CN105132365A - Three-dimensional induction culture method of mesenchymal stem cells - Google Patents
Three-dimensional induction culture method of mesenchymal stem cells Download PDFInfo
- Publication number
- CN105132365A CN105132365A CN201510502814.XA CN201510502814A CN105132365A CN 105132365 A CN105132365 A CN 105132365A CN 201510502814 A CN201510502814 A CN 201510502814A CN 105132365 A CN105132365 A CN 105132365A
- Authority
- CN
- China
- Prior art keywords
- stem cell
- mescenchymal stem
- inducing
- cultivating
- concentration
- 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
- 210000002901 mesenchymal stem cell Anatomy 0.000 title abstract description 16
- 230000006698 induction Effects 0.000 title abstract description 13
- 238000012136 culture method Methods 0.000 title abstract description 7
- 238000000034 method Methods 0.000 claims abstract description 47
- 230000001939 inductive effect Effects 0.000 claims abstract description 42
- 235000010443 alginic acid Nutrition 0.000 claims abstract description 20
- 229920000615 alginic acid Polymers 0.000 claims abstract description 20
- 229910021645 metal ion Inorganic materials 0.000 claims abstract description 17
- 230000008569 process Effects 0.000 claims abstract description 14
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 claims abstract description 6
- 229910001425 magnesium ion Inorganic materials 0.000 claims abstract description 6
- 238000002156 mixing Methods 0.000 claims abstract description 6
- 210000000130 stem cell Anatomy 0.000 claims description 102
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 20
- 235000010413 sodium alginate Nutrition 0.000 claims description 20
- 239000000661 sodium alginate Substances 0.000 claims description 20
- 229940005550 sodium alginate Drugs 0.000 claims description 20
- 102000004887 Transforming Growth Factor beta Human genes 0.000 claims description 18
- 108090001012 Transforming Growth Factor beta Proteins 0.000 claims description 18
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 claims description 17
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 12
- 102000004877 Insulin Human genes 0.000 claims description 10
- 108090001061 Insulin Proteins 0.000 claims description 10
- 102000002070 Transferrins Human genes 0.000 claims description 10
- 108010015865 Transferrins Proteins 0.000 claims description 10
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 claims description 10
- 229960003957 dexamethasone Drugs 0.000 claims description 10
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 10
- 150000002505 iron Chemical class 0.000 claims description 10
- 229960002901 sodium glycerophosphate Drugs 0.000 claims description 10
- REULQIKBNNDNDX-UHFFFAOYSA-M sodium;2,3-dihydroxypropyl hydrogen phosphate Chemical compound [Na+].OCC(O)COP(O)([O-])=O REULQIKBNNDNDX-UHFFFAOYSA-M 0.000 claims description 10
- 150000003839 salts Chemical class 0.000 claims description 9
- 238000004140 cleaning Methods 0.000 claims description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 5
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 abstract description 45
- 239000000243 solution Substances 0.000 abstract description 22
- 230000000694 effects Effects 0.000 abstract description 14
- 230000004069 differentiation Effects 0.000 abstract description 13
- 230000012010 growth Effects 0.000 abstract description 9
- 230000009466 transformation Effects 0.000 abstract description 7
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 abstract description 4
- 229940072056 alginate Drugs 0.000 abstract description 4
- 239000012266 salt solution Substances 0.000 abstract description 4
- 238000012258 culturing Methods 0.000 abstract description 3
- 238000001727 in vivo Methods 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 3
- 239000003795 chemical substances by application Substances 0.000 abstract description 2
- 239000011324 bead Substances 0.000 abstract 2
- 229960001126 alginic acid Drugs 0.000 abstract 1
- 239000000783 alginic acid Substances 0.000 abstract 1
- 150000004781 alginic acids Chemical class 0.000 abstract 1
- 238000003501 co-culture Methods 0.000 abstract 1
- 210000001612 chondrocyte Anatomy 0.000 description 44
- 239000007788 liquid Substances 0.000 description 19
- 210000000845 cartilage Anatomy 0.000 description 10
- 230000009977 dual effect Effects 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 8
- 238000005096 rolling process Methods 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 238000004113 cell culture Methods 0.000 description 6
- 239000011259 mixed solution Substances 0.000 description 6
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 5
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 5
- 229920002683 Glycosaminoglycan Polymers 0.000 description 5
- 210000000988 bone and bone Anatomy 0.000 description 5
- 229910002091 carbon monoxide Inorganic materials 0.000 description 5
- 239000000084 colloidal system Substances 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 210000004102 animal cell Anatomy 0.000 description 4
- 229910001424 calcium ion Inorganic materials 0.000 description 4
- 230000007547 defect Effects 0.000 description 4
- 239000000975 dye Substances 0.000 description 4
- 230000002708 enhancing effect Effects 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 230000003068 static effect Effects 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 description 3
- 229920001287 Chondroitin sulfate Polymers 0.000 description 3
- 102000008186 Collagen Human genes 0.000 description 3
- 108010035532 Collagen Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 3
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 229940059329 chondroitin sulfate Drugs 0.000 description 3
- 229920001436 collagen Polymers 0.000 description 3
- 230000024835 cytogamy Effects 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 210000002744 extracellular matrix Anatomy 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 210000003205 muscle Anatomy 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 210000001188 articular cartilage Anatomy 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 150000001768 cations Chemical class 0.000 description 2
- 230000024245 cell differentiation Effects 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000001079 digestive effect Effects 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 210000003414 extremity Anatomy 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 210000002826 placenta Anatomy 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 230000000452 restraining effect Effects 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 238000005204 segregation Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 230000005477 standard model Effects 0.000 description 2
- 210000003954 umbilical cord Anatomy 0.000 description 2
- 230000003442 weekly effect Effects 0.000 description 2
- QGZCUOLOTMJILH-UHFFFAOYSA-N 2h-tetrazol-2-ium;bromide Chemical compound [Br-].C1=N[NH+]=NN1 QGZCUOLOTMJILH-UHFFFAOYSA-N 0.000 description 1
- 102000016284 Aggrecans Human genes 0.000 description 1
- 108010067219 Aggrecans Proteins 0.000 description 1
- 102100027935 Attractin-like protein 1 Human genes 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 206010007710 Cartilage injury Diseases 0.000 description 1
- 101000997262 Centruroides noxius Potassium channel toxin alpha-KTx 2.1 Proteins 0.000 description 1
- 206010061762 Chondropathy Diseases 0.000 description 1
- 102000002734 Collagen Type VI Human genes 0.000 description 1
