Fe chelators; Transferrin
- C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/135—Platelet-derived growth factor [PDGF]
-
- 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
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/54—Collagen; Gelatin
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Developmental Biology & Embryology (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Rheumatology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a kind of method for efficiently promoting fat stem cell to breed, wherein, methods described is included by using the coated Tissue Culture Plate of gelatin or culture dish and the coated Tissue Culture Flask of gelatin, under the Combined cultures such as recombinant human epidermal growth factor, derivative growth factor of recombined human blood platelet, recombination human basic fibroblast growth factor, insulin, transferrins and hyclone, efficiently promote fat stem cell propagation growth from fat, cell adipose-derived 50mL can cultivate acquisition 10 in 15 days9Fat stem cell above, can meet a large amount of cell numbers of clinical treating disease and medical cosmetology, the pain brought without secondary liposuction and reduction liposuction.According to the above method, present invention also offers a kind of kit for efficiently promoting fat stem cell to breed, grown available for fat stem cell propagation is efficiently promoted.
Description
Technical field
The present invention relates to it is a kind of efficiently promote fat stem cell breed method and by methods described obtain it is efficient
The kit for promoting fat stem cell to breed.Especially, the present invention relates to use the coated Tissue Culture Plate of gelatin or culture dish
With the coated Tissue Culture Flask of gelatin, in recombinant human epidermal growth factor, derivative growth factor of recombined human blood platelet, recombined human alkali
Property the Combined culture such as fibroblast growth factor, insulin, transferrins and hyclone under, efficiently promote fat dry from fat
Cell propagation growth.The fat stem cell that the present invention is obtained can be used for the necks such as medical cosmetology, including skin wrinkle, nti-freckle and chest enlarge
Domain.
Background technology
Due to the fast development of biotechnology, medical cosmetology and cosmetics are applied to biotechnology and develop product,
Particularly in recent years, by cell technology application beauty industry, and the field gradually approved as everybody.Trained such as from skin
Fat stem cell obtained in foster fibroblast and fat etc., all carries out clinical research in medical cosmetology and answers
With to direct injections such as wrinkle of skin, spot and chest enlarges so that these cells can differential growth in skin, skin is played
Filling and repair function.Fat stem cell is a kind of adult stem cell of current study hotspot, and the ability of cell proliferation is relatively good,
In vitro culture can keep stable growing multiplication activity, and with multinomial differentiation potential, also be applied in beauty therapeutic.But
When usual fat stem cell cultivates propagation in vitro, the speed of growth is slow, reaches 108Above cell number need more than 20 days when
Between, and be not met by needed for multiple beauty.Many second of beauty of beauty person may need to carry out liposuction again, but this pain
Chu Feichang pains, common people are difficult to receive.Even with local anaesthesia, but pain is still difficult to stand later.
In summary, fat stem cell cultural method acquisition fat stem cell number conventional at present is limited, fat stem cell
Propagation is slow-growing, and more than 30 days are needed by the generation of Secondary Culture 3, and obtained fat stem cell quantity is less than 108.And fat
Stem cell passage amplification easily occurs breaking up and apoptosis no more than 5 generations, otherwise fat stem cell, cell propagation and the function of repairing
Substantially also decline, the cell with cells and characteristic of stem is substantially reduced, it is impossible to ensure the activity of fat stem cell after beauty.The present invention
Using the coated Tissue Culture Plate of gelatin or culture dish and the coated Tissue Culture Flask of gelatin, recombinant human epidermal growth factor,
Derivative growth factor of recombined human blood platelet, recombination human basic fibroblast growth factor, insulin, transferrins and hyclone etc.
Under Combined culture, promote fat stem cell high efficiently multiplying, substantial amounts of fat stem cell can be obtained, meet beauty person's repeatedly many treatments
The carry out beauty of journey, the beautification functions such as optimal smoothing wrinkle, nti-freckle and chest enlarge will be produced to skin.
The content of the invention
In order to efficiently promote fat stem cell to breed, the fat stem cell for obtaining a large amount of high activities will be helpful to improve medical science
The effect of beauty.The present invention is according to survival and growth conditions, the Tissue Culture Plate of gelatin bag or culture in fat stem cell body
Ware and the coated Tissue Culture Flask of gelatin, in recombinant human epidermal growth factor, derivative growth factor of recombined human blood platelet, recombined human
Under the Combined cultures such as Basic Fibroblast Growth Factor, insulin, transferrins and hyclone, fat stem cell is promoted efficiently to increase
Grow, substantial amounts of active fat stem cell can be obtained.So, the fat stem cell of preparation repeatedly many courses for the treatment of can enter to practise medicine
Beauty, reduction thus the liposuction again of fat stem cell deficiency needs are learned, and reduces the pain that liposuction is brought again.
Gelatin has the undifferentiated state for maintaining the human stem cell through Trypsin Induced.It is grown in the stem cell on gelatin
Versatility mark is expressed, Multidirectional Differentiation Forming ability maintains normal karyotype simultaneously.We have found that in gelatin in early-stage Study
On cultivated the human adipose-derived stem cell in 10 generations and detected, as a result show that these cells still express high-caliber CD90, CD73
And CD105.Meanwhile, compared with the human adipose-derived stem cell grown on matrigel and in normal blake bottle, the people's fat grown on gelatin
Fat stem cell can form the alkaline phosphatase positive colony of similar size and quantity, thus with good stem cell properties.
Recombinant human epidermal growth factor, derivative growth factor of recombined human blood platelet, recombination human basic fibroblast growth factor,
The cell factor such as insulin and transferrins in terms of the propagation of fat stem cell, migration, aggregation and differentiation to having promotion to make
With.We have found that these cell factors can have in the result that streaming instrument is detected and synergistically promote fat stem cell by static
State enter propagation state effect, while keep fat stem cell be in undifferentiated state, with Multidirectional Differentiation ability and self more
New cells and characteristic of stem.
