CN116769702A - Improved culture medium with definite chemical components for inducing in-vitro matrix protein generation and application thereof - Google Patents
Improved culture medium with definite chemical components for inducing in-vitro matrix protein generation and application thereof Download PDFInfo
- Publication number
- CN116769702A CN116769702A CN202310717527.5A CN202310717527A CN116769702A CN 116769702 A CN116769702 A CN 116769702A CN 202310717527 A CN202310717527 A CN 202310717527A CN 116769702 A CN116769702 A CN 116769702A
- Authority
- CN
- China
- Prior art keywords
- medium
- differentiation medium
- vitro
- extracellular matrix
- matrix protein
- 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
- 238000000338 in vitro Methods 0.000 title claims abstract description 65
- 230000001976 improved effect Effects 0.000 title claims abstract description 46
- 239000000126 substance Substances 0.000 title claims abstract description 39
- 230000001939 inductive effect Effects 0.000 title claims abstract description 35
- 239000001963 growth medium Substances 0.000 title abstract description 56
- 108060003393 Granulin Proteins 0.000 title description 107
- 210000004027 cell Anatomy 0.000 claims abstract description 78
- 230000006698 induction Effects 0.000 claims abstract description 45
- 210000002966 serum Anatomy 0.000 claims abstract description 45
- 210000000329 smooth muscle myocyte Anatomy 0.000 claims abstract description 38
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 33
- 230000014509 gene expression Effects 0.000 claims abstract description 24
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 23
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 claims abstract description 21
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 claims abstract description 21
- 210000002950 fibroblast Anatomy 0.000 claims abstract description 21
- 235000013372 meat Nutrition 0.000 claims abstract description 20
- 230000028327 secretion Effects 0.000 claims abstract description 20
- 210000002744 extracellular matrix Anatomy 0.000 claims abstract description 18
- 238000004113 cell culture Methods 0.000 claims abstract description 17
- 230000004069 differentiation Effects 0.000 claims abstract 19
- 239000002609 medium Substances 0.000 claims description 83
- 230000001114 myogenic effect Effects 0.000 claims description 53
- 239000000203 mixture Substances 0.000 claims description 25
- 239000012592 cell culture supplement Substances 0.000 claims description 16
- 230000001965 increasing effect Effects 0.000 claims description 13
- 102000008186 Collagen Human genes 0.000 claims description 11
- 108010035532 Collagen Proteins 0.000 claims description 11
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 10
- 108010009583 Transforming Growth Factors Proteins 0.000 claims description 5
- 102000009618 Transforming Growth Factors Human genes 0.000 claims description 5
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 claims description 4
- 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 4
- YZXBAPSDXZZRGB-DOFZRALJSA-N arachidonic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O YZXBAPSDXZZRGB-DOFZRALJSA-N 0.000 claims description 4
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 4
- 150000001875 compounds Chemical class 0.000 claims description 4
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 claims description 4
- 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 description 4
- 150000002632 lipids Chemical class 0.000 claims description 4
- SECPZKHBENQXJG-FPLPWBNLSA-N palmitoleic acid Chemical compound CCCCCC\C=C/CCCCCCCC(O)=O SECPZKHBENQXJG-FPLPWBNLSA-N 0.000 claims description 4
- -1 small molecule compounds Chemical class 0.000 claims description 4
- 229960005322 streptomycin Drugs 0.000 claims description 4
- 230000003054 hormonal effect Effects 0.000 claims description 3
- OYHQOLUKZRVURQ-NTGFUMLPSA-N (9Z,12Z)-9,10,12,13-tetratritiooctadeca-9,12-dienoic acid Chemical compound C(CCCCCCC\C(=C(/C\C(=C(/CCCCC)\[3H])\[3H])\[3H])\[3H])(=O)O OYHQOLUKZRVURQ-NTGFUMLPSA-N 0.000 claims description 2
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 claims description 2
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 claims description 2
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 claims description 2
- 108010088751 Albumins Proteins 0.000 claims description 2
- 102000009027 Albumins Human genes 0.000 claims description 2