- 108010043741 Collagen Type VI Proteins 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 108090000079 Glucocorticoid Receptors Proteins 0.000 description 1
- 102100033417 Glucocorticoid receptor Human genes 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
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 1
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102000004067 Osteocalcin Human genes 0.000 description 1
- 108090000573 Osteocalcin Proteins 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 102000016611 Proteoglycans Human genes 0.000 description 1
- 108010067787 Proteoglycans Proteins 0.000 description 1
- 102000013275 Somatomedins Human genes 0.000 description 1
- 208000013201 Stress fracture Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 230000002308 calcification Effects 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 210000003321 cartilage cell Anatomy 0.000 description 1
- 230000004098 cellular respiration Effects 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 210000003035 hyaline cartilage Anatomy 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 230000002608 insulinlike Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 229960004232 linoleic acid Drugs 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 238000011587 new zealand white rabbit Methods 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000011164 ossification Effects 0.000 description 1
- 210000004409 osteocyte Anatomy 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 229940085991 phosphate ion Drugs 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000011946 reduction process Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000008698 shear stress Effects 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 210000000689 upper leg Anatomy 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a three-dimensional induction culture method of mesenchymal stem cells, wherein the culture method comprises the following steps: acquiring mesenchymal stem cells; mixing the mesenchymal stem cells with soluble alginate, and adding a salt solution of divalent metal ions except for magnesium ion, so as to form gel beads; and induction-culturing the mesenchymal stem cells in the gel beads by virtue of an inducing agent. The culture method disclosed by the invention, on the basis of the three-dimensional spatial structure of an alginic acid gel body, provides a microenvironment simulating in vivo growth for the mesenchymal stem cells, so as to expand a growth space of the mesenchymal stem cells as well as substance signal exchange between a culture solution and the cells, promote the growth and the differentiation of the mesenchymal stem cells, improve a transformation rate and enhance cell activity; and meanwhile, compared with a co-culture mode in the prior art, the culture method also avoids a process of isolating two cells.
Description
Technical field
The present invention relates to cell engineering field, particularly a kind of three-dimensional method for inducing and cultivating of mescenchymal stem cell.
Background technology
Articular cartilage tissue defect is a kind of common disease in the present age, the damage caused by tumour, the pathology caused by the age, and various genetic diseases and cartilaginous tissue misgrowth and wound can cause damage and the disappearance of joint cartilage.Joint cartilage self-repairing capability is lower, mainly contains the reason of two aspects: one is that chondrocyte belongs to terminally differentiated cells, and metabolic activity is low, self duplication ability; Two is that cartilaginous tissue does not have blood vessel and nerve.What the most often apply clinically is a kind of therapeutic method of surgery of so-called micro-fracture, the method a fine needle is penetrated damaged cartilage tissue to go directly marrow, lure into medullary cell (containing mescenchymal stem cell) along with blood flows to damaged part, then through original position inducing mesenchymal stem cell formed chondrocyte.But what this restorative procedure was finally formed is a kind of fibrous cartilage, compare with normal hyaline cartilage tissue, the content of II Collagen Type VI that fibrous cartilage contains and cartilage aggrecan is lower, and can not substitute normal cartilage function completely, long-term result for the treatment of is undesirable.
When traditional method effectively can not solve articular cartilage defect disease, and along with the development of biotechnology, be cartilage tissue engineeredly hopeful the difficult problem that can solve regenerative agent of cartilaginous tissue.In cartilage tissue engineered, chondrocyte has two large defects, is first that cell derived is restricted, and is secondly the video picture that chondrocyte can dedifferente when cultivating in vitro, loses chondrocyte phenotype and secretes the ability of specific extracellular matrix.Three-dimensional stent material can maintain chondrocyte's form effectively, but the multiplication rate of chondrocyte on timbering material is cultivated still lower relative to plane, is difficult to the cell obtaining q.s.
And mescenchymal stem cell is a kind of cell with multi-lineage potential, it can directional induction be the various kinds of cell such as insulin-like cell, chondrocyte.And mesenchymal cell is present in human multiple tissue; especially the mescenchymal stem cell of umbilical cord, placenta, marrow, adipose tissue-derived; there are wide material sources, be easy to gather, without advantages such as ethics problems; mass-producing can be induced to differentiate into chondrocyte, for clinical treatment articular cartilage damage provides new technical scheme.But the mode inducing mesenchymal stem cell of mescenchymal stem cell and chondrocyte's Dual culture that adopts is converted into chondrocyte more at present, and not only Induction Transformation rate is low for this Dual culture mode, and cytoactive is lower, and chondrocyte isolation is more difficult, and immunity is low.
Summary of the invention
Technical problem to be solved by this invention is, it is low to there is inductivity in the mode obtaining chondrocyte for Dual culture mescenchymal stem cell and chondrocyte in prior art, cytoactive is poor, the defects such as two kinds of cellular segregation difficulties, there is provided a kind of Induction Transformation rate that can improve mescenchymal stem cell, cell activity enhancing and the three-dimensional method for inducing and cultivating of segregative mescenchymal stem cell.
The technical solution adopted for the present invention to solve the technical problems is: the three-dimensional method for inducing and cultivating providing a kind of mescenchymal stem cell, comprises the following steps:
Obtain mescenchymal stem cell;
After mescenchymal stem cell is mixed with the alginates of solubility, then mix with the divalent-metal ion salts solution except magnesium ion, to forming gel ball;
With inductor, inducing culture is carried out to the mescenchymal stem cell in gel ball.
In the three-dimensional method for inducing and cultivating of mescenchymal stem cell provided by the invention, the forming process of described gel ball is: at the uniform velocity instill after being mixed with the alginates of solubility by described mescenchymal stem cell in the divalent-metal ion salts solution except magnesium ion.
In the three-dimensional method for inducing and cultivating of mescenchymal stem cell provided by the invention, described alginates is sodium alginate, and before mixing with described mescenchymal stem cell, the concentration of sodium alginate is 2%-10%W/V.