Fat stem cell has various biological function, can be divided into various kinds of cell, such as Gegenbaur's cell, cartilage cell, god
Through cell, fibroblast and epidermal cell etc., thus fat stem cell can repair the fibroblast of necrosis in skin
And epidermal cell, participate in the metabolism of skin；Cytokine profiles, such as VEGF can be secreted simultaneously
(vascular endothelial growth factor, VEGF), Basic Fibroblast Growth Factor (basic fibroblast
Growth factor, bFGF), EGF (epithelial growth factor, EGF), HGF
(hepatocyte growth facror, HGF), insulin-like growth factor (insulin-like growth factors,
) and platelet derived growth factor (platelet derived growth factor, PDGF) and nerve growth factor IGF-1
(nerve growth factor, NGF) etc., can promote fibroblast and epidermal cell to activate and expand, produce collagen egg
In vain, hyaluronic acid and elastin laminin etc., participate in the reparation of wrinkle of skin, scar, scar and spot etc., regeneration, filling, nti-freckle and
The effect of anti-oxidant grade, and chest reach the effect of chest enlarge.
The height obtained the invention provides a kind of method for efficiently promoting fat stem cell to breed and by methods described
Effect promotes the kit that fat stem cell is bred.Especially, the present invention relates to plate and Tissue Culture Flask is coated with using gelatin, in weight
Group hEGF, derivative growth factor of recombined human blood platelet, recombination human basic fibroblast growth factor, insulin, turn
Under the Combined culture such as ferritin and hyclone, efficiently promote fat stem cell propagation growth from fat.
The autologous fat that fat stem cell prepared by the present invention can be obtained from beauty person's thigh or belly liposuction, washing digestion
After obtain karyocyte, after primary culture in vitro and Secondary Culture obtain significant quantities of fat stem cell, can be placed in 4 DEG C preserve fortune
It is defeated, it is also possible to the frozen stock solution of 40% fat stem cell culture solution, 10% dimethyl sulfoxide (DMSO) and 50% hyclone composition, preserve
In -196 DEG C of liquid nitrogen containers, in case being used after recovering from now on.
The coated Tissue Culture Plate of gelatin of the present invention, culture dish or Tissue Culture Flask, to use 1-20% (quality
Volume ratio) gelatin 4 DEG C of refrigerator overnights after 4 DEG C of refrigerator overnights or 37 DEG C are incubated 1 hour, wrapped in cell culture container
Quilt.Gelatin solution is absorbed after coating, the aeration-drying in Biohazard Safety Equipment or super-clean bench is stored in 4 DEG C of refrigerators standby after drying.
The present invention can cultivate acquisition 10 from 50mL fat in 15 days9Fat stem cell above.The present invention is prepared into
The fat stem cell arrived through flow cytomery, height expression CD90+ be >=90%, CD73+ be >=90% and CD105+ be >=
90%, low expression CD34+ are that≤5%, HLA-DR+ is that≤5% and CD14+ is≤5%；；It is further preferred that autologous fat stem cell is high
It is that 99.08%, CD105+ is 99.12% and CD73+ is 99.39% to express CD90+, and low expression CD34+ is 0.12%, HLA-DR
+ it is 0.51% and CD14+ is 0.24%.
The fat stem cell high expressing cell factor prepared by the present invention, i.e., the VEGF secreted in fat stem cell culture supernatant
It is more than more than 500pg/mL, bFGF more than 500pg/mL, EGF more than 1500pg/mL, IGF-1 more than 10ng/mL and PDGF
1000pg/mL。
Present invention also offers a kind of kit for efficiently promoting fat stem cell to breed, it is characterised in that it is by as follows
Composition is constituted：
1) 1000ml cell culture mediums；
2) 1 piece of coated Tissue Culture Plate of gelatin or culture dish；
3) 8 coated Tissue Culture Flasks of gelatin；
4) 5-50ng/mL recombinant human epidermal growth factors；
5) 5-50ng/mL derivative growth factor of recombined human blood platelet；
6) 5-50ng/m recombination human basic fibroblasts growth factor；
7) 10-100ng/mL insulin；
8) 10-100ng/mL transferrins；
9) 2-10% hyclones；
10) 100ml pancreatin；
11) operation instructions；
The coated Tissue Culture Plate of the culture medium of the reagent kit product, gelatin or culture dish and the coated cell culture of gelatin
Bottle is stored under the conditions of 4 DEG C, and the shelf-life is 1 year；Cell factor, hyclone and pancreatin are maintained under the conditions of -80 DEG C, are guaranteed the quality
Phase is 1 year.
It preferably with the addition of whole cell factors, i.e. 5-50ng/mL recombinant human epidermal growth factors, 5-50ng/mL weights
Group vectors containing human platelet-derived growth, 5-50ng/m recombination human basic fibroblasts growth factor, 10-100ng/mL insulin, 10-
100ng/mL transferrins and 2-10% hyclones.
By the method for the present invention, preparation can be prepared into by cultivating obtained fat stem cell, for wrinkle of skin, scar
Trace, scar and chest carry out injection beauty, and wrinkle of skin, scar, scar and spot etc. are produced and repairs, regenerate, filling, dispelling
Spot and the effect of anti-oxidant grade, and chest reach the effect of chest enlarge.
Brief description of the drawings
Fig. 1 is represented as the human adipose-derived stem cell proliferation activity analysis prepared by embodiment 1.
Fig. 2 is represented as the cell streaming figure of the fat stem cell Phenotypic examination prepared by embodiment 1.
Fig. 3 is represented as the fat stem cell culture supernatant levels of cytokine secretion figure prepared by embodiment 1.
Embodiment
The height obtained the invention provides a kind of method for efficiently promoting fat stem cell to breed and by methods described
Effect promotes the kit that fat stem cell is bred.Especially, the present invention relates to use the coated Tissue Culture Plate of gelatin or culture
Ware and the coated Tissue Culture Flask of gelatin, in recombinant human epidermal growth factor, derivative growth factor of recombined human blood platelet, recombined human
Under the Combined cultures such as Basic Fibroblast Growth Factor, insulin, transferrins and hyclone, efficiently fat is promoted from fat
Stem cells hyperplasia grows, so as to be prepared into autologous fat stem cell preparation.