- ZAKOWWREFLAJOT-CEFNRUSXSA-N D-alpha-tocopherylacetate Chemical compound CC(=O)OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C ZAKOWWREFLAJOT-CEFNRUSXSA-N 0.000 claims description 2
- 102000004877 Insulin Human genes 0.000 claims description 2
- 108090001061 Insulin Proteins 0.000 claims description 2
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 claims description 2
- 102000015696 Interleukins Human genes 0.000 claims description 2
- 108010063738 Interleukins Proteins 0.000 claims description 2
- 239000002211 L-ascorbic acid Substances 0.000 claims description 2
- 235000000069 L-ascorbic acid Nutrition 0.000 claims description 2
- TUNFSRHWOTWDNC-UHFFFAOYSA-N Myristic acid Natural products CCCCCCCCCCCCCC(O)=O TUNFSRHWOTWDNC-UHFFFAOYSA-N 0.000 claims description 2
- 239000005642 Oleic acid Substances 0.000 claims description 2
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 claims description 2
- 235000021314 Palmitic acid Nutrition 0.000 claims description 2
- 235000021319 Palmitoleic acid Nutrition 0.000 claims description 2
- 235000021355 Stearic acid Nutrition 0.000 claims description 2
- 108090000901 Transferrin Proteins 0.000 claims description 2
- 102000004338 Transferrin Human genes 0.000 claims description 2
- DTOSIQBPPRVQHS-PDBXOOCHSA-N alpha-linolenic acid Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(O)=O DTOSIQBPPRVQHS-PDBXOOCHSA-N 0.000 claims description 2
- 235000020661 alpha-linolenic acid Nutrition 0.000 claims description 2
- 229940114079 arachidonic acid Drugs 0.000 claims description 2
- 235000021342 arachidonic acid Nutrition 0.000 claims description 2
- 229960005070 ascorbic acid Drugs 0.000 claims description 2
- 229940107161 cholesterol Drugs 0.000 claims description 2
- 235000012000 cholesterol Nutrition 0.000 claims description 2
- SECPZKHBENQXJG-UHFFFAOYSA-N cis-palmitoleic acid Natural products CCCCCCC=CCCCCCCCC(O)=O SECPZKHBENQXJG-UHFFFAOYSA-N 0.000 claims description 2
- ZAKOWWREFLAJOT-UHFFFAOYSA-N d-alpha-Tocopheryl acetate Natural products CC(=O)OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C ZAKOWWREFLAJOT-UHFFFAOYSA-N 0.000 claims description 2
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 claims description 2
- 229940031098 ethanolamine Drugs 0.000 claims description 2
- 229940125396 insulin Drugs 0.000 claims description 2
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 claims description 2
- 229960004488 linolenic acid Drugs 0.000 claims description 2
- KQQKGWQCNNTQJW-UHFFFAOYSA-N linolenic acid Natural products CC=CCCC=CCC=CCCCCCCCC(O)=O KQQKGWQCNNTQJW-UHFFFAOYSA-N 0.000 claims description 2
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 claims description 2
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 claims description 2
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 claims description 2
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 claims description 2
- 235000021313 oleic acid Nutrition 0.000 claims description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 2
- 229920000053 polysorbate 80 Polymers 0.000 claims description 2
- FASDKYOPVNHBLU-ZETCQYMHSA-N pramipexole Chemical compound C1[C@@H](NCCC)CCC2=C1SC(N)=N2 FASDKYOPVNHBLU-ZETCQYMHSA-N 0.000 claims description 2
- 229960003089 pramipexole Drugs 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 229960001471 sodium selenite Drugs 0.000 claims description 2
- 239000011781 sodium selenite Substances 0.000 claims description 2
- 235000015921 sodium selenite Nutrition 0.000 claims description 2
- 229940042585 tocopherol acetate Drugs 0.000 claims description 2
- 239000012581 transferrin Substances 0.000 claims description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims 2
- 102000013275 Somatomedins Human genes 0.000 claims 1
- 239000012913 medium supplement Substances 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 13
- 230000000694 effects Effects 0.000 abstract description 7
- 230000035800 maturation Effects 0.000 abstract 1
- 230000014616 translation Effects 0.000 description 38
- 238000004519 manufacturing process Methods 0.000 description 22
- 238000013424 sirius red staining Methods 0.000 description 11
- 229920001436 collagen Polymers 0.000 description 10
- 239000013641 positive control Substances 0.000 description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 239000007640 basal medium Substances 0.000 description 8