In the three-dimensional method for inducing and cultivating of mescenchymal stem cell provided by the invention, described divalent-metal ion salts solution is calcium chloride solution, and the concentration of calcium chloride is 1%-5%W/V.
In the three-dimensional method for inducing and cultivating of mescenchymal stem cell provided by the invention, before inducing culture is carried out to the mescenchymal stem cell in described gel ball, also comprise the step of cleaning gel ball.
In the three-dimensional method for inducing and cultivating of mescenchymal stem cell provided by the invention, adopt basic medium cleaning gel ball.
In the three-dimensional method for inducing and cultivating of mescenchymal stem cell provided by the invention, described inductor comprises the ITS+Premix of TGF-β, 7-10mmol/l dexamethasone of 5-15ng/ml, 50-100ug/ml xitix, 5-15mmol/l Sodium Glycerophosphate and 50-150ng/ml.
In the three-dimensional method for inducing and cultivating of mescenchymal stem cell provided by the invention, described inductor also comprises Regular Insulin and Transferrins,iron complexes, and the concentration of described Regular Insulin is 5-10mg/ml, and the concentration of Transferrins,iron complexes is 5-10mg/ml.
In the three-dimensional method for inducing and cultivating of mescenchymal stem cell provided by the invention, described inductor also comprises glutamine, and the concentration of described glutamine is 1-10mmoL/L.
In the three-dimensional method for inducing and cultivating of mescenchymal stem cell provided by the invention, the inducing culture process of the mescenchymal stem cell in described gel ball is carried out in bio-reactor.
Implement the three-dimensional method for inducing and cultivating of mescenchymal stem cell provided by the invention, following beneficial effect can be reached: by utilizing the porous three-dimensional space structure of Lalgine colloid, for mescenchymal stem cell provides marrow three-dimensional microenvironment in an analogue body, expand the growing space of mescenchymal stem cell, and exchange with nutrient solution and intercellular PM signals, promote the Growth and Differentiation of mescenchymal stem cell, improve transformation efficiency, cell activity enhancing; Meanwhile, compared to Dual culture mode of the prior art, the present invention also can save the process of separation two kinds of cells.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, the invention will be further described, in accompanying drawing:
Fig. 1 is in test experience three provided by the invention, according to the canonical plotting made by the concentration of Chs standard model working fluid and OD value thereof.
Embodiment
In order to solve in prior art the mode utilizing mescenchymal stem cell and chondrocyte's Dual culture to obtain chondrocyte, to there is induction duration long, low conversion rate, cellular segregation difficulty, immunological safety is low, need the defects such as chondrocyte source, innovative point of the present invention is to provide a kind of independent inducing mesenchymal stem cell to change into the method for chondrocyte, by utilizing the three-dimensional structure of Lalgine gel, analog cell living environment in vivo, for mescenchymal stem cell provides sufficient growing space, increase the contact area of mescenchymal stem cell and nutrient solution, thus achieve the transformation efficiency improving mescenchymal stem cell, cell activity enhancing also can remove the object of isolated cell step from.
The three-dimensional method for inducing and cultivating of mescenchymal stem cell provided by the invention, comprises the following steps:
One, mescenchymal stem cell is obtained;
Two, after mescenchymal stem cell being mixed with the alginates of solubility, add in the divalent-metal ion salts solution except magnesium ion, to forming gel ball;
Three, after cleaning gel ball, with inductor, inducing culture 7-28 days is carried out to the mescenchymal stem cell in gel ball.
In step one, mescenchymal stem cell can derive from umbilical cord, placenta, marrow etc., preferably, from marrow, obtains mescenchymal stem cell.Preferably, the density of mescenchymal stem cell is 5-15 × 10
6individual/ml.
In step 2, crosslinked and after losing rheological under the effect of the divalent-metal ion of alginates polymer beyond demagging, the flowing of water molecules receives suppression, and then it is high and have the gelinite of through hole to create water content, and the gelinite of alginates is not thermal reversion, there is good stability and biocompatibility, therefore, stable three dimensional growth space can be provided for mescenchymal stem cell, analog cell growing environment in vivo, expand the growing space of mescenchymal stem cell, and increase the contact area of mescenchymal stem cell and nutrient solution, be conducive to the attaching of cell, growth and differ entiation, and the present invention mixed with alginates by mescenchymal stem cell before formation Lalgine gel ball, after making gel ball formation, mescenchymal stem cell can be evenly distributed in gel ball, is more conducive to the cultivation of mescenchymal stem cell.
It should be noted that being swift in response due to metal ion and alginates, by improving gelling temperature, calcium ion total amount or reduce alginate solution concentration and accelerate gel process; And the alginates that the curing speed that slows down, raising alginate solution concentration, calcium ion total amount and use molecular mass are higher, its mechanical property can be strengthened, be more conducive to the differentiation of mescenchymal stem cell.In addition, the dropping order of alginates and metal ion salt solution and rate of addition can affect the character of colloid, and rate of addition is too fast, and the colloid of generation is the gel structure of strip gel and interruption, mescenchymal stem cell skewness; Preferably, the present invention at the uniform velocity instills in metal ion salt solution after adopting and being mixed with alginates by mescenchymal stem cell, and the gel ball size formed with this is comparatively even, also can not affect mescenchymal stem cell being uniformly distributed in colloid.
Wherein, univalent cation and Mg
2+gel can not be formed, and the gel that Ba2+ and Sr2+ is formed compares Ca
2+the gellifying property formed is stronger.Other polyvalent cations, as Pb
2+, Cu
2+, Cd
2+, Co
2+, Ni
2+, Zn
2+and Mn
2+sodium alginate cross-linking gel can be formed Deng also, but limited because having its application of toxicity; Therefore, preferably, divalent-metal ion is calcium ion, and divalent-metal ion salts solution is calcium chloride solution, CaCl
2concentration be 1%-5%W/V, calcium ion concn is larger, and gel ball diameter is larger, and the pore texture of formation is more, and the penetrating power of gel ball is also stronger, be more conducive to cell attachment growth, differentiation; Alginates is sodium alginate or alginate calcium etc., and be preferably sodium alginate soln, the concentration of sodium alginate is 3%-10%W/V.In the present invention, unit W/V is as being mg/ml without particularly pointing out.