The fat stem cell obtains adipose tissue purification culture from thigh or belly liposuction and obtained.Specific implementation step
It is rapid as follows：Adipose tissue 50mL is obtained by liposuction on belly or thigh, is rinsed repeatedly with physiological saline 5 times, fat is absorbed as far as possible
Middle oil and haemocyte；
Using 1: 1 0.25% (mass volume ratio) pancreatin and 0.1% (mass volume ratio) type i collagen enzyme 20-50mL,
Digest 60 minutes, vibrated 2 minutes with vortex oscillation instrument every 15 minutes in 37 DEG C of incubators；1500rpm centrifuges 10 points after digestion
Clock, suction abandons after the grease of upper strata that to add physiological saline resuspended, is filtered with 100 μm of cell sieves；Cell suspension 1000rpm centrifuges 10 points
Clock, cell precipitation with physiological saline it is resuspended washing 2 times after add cell culture fluid (recombinant human epidermal containing 5-50ng/mL grow because
Son, 5-50ng/mL derivative growth factor of recombined human blood platelet, 5-50ng/m recombination human basic fibroblasts growth factor, 10-
100ng/mL insulin, 10-100ng/mL transferrins and 2-10% hyclones) it is resuspended, press 1- after tongue disk orchid dyeing counting
10×106Cell/ml is added in the coated Tissue Culture Plate of gelatin or culture dish and cultivated, in 37 DEG C, 5%CO2Trained in incubator
Support 24-72 hours；
Culture is inhaled after 24-72 hours with 5ml pipettes abandons nutrient solution, adds cell culture fluid (recombined human containing 5-50ng/mL
EGF, 5-50ng/mL derivative growth factor of recombined human blood platelet, the growth of 5-50ng/mL recombination human basic fibroblasts
The factor, 10-100ng/mL insulin, 10-100ng/mL transferrins and 2-10% hyclones) carry out changing liquid, cell culture
Plate or culture dish continue to place 37 DEG C, 5%CO2Continue to cultivate in incubator；
Digestion is carried out when fat stem cell growth fusion reaches more than 80% and expands bottle, and nutrient solution is absorbed, 1 × PBS is taken
(pH value is 7.4) adds the pancreatin of 0.5ml 0.25% into blake bottle after washed once, digestion adds 200 μ l tires after 2-3 minutes
Cow's serum terminates digestion, adds physiological saline, is blown and beaten, is drawn in 15ml centrifuge tubes, then washed with physiology salt with 5mL pipettes
Wash once, be added in same 15mL centrifuge tubes, 1000 rpms of centrifugations collect fat stem cell in 10 minutes；Used after centrifugation
1mL fresh mediums are resuspended, count, and the fresh cell culture fluid (recombined humans containing 5-50ng/mL of 10mL are added with 5mL pipettes
EGF, 5-50ng/mL derivative growth factor of recombined human blood platelet, 5-50ng/m recombination human basic fibroblasts growth because
Son, 10-100ng/mL insulin, 10-100ng/mL transferrins and 2-10% hyclones), it is added to 1 bottle of collagen coated
Continue in blake bottle in 37 DEG C, 5%CO2Cultivated in incubator, a fresh medium was changed every 2 days；Treat that fat stem cell is given birth to
When long fusion reaches more than 80%, by above-mentioned through collected by trypsinisation fat stem cell, continuous passage 3 be commissioned to train it is foster, obtain 109With
On fat stem cell, it is resuspended with 4ml physiological saline, tongue disk orchid dyeing counting, that is, obtain significant quantities of fat stem cell, place 4 DEG C
Under the conditions of save backup.
The coated Tissue Culture Plate of gelatin of the present invention, culture dish or Tissue Culture Flask, to use 1-20% (quality
Volume ratio) gelatin 4 DEG C of refrigerator overnights after 4 DEG C of refrigerator overnights or 37 DEG C are incubated 1 hour, wrapped in cell culture container
Quilt.Gelatin solution is absorbed after coating, the aeration-drying in Biohazard Safety Equipment or super-clean bench is stored in 4 DEG C of refrigerators standby after drying.
Culture plate, culture dish and blake bottle are commercially available, and culture plate can be 24 orifice plates, 12 orifice plates and 6 holes
Plate；Culture dish can be 35mm, 60mm and 90mm culture dish；Blake bottle can be 25cm2、75cm2、175cm2And 225cm2Training
Support bottle；More preferably culture plate can be 6 orifice plates；Culture dish can be 60mm culture dish；Blake bottle can be 175cm2Culture
Bottle；
Cell culture fluid of the present invention is commercially available, including DMEM high glucose mediums, α-MEM training
Foster, EGM-2 culture mediums and DMEM/F12 (1: 1) culture medium etc., more preferred with α-MEM cultures and EGM-2 culture mediums.
Tongue is taken to expect 3 × 10 after blue dyeing counting6Fat stem cell, divides three groups, first group has been respectively added to 20 μ L
The anti-human CD90 monoclonal antibodies of FITC mark mouse, the 20 μ L PE mark anti-human CD105 monoclonal antibodies of mouse and 20 μ L Percp mark mouse antihuman CD 34 lists
It is anti-；Second group has been respectively added to the anti-human CD73 monoclonal antibodies of 20 μ L FITC mark mouse, the 20 μ L PE mark anti-human HLA-DR monoclonal antibodies of mouse
The anti-human CD14 monoclonal antibodies of mouse are marked with 20 μ L Percp；3rd group is Isotype control, has been respectively added to 20 μ L FITC mark mouse
IgG1,20 μ L PE mark mouse IgG1 and 20 μ L PerCP mark mouse IgG1；It is placed in 4 DEG C of refrigerators to dye 30 minutes, then uses 1mL
1 × phosphate buffer wash three times, finally with the cell after the 0.5mL resuspended washings of 1 × PBS, gained washing after it is thin
Born of the same parents use FC500 flow cytomeries；Fat stem cell height expression CD90+ is that >=90%, CD105+ is that >=90% and CD73+ is
>=90%, low expression CD34+ are that≤5%, HLA-DR+ is that≤5% and CD14+ is≤5%.