- 238000011002 quantification Methods 0.000 description 8
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 7
- 239000001044 red dye Substances 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 6
- 239000012091 fetal bovine serum Substances 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 102000012422 Collagen Type I Human genes 0.000 description 5
- 108010022452 Collagen Type I Proteins 0.000 description 5
- 230000004663 cell proliferation Effects 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 238000009472 formulation Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 230000035755 proliferation Effects 0.000 description 5
- 210000002460 smooth muscle Anatomy 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- 239000002033 PVDF binder Substances 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 3
- 229930040373 Paraformaldehyde Natural products 0.000 description 3
- 239000006180 TBST buffer Substances 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 230000010261 cell growth Effects 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 229920002866 paraformaldehyde Polymers 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 239000013589 supplement Substances 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 238000010023 transfer printing Methods 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 2
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 2
- 238000011481 absorbance measurement Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 102000035118 modified proteins Human genes 0.000 description 2
- 108091005573 modified proteins Proteins 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 102000000503 Collagen Type II Human genes 0.000 description 1
- 108010041390 Collagen Type II Proteins 0.000 description 1
- 102000001187 Collagen Type III Human genes 0.000 description 1
- 108010069502 Collagen Type III Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000016359 Fibronectins Human genes 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 102000014429 Insulin-like growth factor Human genes 0.000 description 1
- 102000008934 Muscle Proteins Human genes 0.000 description 1
- 108010074084 Muscle Proteins Proteins 0.000 description 1
- 102000016611 Proteoglycans Human genes 0.000 description 1
- 108010067787 Proteoglycans Proteins 0.000 description 1
- 102000009523 Transcription Factor 4 Human genes 0.000 description 1
- 108010048992 Transcription Factor 4 Proteins 0.000 description 1
- 102000013127 Vimentin Human genes 0.000 description 1
- 108010065472 Vimentin Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 210000001789 adipocyte Anatomy 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 239000012598 cell culture matrix Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 210000004087 cornea Anatomy 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 235000019197 fats Nutrition 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000012606 in vitro cell culture Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 210000003041 ligament Anatomy 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000000663 muscle cell Anatomy 0.000 description 1
- 210000001087 myotubule Anatomy 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000013587 production medium Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000008263 repair mechanism Effects 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- 210000005048 vimentin Anatomy 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides an improved culture medium with definite chemical components for inducing in-vitro extracellular matrix secretion and application thereof, which can help smooth muscle cells and fibroblasts to carry out extracellular matrix induction differentiation in vitro more safely, cheaply and efficiently. Compared with the existing serum-containing extracellular matrix secretion culture medium, the improved culture medium can remarkably improve the secretion level of the extracellular matrix of cells in the differentiation and maturation stage, remarkably improve the gene expression level of Col 1A2 and alpha-SMA and remarkably improve the protein expression level of alpha-SMA, can achieve the effect of the existing serum-containing extracellular matrix secretion culture medium in a certain concentration range, and provides a more efficient, low-cost and safe method for forming more extracellular matrix proteins for cell culture meat.