In step 3, after treating that gel ball is formed, remove unnecessary metal ion salt solution, and repeatedly clean by NaCl solution, again with basic medium cleaning, remove the divalent-metal ion on gel ball surface, avoid divalent-metal ion to produce toxic action in cell cultivation process; Then, then by the gel ball containing mescenchymal stem cell be placed in the culture vessel being added with induction broth and carry out inducing culture.
Wherein, culture vessel can be static Tissue Culture Plate, and also can be bio-reactor, can realize dynamic cultivation, also can carry out static cultivation in cold situation, bio-reactor can be roller system, comprises rolling bottle and Rotary Machine; Also can be shaking flask, filling system etc., can select according to different supplier induced demand.Dynamic cultivation not only can realize culturing cell on a large scale, obtain the cell of q.s, but also more accurately can control reaction conditions, be convenient to following large-scale production, secondly, dynamic cultivation can improve the efficiency of mass transfer and biography oxygen in culturing process, effectively can overcome the mass transfer limitations of static cultivation, by bio-reactor in conjunction with three dimensional culture system, effectively can improve the proliferation and differentiation of mescenchymal stem cell, and improve the activity of cell, in bio-reactor, dynamic cultivation condition shear-stress can promote that mescenchymal stem cell forms chondrocyte to a certain extent, improve Induction Transformation rate.
Further, in inducing culture process, inductor of the present invention comprises TGF-β, dexamethasone, xitix, Sodium Glycerophosphate and ITS+Premix.
Wherein, TGF-β (transforminggrowthfactor-β, TGF-β transforming growth factor-beta) be that gang extensively exists, structure relevant, intimate multifunction activity peptide, there is the polypeptide growth factor of several functions, the reproduction restraint of mescenchymal stem cell can be promoted, promote the synthesis of extracellular matrix, promote that collagen produces, stimulate synthesis and the extracellular matrix secretion albumen of chondrocyte, promote the propagation of osteocyte.TGF-β has three kinds of isomer, be respectively TGF-β 1, β 2 and β 3, TGF-β 2, β 3 comparatively β 1 can more fast and effeciently promote that mesenchymal stem cells into chondrocytes breaks up, but between TGF-β 2, β 3, the level of induced dry-cell is substantially identical.Because the biological function of TGF-β in osteogenesis is dual regulation, can the proliferation and differentiation effect of irritation cell, also can carry out restraining effect, in general, lower concentration TGF-β acts as a spur, and promotes the proliferation and differentiation of cell; High density plays restraining effect, and therefore, preferably, the concentration of TGF-β is 5-15ng/ml.
Dexamethasone, be purchased from Sigma company, the expression of alkaline phosphatase, Bone Gla protein and NTx etc. can be stimulated, increase the activity of alkaline phosphatase significantly, alkaline phosphatase is enzyme necessary in bone forming process, and the expression of alkaline phosphatase represents osteoplastic situation, the glucocorticoid receptor being positioned at mescenchymal stem cell surface can be activated, promote mesenchymal stem cells into chondrocytes differentiation, meanwhile, improve the differentiation rate of mesenchymal stem cells into chondrocytes; Preferably, the concentration of dexamethasone is 7-10mmol/ml.
Xitix is also known as vitamins C, it is the one of water-soluble vitamins, participate in redox processes, play an important role in the oxidation of biology and reduction process and cellular respiration, simultaneously, xitix is that collage synthesis is necessary, also the synthesis of adjustable ATP enzyme and ALP activity and non-collagen stroma protein; Xitix, to the promoter action of Chondrocyte Differentiation, mainly by increasing the accumulation of collagen, then increases the expression of alkaline phosphatase in chondrocyte; In addition, also can suppress or reduce apoptotic generation in Induction Process as a kind of antioxidant.Preferably, the concentration of xitix is 50-100ug/ml.
Sodium Glycerophosphate can provide phosphate ion for chondrocyte, promotes deposition and the calcification of physiological calcium salt simultaneously, is the prerequisite that bone marrow matrix mescenchymal stem cell produces Mineral nodules; Preferably, the concentration of Sodium Glycerophosphate is 5-15mmol/ml.
ITS+Premix adds basic medium as a kind of somatomedin, is purchased from BD company, and its composition includes bovine serum albumin and linolic acid etc., can promote the proliferate of cell; Preferably, the concentration of ITS+Premix is 50-150ng/ml.
Further, induced liquid provided by the invention also comprises Regular Insulin and Transferrins,iron complexes; Regular Insulin can reduce protein degradation, increase the absorption of amino acid and glucose, for Growth of Cells and differentiation provide energy, mescenchymal stem cell is impelled to be in low sugar environment, keep cytoactive, be conducive to the differentiation of mesenchymal stem cells into chondrocytes, preferably, the concentration range of Regular Insulin is 5-10mg/ml; And Transferrins,iron complexes can intervene ion metabolism in chondrocyte, promote the generation of chondrocyte, preferably, the concentration range of Transferrins,iron complexes is 5-10mg/ml.
Further, induced liquid provided by the invention also comprises glutamine, and glutamine participates in synthesis and the nucleic acid metabolism of protein, can be used as the energy derive in induced liquid, in addition, glutamine, by cell compatibilization, also can promote the growth and differ entiation of cell; Preferably, the concentration range of glutamine is 1-10mmol/l.