The fat stem cell prepared can be in 40% fat stem cell culture solution, 10% dimethyl sulfoxide (DMSO) and 50% tire ox
The frozen stock solution of serum composition, is stored in -196 DEG C of liquid nitrogen containers, in case being used after recovering from now on.After fat stem cell recovery, still
Height expresses CD90, CD73 and CD105, low expression CD34, HLA-DR and CD14, with fat stem cell phenotypic characteristic.
The fat stem cell high expressing cell factor prepared by the present invention, fat stem cell culture supernatant is inhaled by enzyme linked immunological
Attached to determine (enzyme linked immunosorbent assay, ELISA) detection secrete cytokines concentration, VEGF is more than
500pg/mL, bFGF are more than 500pg/mL, EGF and are more than 1500pg/mL, IGF-1 more than 10ng/mL and PDGF more than 1000pg/
mL。
Present invention also offers a kind of kit for efficiently promoting fat stem cell to breed, it is characterised in that it is by as follows
Composition is constituted：
1) 1000ml cell culture mediums；
2) 1 piece of coated Tissue Culture Plate of gelatin or culture dish；
3) 8 coated Tissue Culture Flasks of gelatin；
4) 5-50ng/mL recombinant human epidermal growth factors；
5) 5-50ng/mL derivative growth factor of recombined human blood platelet；
6) 5-50ng/m recombination human basic fibroblasts growth factor；
7) 10-100ng/mL insulin；
8) 10-100ng/mL transferrins；
9) 2-10% hyclones；
10) 100ml pancreatin；
11) operation instructions；
The coated Tissue Culture Plate of the culture medium of the reagent kit product, gelatin or culture dish and the coated cell culture of gelatin
Bottle is stored under the conditions of 4 DEG C, and the shelf-life is 1 year；Cell factor, hyclone and pancreatin are maintained under the conditions of -80 DEG C, are guaranteed the quality
Phase is 1 year.
It preferably with the addition of whole cell factors, i.e. 5-50ng/mL recombinant human epidermal growth factors, 5-50ng/mL weights
Group vectors containing human platelet-derived growth, 5-50ng/m recombination human basic fibroblasts growth factor, 10-100ng/mL insulin, 10-
100ng/mL transferrins and 2-10% hyclones.
Preparation can be prepared into the invention provides fat stem cell, is entered for wrinkle of skin, scar, scar and chest
Row injection beauty, according to the wrinkle depth and scar and scar size, the fat stem cell number of injection is different, what full face was used
Fat stem cell number is generally 5-50 × 106, 1 course for the treatment of is 4 times, is spaced 1 week, can reach more preferably cosmetic result.According to chest
Size, the fat stem cell number of injection also different chest, the fat stem cell number that chest enlarge is used is generally each breast 100
×106More than, 1 course for the treatment of is 2 times, is spaced 2 weeks, can reach more preferable chest enlarge effect.
Hereinafter, the specific embodiment of the present invention is illustrated, but the technical scope of the present invention is not limited to these examples.
The preparation of the human adipose-derived stem cell of embodiment 1
Beauty volunteer obtains adipose tissue 50mL (signing informed consent form with it) from liposuction on belly or thigh, uses
50mL physiological saline is rinsed 5 times repeatedly, and oil and haemocyte in fat are absorbed as far as possible.Use 1: 1 0.25% (mass volume ratio)
Pancreatin (Beijing Suo Laibao companies) and 0.1% (mass volume ratio) type i collagen enzyme (Sigma Co., USA) 50mL, in 37 DEG C of trainings
Support in case and digest 60 minutes, vibrated 2 minutes with vortex oscillation instrument every 15 minutes；1500rpm is centrifuged 10 minutes after digestion, and suction is abandoned
Physiological saline is added after the grease of upper strata resuspended, filtered with 100 μm of cell sieves (U.S. company BD)；Cell suspension 1000rpm is centrifuged
10 minutes, cell precipitation added EGM-2 culture mediums (Lonza companies of the U.S.) after being washed 2 times with physiological saline is resuspended and (contains 10ng/
ML EGF, R＆D companies of the U.S.；5ng/mL PDGF, R＆D companies of the U.S.；10ng/mL bFGF, R＆D companies of the U.S.；10/mL pancreas islet
Element, Sigma Co., USA；5ng/mL transferrins, R＆D companies of the U.S.；With 5% hyclone, Life companies of the U.S.) it is resuspended,
1-10 × 10 are pressed after tongue disk orchid dyeing counting6Cell/ml is added in the coated Tissue Culture Plate of gelatin or culture dish and cultivated, in
37 DEG C, 5%CO2Cultivated 24-72 hours in incubator；
Culture is inhaled after 24-72 hours with 5ml pipettes abandons nutrient solution, addition cell culture fluid (EGF containing 10ng/mL,
5ng/mL PDGF, 10ng/mL bFGF, 10/mL insulin, 5ng/mL transferrins and 5% hyclone) carry out changing liquid, carefully
Born of the same parents' culture plate or culture dish continue to place 37 DEG C, 5%CO2Continue to cultivate in incubator；
Digestion is carried out when fat stem cell growth fusion reaches more than 80% and expands bottle, and suction abandons nutrient solution, takes 1 × PBS (pH
It is worth to add the pancreatin of 0.5ml 0.25% after 7.4) washed once into blake bottle, digestion adds 200 μ l tire ox bloods after 2-3 minutes
It is clear to terminate digestion, physiological saline is added, is blown and beaten, is drawn in 15ml centrifuge tubes with 5mL pipettes, then with brine one
It is secondary, it is added in same 15mL centrifuge tubes, 1000 rpms of centrifugations collect fat stem cell in 10 minutes；1mL is used after centrifugation
Fresh medium is resuspended, counts, and fresh cell culture fluid (EGF containing 10ng/mL, the 5ng/mL of 10mL is added with 5mL pipettes
PDGF, 10ng/mL bFGF, 10/mL insulin, 5ng/mL transferrins and 5% hyclone), it is added to 1 bottle of collagen coating
Blake bottle in continue in 37 DEG C, 5%CO2Cultivated in incubator, a fresh medium was changed every 2 days；Treat fat stem cell
Growth fusion is when reaching more than 80%, by above-mentioned through collected by trypsinisation fat stem cell, and continuous passage 3 is commissioned to train foster, obtains 109
Fat stem cell above, tongue disk orchid dyeing counting resuspended with 4ml physiological saline, that is, obtain significant quantities of fat stem cell, place 4
Saved backup under the conditions of DEG C.