Description
Technical Field
The invention belongs to the technical field of stem cells and animal cell culture meat, and particularly relates to an improved culture medium with definite chemical components for inducing in-vitro matrix protein generation and application thereof.
Background
The cell culture meat is used for producing edible meat through in vitro cell culture according to the growth and damage repair mechanism of animal meat, and is a future food production technology with the potential of realizing efficient and green production of meat. The technology can make up for the gap of the increasing meat demand, and avoids the risks of the traditional meat in aspects such as resource utilization, environmental protection, animal welfare, epidemic control and the like.
Production of cell culture meat requires preferential production of muscle proteins, fats and matrix proteins. The edible part of conventional meat is not formed solely by superposition of cell numbers, but rather by a complex spatial arrangement of extracellular matrix components, forming a specific texture of the meat. Wherein, the matrix protein is a collagen fiber network structure which mainly comprises type I and type III collagen and fibronectin and various proteoglycans, and wraps muscle cells and fat cells, thereby playing an important role in forming a complex space structure of meat. In addition, matrix proteins are also an important source of meat aroma and juicy flavor. The ability of fibroblasts, myofibers, smooth muscle cells to induce production of matrix proteins in vitro can be used for the production of cell culture matrix proteins. Therefore, the selection of a good culture medium for promoting the generation of myogenic cell matrix proteins is a key ring of cell culture protein production, directly influences the formation and quality of the proteins, and influences the nutritional value and the organoleptic properties of cell culture meat.
The formula of the culture medium for inducing the generation of matrix protein by the in vitro myogenic cells, which is commonly used at present, is that a DMEM basic culture medium is added with 2vol% of fetal calf serum and 5ng/mL of transforming growth factor, and 1vol% of diabody can be added to prevent microbial pollution. The medium is capable of supporting the basic cell matrix protein production process. However, the culture medium has the problems of undefined chemical components, unstable components of different batches, easy pathogen pollution such as viruses, high cost, limited supply and the like due to the addition of the fetal bovine serum. This means that the serum-containing medium cannot be used for producing cell culture meat in a large scale with high efficiency, stability and low cost, and seriously hinders the progress of industrialization of the cell culture meat. Therefore, it is important to develop a serum-free medium with definite chemical composition, which can promote the high-efficiency secretion of matrix proteins by myogenic cells.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention aims to replace the traditional culture medium for inducing the generation of the myogenic cell matrix protein and provide a set of culture medium for generating the myogenic cell matrix protein in vitro with definite chemical components and an application method thereof.
It is a first object of the present invention to provide an improved medium of defined chemical composition for inducing matrix protein production in vitro, said myogenic cells being smooth muscle cells or fibroblasts, said improved medium comprising a basal medium and a cell culture supplement, said improved medium being serum-free.
The serum-free means that no animal serum including horse serum, fetal bovine serum, human serum and the like is added.
The improved culture medium replaces serum in the traditional myogenic cell matrix protein production medium by adding cell culture supplement factors.
Further, the basal medium is selected from one of DMEM medium, MEM medium, DMEM/F12 medium, F10 medium and F12 medium.
Further, the cell culture supplement factor comprises one or more of hormonal compounds, protein substances, lipids and small molecule compounds.
Further, the hormone compound is insulin;
the protein substance is selected from one or more of transferrin, albumin, insulin-like growth factor, interleukin and transforming growth factor;
the lipid is selected from one or more of Tween 80, arachidonic acid, cholesterol, vitamin E acetate, linoleic acid, linolenic acid, tetradecanoic acid, oleic acid, palmitic acid, palmitoleic acid, pramipexole F68 and octadecanoic acid;
the small molecule compound is selected from one or a combination of more of sodium selenite, ethanolamine and L-ascorbic acid.