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Embodiment 1
The three-dimensional method for inducing and cultivating of mescenchymal stem cell provided by the invention, comprises the following steps:
1, the separation of mescenchymal stem cell;
By air Injection new zealand white rabbit auricular vein, make it dead.The four limbs of White Rabbit smeared by alcohol with 75%, sterilize and reduce the pollution of the rabbit hair.Cut femur and the spinal joints of rabbit four limbs, removing skin histology retains the muscle reticular tissue of joint, and the muscle tissue at all the other positions cuts off.The tissue processed is immersed in the alcohol of 75%, takes out after 30min, clean with aseptic PBS, be placed in super clean bench.Cut away the muscle of joint with aseptic operation cutter, joint is exposed.Cut off joint with bone shears, bone chamber is exposed.Use disposable syringe to draw aseptic PBS, repeatedly rinse bone chamber, the marrow in bone chamber is all flushed in disposable plate.Liquid rotating is moved on 200 aseptic object screen clothes, cross and filter large tissue, obtain suspension.Suspension is slowly joined the 50ml centrifuge tube of the Ficoll lymph parting liquid that 15ml is housed in advance, the centrifugal 30min of 2790rpm.After centrifugal end, with sharp suction pipe imbitition middle white nepheloid layer cell, after cell being transferred to the large square vase of T150, adding 50ml growth of mesenchymal stem cells substratum, after 3 days, change liquid.Go down to posterity after cytogamy degree reaches 90%, trysinization 3-5min, absorption suspension is transferred in new square vase to be cultivated, and this cell is designated as P2 cell, and partial freeze is preserved, and rest part continues to go down to posterity.
2, the Secondary Culture of mescenchymal stem cell, obtains P3 for mescenchymal stem cell;
Cytogamy degree gets final product had digestive transfer culture when reaching about 90%.Sucking-off substratum, cleans cell surface with aseptic PBS, washes away residual substratum.Add 5ml pancreatin, leave standstill 5min, the most cell of microscopic examination becomes spherical, and when can float, the substratum added containing FBS stops digestion.Sucking-off cell suspension, blood counting chamber counts, then with 1.0 × 10
4individual/cm
2density be inoculated in T150 square vase.Square vase is placed in 37 DEG C, 5%CO
2cultivate in animal cell culture case, treating that cytogamy degree reaches 90% can had digestive transfer culture again; Adopt P3 for mescenchymal stem cell in the present embodiment.
3, embed P3 for mescenchymal stem cell, namely mix with solubility marine alga salt;
Get P3 in step 2 for mescenchymal stem cell, and resuspended adjustment cell density is 2 × 10
7individual/ml, use syringe to draw isopyknic aseptic 4%W/V sodium alginate, with cell suspension Homogeneous phase mixing, now cell density is l × 10
7individual/ml, sodium alginate final concentration is 2%W/V.Use syringe to draw mescenchymal stem cell and sodium alginate mixed solution, load onto aseptic syringe needle, the air in Inside Syringe.Slow pushing syringe, at the uniform velocity instills mescenchymal stem cell and sodium alginate mixed solution the 1%W/VCaCl that 100ml is housed afterwards
2in the beaker of solution.Ensure that needle position maintains an equal level with beaker rim of a cup all the time as far as possible, at the uniform velocity instill mescenchymal stem cell and sodium alginate mixed solution.After question response 10min, the sodium alginate containing mescenchymal stem cell is own through being cross-linked to form gel ball.
4, clean gel ball, and inducing culture is carried out to the mescenchymal stem cell in gel ball;
Sucking-off CaCl
2solution, with 10ml aseptic 0.9%NaCl solution washing gel ball, be divided in rolling bottle after not washing twice containing the α-MEM of FBS with 10ml again, add 50ml chondrocyte induction substratum in rolling bottle, be placed in by rolling bottle on Rotary Machine, rotating speed is 50rpm, full dose changes liquid 3 times weekly, half amount changes liquid 1 time, is placed in 37 DEG C, 5%CO
2cultivate 28 days in animal cell culture case.
Wherein, containing induced liquid in chondrocyte induction substratum, induced liquid composition is: the ITS+Premix of TGF-β, 10mmol/l dexamethasone of 10ng/ml, 70ug/ml xitix, 10mmol/l Sodium Glycerophosphate and 100ng/ml.
Embodiment 2
Be with the difference of embodiment 1, the step 3 in this embodiment 2 is:
Get P3 in step 2 for mescenchymal stem cell, and resuspended adjustment cell density is 2 × 10
7individual/ml, use syringe to draw isopyknic aseptic 20%W/V sodium alginate, with cell suspension Homogeneous phase mixing, now cell density is l × 10
7individual/ml, sodium alginate final concentration is 10%W/V.Use syringe to draw mescenchymal stem cell and sodium alginate mixed solution, load onto aseptic syringe needle, the air in Inside Syringe.Slow pushing syringe, at the uniform velocity instills mescenchymal stem cell and sodium alginate mixed solution the 5%W/VCaCl that 100ml is housed afterwards
2in the beaker of solution.Ensure that needle position maintains an equal level with beaker rim of a cup all the time as far as possible, at the uniform velocity instill mescenchymal stem cell and sodium alginate mixed solution.After question response 10min, the sodium alginate containing mescenchymal stem cell is own through being cross-linked to form gel ball.
Embodiment 3
Be with the difference of embodiment 1, what adopt in step 4 is static cultivation system, is specially:
Step 4: sucking-off CaCl
2solution, with 10ml aseptic 0.9%NaCl solution washing gel ball, then be divided in rolling bottle after not washing twice containing the α-MEM of FBS with 10ml, add 50ml chondrocyte induction substratum in rolling bottle, full dose changes liquid 3 times weekly, and half amount changes liquid 1 time, is placed in 37 DEG C, 5%CO
2cultivate 28 days in animal cell culture case.
Wherein, containing induced liquid in chondrocyte induction substratum, induced liquid composition is: the ITS+Premix of TGF-β, 10mmol/l dexamethasone of 10ng/ml, 70ug/ml xitix, 10mmol/l Sodium Glycerophosphate and 100ng/ml.
Embodiment 4
Be with the difference of step 2, the induced liquid composition adopted in this embodiment step 4 is:
The concentration of TGF-β is 10ng/ml, and the concentration of dexamethasone is 10mmol/l, and the concentration of xitix is 70ug/ml, the concentration of Sodium Glycerophosphate is 10mmol/l, the concentration of ITS+Premix is 100ng/ml, and the concentration of Regular Insulin is 6.25mg/ml, and the concentration of Transferrins,iron complexes is 6.25mg/ml.
Embodiment 5
Be with the difference of step 2, the induced liquid composition adopted in this embodiment step 4 is:
The concentration of TGF-β is 5ng/ml, the concentration of dexamethasone is 7mmol/l, the concentration of xitix is 50ug/ml, the concentration of Sodium Glycerophosphate is 5mmol/l, the concentration of ITS+Premix is 50ng/ml, the concentration of Regular Insulin is 5mg/ml, and the concentration of Transferrins,iron complexes is 5mg/ml, and the concentration of glutamine is 1mmoL/L.