The gelatin of embodiment 2 is coated with the preparation of plate and Tissue Culture Flask
5g gelatin is weighed, is dissolved with 1 × PBS of 100mL (pH=7.4) in 37 DEG C of water-baths, and is filtered with 0.45 μm of filter
It is degerming, that is, it is configured to 5% gelatin solution.6 orifice plates add 500 μ L gelatin solutions, 90mm per hole and add 2mL gelatin solutions and 175cm2Training
Support bottle and add 4mL gelatin solutions, 4 DEG C of refrigerator overnights after 4 DEG C of refrigerator overnights or 37 DEG C are incubated 1 hour, in cell culture container
It is coated with.Gelatin solution is absorbed after coating, the aeration-drying in Biohazard Safety Equipment or super-clean bench, good seal is stored in 4 after drying
DEG C refrigerator is standby.
The human adipose-derived stem cell proliferation activity of embodiment 3 is analyzed
The fat stem cell prepared in Example 1 and the fat stem cell prepared by comparative example carry out proliferation activity
Analysis.The fat stem cell body that comparative example is delivered according in November, 2016 such as Strassburg S on cellular biochemistry magazine
Outer support lymphatic vessel generation parameter article carry out culture obtain fat stem cell (Strassburg S, Torio-Padron N,
Finkenzeller G, Frankenschmidt A, Stark GB.Adipose-Derived Stem Cells Support
Lymphangiogenic Parameters In Vitro.J Cell Biochem.2016Nov；117(11)：2620-9.)
Using CCK-8 (the green skies company in Shanghai) kit detect, according to operation instructions respectively the 1st day, the 2nd day,
3rd day, the 4th day and the 5th day detection cell-proliferation activity.From Fig. 1 it is recognised that the fat stem cell for preparing of the present invention with it is right
Ratio is prepared and compared, and cell proliferation rate faster, between the two and with significant difference (* * P=0.012), shows the present invention
The fat stem cell of preparation has higher proliferation activity.
The human adipose-derived stem cell flow cytometer detection of embodiment 4
3 × 10 prepared in Example 16Fat stem cell, divides three groups, first group has been respectively added to 20 μ L FITC
Mark the anti-human CD90 monoclonal antibodies of mouse, the 20 μ L PE mark anti-human CD105 monoclonal antibodies of mouse and 20 μ L Percp mark mouse antihuman CD 34 monoclonal antibodies；
Second group has been respectively added to the anti-human CD73 monoclonal antibodies of 20 μ L FITC mark mouse, the 20 μ L PE mark anti-human HLA-DR monoclonal antibodies of mouse and 20
μ L Percp mark the anti-human CD14 monoclonal antibodies of mouse；3rd group is Isotype control, be respectively added to 20 μ L FITC marks mouse IgG1,
20 μ L PE mark mouse IgG1 and 20 μ L PerCP mark mouse IgG1；Be placed in 4 DEG C of refrigerators and dye 30 minutes, then with the 1 of 1mL ×
PBS (pH value is 7.4) is washed 3 times, finally with the cell after the 0.5mL resuspended washings of 1 × PBS, and the cell after gained washing is used
FC500 flow cytomeries (Beckman Coulter Inc. of the U.S.).Result shows the human adipose-derived stem cell height expression in Fig. 2
CD90+ is that 99.08%, CD105+ is 99.12% and CD73+ is 99.39%, and low expression CD34+ is that 0.12%, HLA-DR+ is
0.51% and CD14+ is 0.24%, shows to meet fat stem cell phenotype marker.
The human adipose-derived stem cell cytokine secretion of embodiment 5 is detected
Human adipose-derived stem cell comparative example into the culture supernatant and embodiment 3 in the 5th generation is cultivated in extraction embodiment 1 to prepare
Fat stem cell (is purchased from U.S. to the culture supernatant in the 5th generation using VEGF, bFGF, EGF, IGF-1 and PDGF ELISA kit
Ebioscience companies of state) detected, according to ELISA operation instructions, take 50 μ l culture supernatants to be added in ELISA Plate
Row detection, by ELIASA in 450nm wavelength measurements, 620nm wavelength references carry out detection absorbance (OD values).Pass through standard items
Standard curve is drawn out, and detects the content of cell factor in each supernatant.
As shown in result in Fig. 3, fat stem cell culture supernatant cytokine concentrations are above comparative example, two in the present invention
Between person have significant difference, wherein VEGF be 672.5pg/mL, bFGF be 1767.8pg/mL, EGF be 780.3pg/mL,
IGF-1 is 10045.6pg/mL and PDGF is 1324.1pg/mL, shows that the fat stem cell for preparing of the present invention has higher thin
Intracellular cytokine secretion capacity.