Further, in the improved culture medium, the total concentration range of the cell culture supplement factor is not less than 5.06mg/mL;
preferably, the total concentration range of the cell culture supplement factor addition is 5.06mg/mL-20.25mg/mL;
further, the concentration of any cell culture supplement factor is not lower than 1ng/mL;
preferably, the concentration of any cell culture supplement factor ranges from 1ng/mL to 20mg/mL;
it is a second object of the present invention to provide the use of the improved culture medium as described above for inducing myogenic cells to produce extracellular matrix secretion in vitro.
In a particular embodiment, the myogenic cells are smooth muscle cells or fibroblasts.
Further, the whole process of inducing the generation of matrix protein in vitro adopts an improved culture medium with a unique formula.
Furthermore, the improved culture medium can induce the secretion of myogenic cells in vitro to produce extracellular matrix proteins, and concretely can improve the secretion efficiency, secretion capacity and secretion level of smooth muscle cells and fibroblasts in the process of generating the extracellular matrix proteins.
Furthermore, the improved culture medium can improve the secretion efficiency of matrix proteins of smooth muscle cells and fibroblasts in the process of in vitro induction of matrix proteins, and the induced smooth muscle cells and fibroblasts have higher absorbance values after being quantified by sirius red staining in a shorter day.
Furthermore, the improved culture medium can improve the gene expression quantity of the matrix protein generation related genes Col 1A2 and alpha-SMA and can improve the protein expression quantity of the matrix protein generation related proteins Collagen I and alpha-SMA.
After smooth muscle cells are induced in vitro for 1, 2, 3, 4 and 5 days by adopting the improved culture medium, the matrix protein generation efficiency is respectively improved by 1.19, 1.34, 1.07 and 1.12 times through the quantitative measurement of sirius red, and the matrix protein generation amount of the induced 2 nd day and 3 rd day is higher than that of the induced 4 th day of the existing serum-containing culture medium. The matrix protein generation related gene Col 1A2 was increased 1.69-fold on day 3 of induction, and α -SMA was increased 1.22-fold on day 6 of induction. On the 5 th day of induction, the expression level of matrix protein generation related protein alpha-SMA is improved by 2.05 times, and the expression level of Collagen I is improved by 1.94 times.
The content of extracellular matrix components of the fibroblasts is obviously better than or equal to that of the conventional serum-containing culture medium on days 1, 2 and 3 after the in-vitro induction of the fibroblasts by adopting the improved culture medium, and the content of matrix protein production on day 2 is higher than that on day 4 after the induction of the conventional serum-containing culture medium.
The culture medium of the invention can be used together with conventional additives of cell culture media, such as penicillin-streptomycin diabodies and the like. The technical scheme of the invention has the beneficial effects that:
the invention relates to an improved culture medium with definite chemical components for generating myogenic cell in vitro matrix protein, which replaces serum in the culture medium by adding cell culture supplement factors, thereby avoiding a plurality of problems existing in serum use. Meanwhile, in the process of inducing the generation of the myogenic cell matrix protein in vitro, the matrix protein generation efficiency and capability can be obviously improved, and the development of the cell culture meat industry is assisted.
Compared with the existing serum-containing culture medium for generating the smooth muscle cell in-vitro matrix protein, the modified culture medium for generating the smooth muscle cell in-vitro matrix protein, which is provided by the invention, has the advantages that after induction, the generated collagen is subjected to sirius red staining quantification, and the light absorption value is obviously higher than that of a serum-containing culture medium group; from the aspect of the expression level of genes related to matrix protein generation, the improved culture medium disclosed by the invention can be used for remarkably improving the gene expression level of Col 1A2 after inducing smooth muscle cells for 3 days; after induction for 5 days, the gene expression level of alpha-SMA was significantly increased. From the aspect of the expression level of matrix protein generation related protein, the improved culture medium provided by the invention can obviously improve the protein expression level of alpha-SMA for smooth muscle cells after induction for 5 days, can improve the generation amount of matrix protein and can accelerate the generation speed of matrix protein. In addition, the concentration range of the cell supplement factor added in the improved culture medium is larger, and the effect of a serum-containing control group can be achieved under the lower concentration.