Embodiment 6
Be with the difference of step 2, the induced liquid composition adopted in this embodiment step 4 is:
The concentration of TGF-β is 15ng/ml, the concentration of dexamethasone is 10mmol/l, the concentration of xitix is 100ug/ml, the concentration of Sodium Glycerophosphate is 15mmol/l, the concentration of ITS+Premix is 150ng/ml, the concentration of Regular Insulin is 10mg/ml, and the concentration of Transferrins,iron complexes is 10mg/ml, and the concentration of glutamine is 10mmoL/L.
For verifying the unusual effect of the three-dimensional culture method of stem cell provided by the invention further, be specifically described by following experiment and experimental data.
Test experience one, fluorescent dye anyway
The cell that detected object: embodiment 1-6 obtains
0,35 day time, in rolling bottle, get gel ball, be positioned in EP pipe (centrifuge tube), often pipe 1 gel ball.Wash with 0.9%NaCl.Prepare work dye liquor in advance: joined in 1ml0.9%NaCl by the CAM fluorescence dye of 1mg/mlPI and 1mg/ml of 2 μ L and mix.Often pipe adds 150-200 μ L working fluid, blows and beats gently, gel ball is suspended in dye liquor.EP pipe is placed in 37 DEG C, 5%CO
2animal cell culture case, lucifuge hatches 30min.Take out EP pipe, sucking-off dye liquor, twice, 0.9%NaCl detergent gel ball.Gel ball is placed on slide glass, fluorescence microscope.Dead cell excites and takes on a red color under green fluorescence irradiates, and viable cell excites in green under blue-fluorescence irradiates, and observation is taken pictures.
Result: in encapsulation process, CaCl
2part necrocytosis can be caused etc. chemical substance.35 days time, in the gel ball of the method, seldom there is dead cell.
Test experience two, MTT (tetrazolium bromide) detect
Detected object
The chondrocyte that test set-embodiment of the present invention 1-6 obtains;
In control group-prior art, by the chondrocyte that mescenchymal stem cell and chondrocyte's Dual culture mode obtain.
The plastosome of viable cell can produce succinodehydrogenase, and it is water insoluble that this enzyme can make MTT be reduced to, and be but dissolved in the first hairpin of DMSO (dimethyl sulfoxide (DMSO)), dead cell then can not.Respectively following operation is performed to experimental group and control group: 3 gel balls getting 0,7,14,21,28 and 35 day, are divided in 3 EP pipes (n=3) respectively, add the substratum that the MTT of the 5mg/ml of 40 μ L and 200 μ L is fresh.And blow and beat gently, gel ball is suspended in dye liquor.Lucifuge in 37 DEG C, 5%CO
2hatch in animal cell culture case 4h reaction terminate after, remove supernatant, gel ball smashed, add the DMSO of 300 μ L, vibration, 10000rpm high speed centrifugation 5min.Draw 200 μ L supernatants, be transferred to 96 orifice plates, use microplate reader to survey light absorption value (OD value) at 490nm place.
Table 1
| OD value | 0d | 7d | 14d | 21d | 28d | 35d |
| Embodiment 1 | 0.61±0.11 | 0.60±0.12 | 0.59±0.13 | 0.57±0.12 | 0.56±0.18 | 0.53±0.11 |
| Embodiment 2 | 0.61±0.11 | 0.68±0.15 | 0.64±0.13 | 0.63±0.19 | 0.61±0.13 | 0.64±0.17 |
| Embodiment 3 | 0.61±0.11 | 0.56±0.14 | 0.54±0.12 | 0.47±0.15 | 0.42±0.13 | 0.40±0.16 |
| Embodiment 4 | 0.61±0.11 | 0.63±0.12 | 0.61±0.13 | 0.59±0.12 | 0.58±0.18 | 0.62±0.11 |
| Embodiment 5 | 0.61±0.11 | 0.65±0.12 | 0.63±0.13 | 0.61±0.12 | 0.59±0.18 | 0.63±0.11 |
| Embodiment 6 | 0.61±0.11 | 0.67±0.12 | 0.64±0.13 | 0.62±0.12 | 0.59±0.18 | 0.63±0.11 |
| Control group | 0.61±0.12 | 0.55±0.13 | 0.51±0.13 | 0.45±0.11 | 0.43±0.16 | 0.39±0.12 |
Detected result: OD value is larger, the content representing first hairpin in solution is higher, also illustrates simultaneously, impels MTT enzymolysis to be that the succinodehydrogenase that first is worn in one's hair is more, and the quantity of secretion succinodehydrogenase viable cell is more.From data in table 1, in embodiment of the present invention 1-3, OD value is all greater than control group, illustrates thus, and the cytoactive that three-dimensional culture method obtains of stem cell provided by the invention is comparatively strong, and quantity is more.
Test experience three, GAG (glycosaminoglycan) assay
Detected object:
The chondrocyte that experimental group-embodiment 1-6 obtains;
In control group-prior art, by the chondrocyte that mescenchymal stem cell and chondrocyte's Dual culture mode obtain.
Respectively following operation is performed to the chondrocyte in experimental group and control group:
1), sample preparation: each 9 of the gel ball getting 0,7,14,21,28 and 35 day, is divided into 3 groups and is placed in EP pipe (n=3).Add 300 μ L papoids, 60 DEG C of water-baths are spent the night.-20 DEG C of freezen protective.During use, take out sample, room temperature is melted, vibration, and the centrifugal 5min of 4000rpm gets supernatant and is transferred to new EP and manages.
2) Chs (chondroitin sulfate, the covalently bound class glycosaminoglycan forming proteoglycan on protein, is extensively present in animal cartilage tissue) standard model working fluid production standard curve, is got.