Embodiment 6 efficiently promotes the preparation of fat stem cell proliferation kit
According to embodiment 1-4, the product is mainly made up of following composition：
1) 1000ml cell culture mediums, are EGM-2 culture mediums；
2) 1 piece of coated Tissue Culture Plate of gelatin or culture dish；
3) 8 coated Tissue Culture Flasks of gelatin；
4) 1ng/mL recombinant human epidermal growth factors；
5) 5ng/mL derivative growth factor of recombined human blood platelet；
6) 10ng/m recombination human basic fibroblasts growth factor；
7) 10ng/mL insulin；
8) 5ng/mL transferrins；
9) 5% hyclone；
10) 100ml pancreatin；
11) operation instructions；
The reagent kit product culture medium is stored under the conditions of 4 DEG C；The coated Tissue Culture Plate of gelatin or culture dish and gelatin
Coated Tissue Culture Flask production method is prepared according to the method described above, is stored under the conditions of 4 DEG C, and the shelf-life is 1 year；Cell because
Son, hyclone and pancreatin are maintained under the conditions of -80 DEG C, and the shelf-life is 1 year.
Claims (10)
1. a kind of method for efficiently promoting fat stem cell to breed, it is characterised in that methods described is included by using gelatin bag
The coated Tissue Culture Flask of Tissue Culture Plate or culture dish and gelatin of quilt is small in recombinant human epidermal growth factor, recombinant human
Plate derivative growth factor, recombination human basic fibroblast growth factor, insulin, the Combined culture of transferrins and hyclone
Under, efficiently promote fat stem cell propagation growth from fat；
Wherein, the fat stem cell comes from 50mL fat, and acquisition 10 can be cultivated in 15 days9Fat stem cell above, fat
Fat stem cell is through flow cytomery, and height expression CD90+ is that >=90%, CD73+ is that >=90% and CD105+ is >=90%, low
It is that≤5%, HLA-DR+ is that≤5% and CD14+ is≤5% to express CD34+；
Fat stem cell culture solution that the fat stem cell prepared can be 40% in percent by volume, percent by volume are
10% dimethyl sulfoxide (DMSO) and percent by volume is stored in -196 DEG C of liquid nitrogen containers in the frozen stock solution for 50% hyclone composition
In；It is preferred that after fat stem cell recovery, still high expression CD90, CD73 and CD105, low expression CD34, HLA-DR and CD14.
2. according to the method described in claim 1, it is characterised in that the coated Tissue Culture Plate of gelatin, culture dish or thin
Born of the same parents' blake bottle, be use quality volume ratio be 1-20% gelatin after 4 DEG C of refrigerator overnights or 37 DEG C are incubated 1 hour 4 DEG C of refrigerators
Overnight, it is coated with cell culture container；Gelatin solution is absorbed after coating, is divulged information in Biohazard Safety Equipment or super-clean bench dry
It is dry, it is stored in 4 DEG C of refrigerators after drying standby.
3. according to the method described in claim 1, it is characterised in that the cell factor concentration recombinates for 1-200ng/mL
HEGF, 1-200ng/mL derivative growth factor of recombined human blood platelet, the life of 1-200ng/m recombination human basic fibroblasts
The long factor, 1-500ng/mL insulin, 1-500ng/mL transferrins and/or 1-20% hyclones.
4. method according to claim 3, it is characterised in that the cell factor concentration, it is therefore preferable to 5-50ng/
ML recombinant human epidermal growth factors, 5-50ng/mL derivative growth factor of recombined human blood platelet, 5-50ng/m recombination human basics are into fibre
Tie up growth factor, 10-100ng/mL insulin, 10-100ng/mL transferrins and/or 2-10% hyclones.
5. the method according to claim any one of 1-4, it is characterised in that adipose tissue 50mL, with physiological saline repeatedly
Rinse 5 times, oil and haemocyte in fat are absorbed as far as possible；
The type i collagen enzyme 20-50mL that the pancreatin and mass volume ratio for the use of 1: 1 mass volume ratio being 0.25% are 0.1%,
Digest 60 minutes, vibrated 2 minutes with vortex oscillation instrument every 15 minutes in 37 DEG C of incubators；1500rpm centrifuges 10 points after digestion
Clock, suction abandons after the grease of upper strata that to add physiological saline resuspended, is filtered with 100 μm of cell sieves；Cell suspension 1000rpm centrifuges 10 points
Clock, cell precipitation with physiological saline it is resuspended washing 2 times after add cell culture fluid it is resuspended, tongue disk orchid dyeing counting after by 1-10 ×
106Cell/ml is added in the coated Tissue Culture Plate of gelatin or culture dish and cultivated, in 37 DEG C, 5%CO2Cultivated in incubator
24-72 hours；
Culture is inhaled after 24-72 hour with 5ml pipettes abandons nutrient solution, adds cell culture fluid progress and changes liquid, Tissue Culture Plate or
Culture dish continues to place 37 DEG C, 5%CO2Continue to cultivate in incubator；
Digestion is carried out when fat stem cell growth fusion reaches more than 80% and expands bottle, nutrient solution is absorbed, and it is 7.4 to take pH value
1 × PBS adds the pancreatin of 0.5ml 0.25% into blake bottle after washed once, digestion adds 200 μ l hyclones after 2-3 minutes
Digestion is terminated, physiological saline is added, is blown and beaten, is drawn in 15ml centrifuge tubes with 5mL pipettes, then with brine one
It is secondary, it is added in same 15mL centrifuge tubes, 1000 rpms of centrifugations collect fat stem cell in 10 minutes；1mL is used after centrifugation
Fresh medium is resuspended, counts, and adds the fresh cell culture fluids of 10mL with 5mL pipettes, is added to the coated training of 1 bottle of collagen
Support and continue in bottle in 37 DEG C, 5%CO2Cultivated in incubator, a fresh medium was changed every 2 days；Treat that fat stem cell grows
Fusion is when reaching more than 80%, by above-mentioned through collected by trypsinisation fat stem cell, and continuous passage 3 is commissioned to train foster, obtains 109More than
Fat stem cell, it is resuspended with 4ml physiological saline, tongue disk orchid dyeing counting, that is, obtain significant quantities of fat stem cell, place 4 DEG C of bars
Saved backup under part；
The jelly that partial fat stem cell constitutes in 40% fat stem cell culture solution, 10% dimethyl sulfoxide (DMSO) and 50% hyclone
Liquid storage is stored in -196 DEG C of liquid nitrogen containers, in case being used after recovering from now on；
Tongue is taken to expect 3 × 10 after blue dyeing counting6Fat stem cell, divides three groups, and first group has been respectively added to 20 μ L FITC marks
Remember the anti-human CD90 monoclonal antibodies of mouse, the 20 μ L PE mark anti-human CD105 monoclonal antibodies of mouse and 20 μ L Percp mark mouse antihuman CD 34 monoclonal antibodies；The
Two groups have been respectively added to the anti-human CD73 monoclonal antibodies of 20 μ L FITC mark mouse, the 20 μ L PE mark anti-human HLA-DR monoclonal antibodies of mouse and 20 μ L
Percp marks the anti-human CD14 monoclonal antibodies of mouse；3rd group is Isotype control, has been respectively added to 20 μ L FITC mark mouse IgG1,20 μ L
PE marks mouse IgG1 and 20 μ L PerCP mark mouse IgG1；It is placed in 4 DEG C of refrigerators to dye 30 minutes, then with 1mL 1 × phosphate
Buffer solution is washed three times, finally with the cell after the 0.5mL resuspended washings of 1 × PBS, and the cell after gained washing is flowed with FC500
Formula cell instrument is detected；Fat stem cell height expression CD90+ >=90%, CD105+ >=90% and CD73+ >=90%, low expression
CD34+≤5%, HLA-DR+≤5% and CD14+≤5%.