Making method:
Precision weighing chondroitin sulfate standard substance are appropriate, make the 0.5mg/ml aqueous solution, get six colorimetric cylinders once to number, No. 0 is blank, add 0.5ml distilled water, liquid 0.10 drawn respectively by 1-5 pipe, 0.20, 0.30, 0.40, 0.50ml is placed in 50ml colorimetric cylinder, mend to 0.5ml with distilled water, add sulfuric acid respectively: each 6ml of sulfuric acid liquid of water (2:1), limit edged shake, then be placed in 95 DEG C of water and heat 60min, after taking-up is chilled to room temperature, measure OD value, as shown in table 2 below with shown in Fig. 1, in Fig. 1, ordinate zou is OD value, X-coordinate is chondroitin sulfate standard concentration.
Table 2
3), get DMMB (1,9-dimethylated methene base the is blue) solution that 50 μ L samples join 2ml, mixing, prevents bubble.Use ultraviolet spectrophotometer to survey 0D value at 525nm place, substitute into typical curve equation y=0.9930x+0.0041, standard deviation R^2=0.9991, calculates GAG content (μ g/ pearl).
Table 3
| Detected object | 0d | 7d | 14d | 21d | 28d | 35d |
| Embodiment 1 | 7.3±0.7 | 8.2±0.6 | 8.9±0.9 | 9.3±0.6 | 10.2±0.5 | 9.1±0.3 |
| Embodiment 2 | 7.3±0.7 | 8.4±0.3 | 9.0±0.5 | 9.5±0.5 | 11.4±0.6 | 10.2±0.4 |
| Embodiment 3 | 7.3±0.7 | 7.6±0.8 | 7.9±0.7 | 8.2±0.5 | 7.8±0.4 | 7.3±0.2 |
| Embodiment 4 | 7.3±0.7 | 8.3±0.3 | 9.1±0.5 | 9.8±0.6 | 11.2±0.5 | 10.1±0.3 |
| Embodiment 5 | 7.3±0.7 | 7.8±0.4 | 8.2±0.7 | 8.5±0.9 | 9.7±0.2 | 8.6±0.6 |
| Embodiment 6 | 7.3±0.8 | 8.5±0.5 | 9.6±0.6 | 10.7±0.9 | 12.2±0.3 | 11.3±0.9 |
| Control group | 7.3±0.6 | 7.5±0.8 | 7.8±0.9 | 8.1±0.5 | 7.6±0.7 | 7.0±0.4 |
As shown in Table 3, the OD value in embodiment 1-6 provided by the invention is all greater than control group, illustrates thus, and GAG contained in chondrocyte in the present invention is more, and chondrocyte's quantity is more, and activity is stronger.
In sum, the three-dimensional method for inducing and cultivating of mescenchymal stem cell provided by the invention, by utilizing the three-D space structure of Lalgine colloid, for mescenchymal stem cell provides a microenvironment simulating tumor growth, expand the growing space of mescenchymal stem cell, and exchange with nutrient solution and intercellular PM signals, promote the Growth and Differentiation of mescenchymal stem cell, improve transformation efficiency, cell activity enhancing; Meanwhile, compared to Dual culture mode of the prior art, not only eliminate the process obtaining chondrocyte source, simple to operate, easily separated mescenchymal stem cell and chondrocyte, and, relatively shorten the inducing culture time of mescenchymal stem cell, to obtain cartilage cell activity comparatively strong, quantity is more.
Below by reference to the accompanying drawings embodiments of the invention are described; but the present invention is not limited to above-mentioned embodiment; above-mentioned embodiment is only schematic; instead of it is restrictive; those of ordinary skill in the art is under enlightenment of the present invention; do not departing under the ambit that present inventive concept and claim protect, also can make a lot of form, these all belong within protection of the present invention.
Claims (10)
1. a three-dimensional method for inducing and cultivating for mescenchymal stem cell, is characterized in that, comprise the following steps:
Obtain mescenchymal stem cell;
After mescenchymal stem cell is mixed with the alginates of solubility, then mix with the divalent-metal ion salts solution except magnesium ion, to forming gel ball;
With inductor, inducing culture is carried out to the mescenchymal stem cell in gel ball.
2. the three-dimensional method for inducing and cultivating of mescenchymal stem cell according to claim 1, it is characterized in that, the forming process of described gel ball is: after being mixed with the alginates of solubility by described mescenchymal stem cell, more at the uniform velocity instills in the divalent-metal ion salts solution except magnesium ion.
3. the three-dimensional method for inducing and cultivating of mescenchymal stem cell according to claim 1, is characterized in that, described alginates is sodium alginate, and before mixing with described mescenchymal stem cell, the concentration of sodium alginate is 2%-10%W/V.
4. the three-dimensional method for inducing and cultivating of mescenchymal stem cell according to claim 1, is characterized in that, described divalent-metal ion salts solution is calcium chloride solution, and the concentration of calcium chloride is 1%-5%W/V.
5. the three-dimensional method for inducing and cultivating of mescenchymal stem cell according to claim 1, is characterized in that, before carrying out inducing culture, also comprises the step of cleaning gel ball to the mescenchymal stem cell in described gel ball.
6. the three-dimensional method for inducing and cultivating of mescenchymal stem cell according to claim 5, is characterized in that, adopts basic medium cleaning gel ball.
7. the three-dimensional method for inducing and cultivating of mescenchymal stem cell according to claim 1, it is characterized in that, described inductor comprises the ITS+Premix of TGF-β, 7-10mmol/l dexamethasone of 5-15ng/ml, 50-100ug/ml xitix, 5-15mmol/l Sodium Glycerophosphate and 50-150ng/ml.
8. the three-dimensional method for inducing and cultivating of mescenchymal stem cell according to claim 7, is characterized in that, described inductor also comprises Regular Insulin and Transferrins,iron complexes, and the concentration of described Regular Insulin is 5-10mg/ml, and the concentration of Transferrins,iron complexes is 5-10mg/ml.
9. the three-dimensional method for inducing and cultivating of mescenchymal stem cell according to claim 8, is characterized in that, described inductor also comprises glutamine, and the concentration of described glutamine is 1-10mmoL/L.