6. method according to claim 5, it is characterised in that the cell culture fluid recombinant human epidermal containing 5-50ng/mL
Growth factor, 5-50ng/mL derivative growth factor of recombined human blood platelet, 5-50ng/m recombination human basic fibroblasts growth factor,
10-100ng/mL insulin, 10-100ng/mL transferrins and 2-10% hyclones.
7. the method according to claim any one of 5-6, it is characterised in that the human adipose-derived stem cell height expression CD90+
For 99.08%, CD105+ be 99.12% and CD73+ is 99.39%, and low expression CD34+ is that 0.12%, HLA-DR+ is 0.51%
It is 0.24% with CD14+.
8. the method according to claim any one of 1-7, it is characterised in that the fat stem cell high expressing cell because
The VEGF secreted in son, i.e. fat stem cell culture supernatant is more than more than 500pg/mL, bFGF more than 500pg/mL, EGF
1500pg/mL, IGF-1 are more than 10ng/mL and PDGF and are more than 1000pg/mL.
9. the method according to any one of claim 8, it is characterised in that the VEGF is that 672.5pg/mL, bFGF are
1767.8pg/mL, EGF are that 780.3pg/mL, IGF-1 are 10045.6pg/mL and PDGF is 1324.1pg/mL.
10. a kind of kit for efficiently promoting fat stem cell to breed prepared according to any one of claim 1-9 methods described,
Characterized in that, it is made up of following composition：
1) 1000ml cell culture mediums；
2) 1 piece of coated Tissue Culture Plate of gelatin or culture dish；
3) 8 coated Tissue Culture Flasks of gelatin；
4) 5-50ng/mL recombinant human epidermal growth factors；
5) 5-50ng/mL derivative growth factor of recombined human blood platelet；
6) 5-50ng/m recombination human basic fibroblasts growth factor；
7) 10-100ng/mL insulin；
8) 10-100ng/mL transferrins；
9) 2-10% hyclones；
10) 100ml pancreatin；
11) operation instructions；
It is preferred that, the coated Tissue Culture Plate of the culture medium of the reagent kit product, gelatin or culture dish and the coated cell training of gelatin
Foster bottle is stored under the conditions of 4 DEG C, and the shelf-life is 1 year；Cell factor, hyclone and pancreatin are maintained under the conditions of -80 DEG C, are protected
The matter phase is 1 year.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710303383.3A CN106957818A (en) | 2017-05-03 | 2017-05-03 | A kind of method for efficiently promoting fat stem cell to breed and its kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710303383.3A CN106957818A (en) | 2017-05-03 | 2017-05-03 | A kind of method for efficiently promoting fat stem cell to breed and its kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106957818A true CN106957818A (en) | 2017-07-18 |
Family
ID=59482967
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710303383.3A Pending CN106957818A (en) | 2017-05-03 | 2017-05-03 | A kind of method for efficiently promoting fat stem cell to breed and its kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106957818A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107714727A (en) * | 2017-10-24 | 2018-02-23 | 成都远山博桥生物科技有限公司 | A kind of enriching fat stem cell culture supernatant preparation, its preparation method and application |
| CN108753705A (en) * | 2018-05-29 | 2018-11-06 | 深圳市嘉祺生物科技有限公司 | A kind of preparation method of fat stem cell and products thereof of high expression collagen |
| CN110540678A (en) * | 2018-05-29 | 2019-12-06 | 怡诺博(北京)生物医学技术有限公司 | Modified sodium alginate hydrogel, cell culture matrix material prepared from same and application of cell culture matrix material |
| CN113544259A (en) * | 2020-01-31 | 2021-10-22 | 瑞帝安有限公司 | Method for differentiating human adipose-derived mesenchymal stem cells into hair papilla cells |
| CN115305233A (en) * | 2022-03-15 | 2022-11-08 | 黄启明 | Preparation and application of daidzein and apigenin composite agarose-collagen hydrogel three-dimensional culture stem cells and extracellular vesicles |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102666840A (en) * | 2009-10-23 | 2012-09-12 | Rnl生物技术株式会社 | Method for inducing migration of adult stem cells derived from adipose tissue |
| CN102757936A (en) * | 2012-06-15 | 2012-10-31 | 江苏瑞思坦生物科技有限公司 | Proliferation accelerator for human adipose-derived stem cells and application method thereof |
| CN103667185A (en) * | 2012-08-31 | 2014-03-26 | 孙勇 | New adipose-derived stem cell technology for plastic surgery and cosmetology |
| CN104560868A (en) * | 2014-11-27 | 2015-04-29 | 广州赛莱拉干细胞科技股份有限公司 | Primary isolation culture method of adipose-derived stem cells |
| CN105112364A (en) * | 2015-08-18 | 2015-12-02 | 广州暨南生物医药研究开发基地有限公司 | Serum-free medium for human adipose-derived stem cells and preparation method thereof |
-
2017
- 2017-05-03 CN CN201710303383.3A patent/CN106957818A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102666840A (en) * | 2009-10-23 | 2012-09-12 | Rnl生物技术株式会社 | Method for inducing migration of adult stem cells derived from adipose tissue |
| CN102757936A (en) * | 2012-06-15 | 2012-10-31 | 江苏瑞思坦生物科技有限公司 | Proliferation accelerator for human adipose-derived stem cells and application method thereof |
| CN103667185A (en) * | 2012-08-31 | 2014-03-26 | 孙勇 | New adipose-derived stem cell technology for plastic surgery and cosmetology |