10. the three-dimensional method for inducing and cultivating of mescenchymal stem cell according to claim 1, is characterized in that, carries out in bio-reactor the inducing culture process of the mescenchymal stem cell in described gel ball.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510502814.XA CN105132365A (en) | 2015-08-17 | 2015-08-17 | Three-dimensional induction culture method of mesenchymal stem cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510502814.XA CN105132365A (en) | 2015-08-17 | 2015-08-17 | Three-dimensional induction culture method of mesenchymal stem cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105132365A true CN105132365A (en) | 2015-12-09 |
Family
ID=54717938
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510502814.XA Pending CN105132365A (en) | 2015-08-17 | 2015-08-17 | Three-dimensional induction culture method of mesenchymal stem cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105132365A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108624581A (en) * | 2018-05-15 | 2018-10-09 | 中国科学院苏州生物医学工程技术研究所 | A kind of microballoon and brainpower insufflation system of mescenchymal stem cell materials for binding biological |
| CN111195370A (en) * | 2018-11-16 | 2020-05-26 | 上海交通大学医学院附属第九人民医院 | High-magnesium microenvironment bone marrow stem cell microsphere carrier and preparation method and application thereof |
| CN111321118A (en) * | 2020-01-14 | 2020-06-23 | 河南省银丰生物工程技术有限公司 | Method for in-vitro amplification of cord blood hematopoietic stem cells |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1837358A (en) * | 2006-04-17 | 2006-09-27 | 大连理工大学 | A method for expanding hematopoietic stem cells under three-dimensional conditions |
| CN101407786A (en) * | 2008-09-10 | 2009-04-15 | 广州市创伤外科研究所 | Method for cultivating stereo proliferative cartilage cell |
| CN103849593A (en) * | 2012-12-06 | 2014-06-11 | 中国科学院大连化学物理研究所 | 3D (Three-Dimensional) co-culture method for magnetic separated cells |
-
2015
- 2015-08-17 CN CN201510502814.XA patent/CN105132365A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1837358A (en) * | 2006-04-17 | 2006-09-27 | 大连理工大学 | A method for expanding hematopoietic stem cells under three-dimensional conditions |
| CN101407786A (en) * | 2008-09-10 | 2009-04-15 | 广州市创伤外科研究所 | Method for cultivating stereo proliferative cartilage cell |
| CN103849593A (en) * | 2012-12-06 | 2014-06-11 | 中国科学院大连化学物理研究所 | 3D (Three-Dimensional) co-culture method for magnetic separated cells |
Non-Patent Citations (3)
| Title |
|---|
| 张东等: "藻酸盐三维细胞培养在骨组织工程中应用的研究进展", 《生物技术通报》 * |
| 王琦: "间充质干细胞在海藻酸钠水凝胶中生长、分化及其与软骨细胞共培养的研究", 《中国优秀硕士学位论文全文数据库基础科学辑》 * |
| 陈松等: "TGF-β3、BMP-2及地塞米松诱导兔滑膜MSCs成软骨分化的研究", 《中国修复重建外科杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108624581A (en) * | 2018-05-15 | 2018-10-09 | 中国科学院苏州生物医学工程技术研究所 | A kind of microballoon and brainpower insufflation system of mescenchymal stem cell materials for binding biological |
| CN111195370A (en) * | 2018-11-16 | 2020-05-26 | 上海交通大学医学院附属第九人民医院 | High-magnesium microenvironment bone marrow stem cell microsphere carrier and preparation method and application thereof |
| CN111321118A (en) * | 2020-01-14 | 2020-06-23 | 河南省银丰生物工程技术有限公司 | Method for in-vitro amplification of cord blood hematopoietic stem cells |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yang et al. | Degradable photothermal bioactive glass composite hydrogel for the sequential treatment of tumor-related bone defects: From anti-tumor to repairing bone defects | |
| Song et al. | The homing of bone marrow MSCs to non-osseous sites for ectopic bone formation induced by osteoinductive calcium phosphate | |
| Wu et al. | In situ controlled release of stromal cell-derived factor-1α and antimiR-138 for on-demand cranial bone regeneration | |
| EP0175286B1 (en) | In vitro cell culture system and method | |
| Rocha et al. | Encapsulation of adipose-derived stem cells and transforming growth factor-β1 in carrageenan-based hydrogels for cartilage tissue engineering | |
| Ishaug-Riley et al. | Three-dimensional culture of rat calvarial osteoblasts in porous biodegradable polymers | |
| Malda et al. | Low oxygen tension stimulates the redifferentiation of dedifferentiated adult human nasal chondrocytes | |
| Lu et al. | Bone tissue engineering by using a combination of polymer/Bioglass composites with human adipose-derived stem cells | |
| CN106754668B (en) | Stem cell culture solution and injection | |
| CN104147641B (en) | A kind of for personalized bone renovating material and its preparation method | |
| CN105132368A (en) | Cell inducing culture medium and method for inducing and culturing mesenchyma stem cells | |
| CN102985534A (en) | Culture method for massive expansion of hair follicle stem cells in vitro | |
| CN105288737A (en) | Tissue engineering cartilage composite scaffold and preparation method thereof | |
| CN102416201A (en) | Preparation method and application of a transforming growth factor composite scaffold for cartilage repair in vivo | |
| Li et al. | Fabrication and evaluation of bone morphogenetic protein-2 microspheres coated black phosphorus nanosheets@ polylactic-glycolic acid copolymers scaffold: A multifunctional antibacterial photothermal scaffold for bone regeneration | |
| CN110475856A (en) | Use the cell culture of nanofiber | |
| CN109758606A (en) | A kind of rgd peptide modification chitosan/hydroxyapatite compound rest and preparation method thereof | |
| CN104046587A (en) | Method for adjusting and controlling in vitro three-dimensional directed differentiation of stem cells | |
| CN105013014A (en) | Preparation method and application of acellular matrix biological material | |
| CN106350482A (en) | Culture system and application thereof as well as method for culturing cartilage cells | |
| CN105132365A (en) | Three-dimensional induction culture method of mesenchymal stem cells | |
| Findeisen et al. | Cell spheroids are as effective as single cells suspensions in the treatment of critical-sized bone defects | |
| Chen et al. | 3D-printed mesoporous bioactive glass scaffolds for enhancing bone repair via synergetic angiogenesis and osteogenesis | |
| CN101920045B (en) | A kind of gelatin-chitosan-hyaluronic acid-heparan sulfate composite three-dimensional scaffold and its preparation method | |
| US20150344842A1 (en) | Method for production of decellularized biological material and the decellularized biological material prepared therefrom |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20151209 |