| CN104560868A (en) * | 2014-11-27 | 2015-04-29 | 广州赛莱拉干细胞科技股份有限公司 | Primary isolation culture method of adipose-derived stem cells |
| CN105112364A (en) * | 2015-08-18 | 2015-12-02 | 广州暨南生物医药研究开发基地有限公司 | Serum-free medium for human adipose-derived stem cells and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 巴音桑: "儿童脂肪干细胞的成骨诱导及其复合胶原羟基磷灰石支架的生物相容性的实验研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107714727A (en) * | 2017-10-24 | 2018-02-23 | 成都远山博桥生物科技有限公司 | A kind of enriching fat stem cell culture supernatant preparation, its preparation method and application |
| CN108753705A (en) * | 2018-05-29 | 2018-11-06 | 深圳市嘉祺生物科技有限公司 | A kind of preparation method of fat stem cell and products thereof of high expression collagen |
| CN110540678A (en) * | 2018-05-29 | 2019-12-06 | 怡诺博(北京)生物医学技术有限公司 | Modified sodium alginate hydrogel, cell culture matrix material prepared from same and application of cell culture matrix material |
| CN110540678B (en) * | 2018-05-29 | 2022-09-06 | 怡诺博(北京)生物医学技术有限公司 | Modified sodium alginate hydrogel, cell culture matrix material prepared from same and application of cell culture matrix material |
| CN113544259A (en) * | 2020-01-31 | 2021-10-22 | 瑞帝安有限公司 | Method for differentiating human adipose-derived mesenchymal stem cells into hair papilla cells |
| CN115305233A (en) * | 2022-03-15 | 2022-11-08 | 黄启明 | Preparation and application of daidzein and apigenin composite agarose-collagen hydrogel three-dimensional culture stem cells and extracellular vesicles |
| CN115305233B (en) * | 2022-03-15 | 2024-05-10 | 黄启明 | Preparation and application of soybean aglycone and apigenin composite agarose-collagen hydrogel three-dimensional culture stem cells and extracellular vesicles |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106957818A (en) | A kind of method for efficiently promoting fat stem cell to breed and its kit | |
| CN104263697B (en) | A kind of method that inducing culture and induction human adipose mesenchymal stem cells generate insulin secretory cell | |
| Pill et al. | Vascularization mediated by mesenchymal stem cells from bone marrow and adipose tissue: a comparison | |
| Cheng et al. | The influence of spheroid formation of human adipose-derived stem cells on chitosan films on stemness and differentiation capabilities | |
| Hegab et al. | Isolation and characterization of murine multipotent lung stem cells | |
| CN108251359A (en) | A kind of mesenchymal stem cell serum-free culture medium and cultural method | |
| Shen et al. | Implications of adipose-derived stromal cells in a 3D culture system for osteogenic differentiation: an in vitro and in vivo investigation | |
| Colazzo et al. | Shear stress and VEGF enhance endothelial differentiation of human adipose-derived stem cells | |
| CN105555282B (en) | Composition for preventing and treating liver fibrosis or cirrhosis comprising mesenchymal stem cells derived from human embryonic stem cells as an active ingredient | |
| Chang et al. | Multiple directional differentiation difference of neonatal rat fibroblasts from six organs | |
| CN101148656B (en) | Construction method for tissue engineering liver unit | |
| Weinzierl et al. | Bone engineering with adipose tisssue derived stromal cells | |
| Hutton et al. | Vascular morphogenesis of adipose-derived stem cells is mediated by heterotypic cell-cell interactions | |
| CN101748096A (en) | Sub totipotential stem cell and preparation method and application thereof | |
| Frerich et al. | Comparison of different fabrication techniques for human adipose tissue engineering in severe combined immunodeficient mice | |
| Lin et al. | Fibroblast growth factor-2 facilitates rapid anastomosis formation between bioengineered human vascular networks and living vasculature | |
| CN112292447B (en) | Umbilical cord mesenchymal stem cell and preparation method of cell membrane thereof | |
| CN108795855A (en) | A kind of serum free medium of mescenchymal stem cell | |
| Twardowski et al. | Cardiac fibroblasts support endothelial cell proliferation and sprout formation but not the development of multicellular sprouts in a fibrin gel co-culture model | |
| Freymann et al. | Effect of human serum and 2 different types of platelet concentrates on human meniscus cell migration, proliferation, and matrix formation | |
| KR102676761B1 (en) | Method for producing mesenchymal stem cells | |
| CN101821383A (en) | A kind of primary mesenchymal stem cells and application thereof that is used for substratum, method and the acquisition of the external mass-producing cultivation of human adult primary mesenchymal stem cells | |
| CN109749993A (en) | A kind of cultural method of umbilical cord mesenchymal stem cells | |
| Melero‐Martin et al. | An in vivo experimental model for postnatal vasculogenesis | |
| Pytlík et al. | The cultivation of human multipotent mesenchymal stromal cells in clinical grade medium for bone tissue engineering |
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 | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20170718 |
|
| WD01 | Invention patent application deemed withdrawn after publication |