Compared with the conventional serum-containing culture medium for in-vitro induction of matrix protein production by the fibroblasts, the modified culture medium with definite chemical components for in-vitro induction of matrix protein production can remarkably improve the secretion efficiency of the collagen, can improve the production amount of the matrix protein and can accelerate the production speed of the matrix protein after the collagen produced by the modified culture medium is subjected to sirius red staining quantification on days 1, 2 and 3 after induction, and the absorbance value of the collagen is remarkably superior to or equal to that of the conventional serum-containing culture medium, and the absorbance value of the collagen on day 2 after induction is higher than that of the serum-containing culture medium on day 4 after induction.
Drawings
FIG. 1 is a photograph of a bright field photograph of a conventional serum-containing medium for inducing the production of myogenic cell in vitro matrix proteins and a modified medium with definite chemical composition according to the present invention for inducing smooth muscle cell in vitro matrix proteins at days 0, 1, 2, 3, 4, 5.
FIG. 2 is a photograph of a bright field of a smooth muscle cell after red staining of cells on days 0, 1, 2, 3, 4, 5, using a conventional serum-containing medium for inducing the production of myogenic cell in vitro matrix proteins and a modified medium with definite chemical composition according to the present invention.
FIG. 3 is a statistical plot of absorbance measurements at 550nm after staining for 0, 1, 2, 3, 4, 5 days of cell sirius red, using a prior serum-containing medium for inducing myogenic cell in vitro matrix protein production and a modified medium of the present invention with a clear chemical composition.
FIG. 4 is a statistical plot of the expression of the present serum-containing medium and the modified medium of the present invention for inducing the production of the matrix protein in vitro of myogenic cells on days 0, 3, 5 CoL A2, α -SMA genes.
FIG. 5 shows the development of bands and grey scale analysis statistics of expression of the Collagen from the serum-containing medium of the present invention and the modified medium of the present invention for inducing the production of the matrix protein from the smooth muscle cells in vitro on days 0, 3 and 5.
FIG. 6 is a photograph of day 5 of bright field induction of the effect of different concentrations of cell culture supplementation factors in modified medium of defined chemical composition for inducing myogenic matrix protein production in vitro according to the invention on smooth muscle matrix protein production.
FIG. 7 is a photograph showing the effect of different cell culture supplement factor concentrations in modified medium with definite chemical composition for inducing myogenic matrix protein production in vitro on smooth muscle matrix protein production effect, and after induced sample collection on day 5 sirius red staining.
FIG. 8 quantitative statistical graphs of the red staining of sirius for sample collection on day 5 induced by the influence of different cell culture supplement factor concentrations in modified medium with definite chemical composition for inducing myogenic matrix protein production in vitro on smooth muscle matrix protein production effect.
FIG. 9 is a photograph of the bright field of day 0, 1, 2, 3, 4, 5 of the prior serum-containing medium and the modified medium of the present invention with definite chemical composition for inducing the production of matrix proteins in vitro of myogenic cells.
FIG. 10 is a photograph of a prior serum-containing medium for inducing the production of myogenic cell in vitro matrix protein and a modified medium with definite chemical composition according to the present invention for inducing the production of fibroblast in vitro matrix protein after red staining of cells of day 0, 1, 2, 3, 4, 5.
FIG. 11 is a statistical plot of absorbance measurements at 550nm after staining cell sirius red on days 0, 1, 2, 3, 4, 5 for induction of myogenic cell in vitro matrix protein production using a prior serum-containing medium and a modified medium of defined chemical composition of the invention.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to limit the invention in any way.
Example 1 smooth muscle cell in vitro induced matrix protein production and quantification of sirius red staining
The experiment is divided into two groups, namely a modified culture medium with definite components for inducing the generation of matrix proteins in vitro of the myogenic cells and a serum-containing culture medium method for inducing the generation of matrix proteins in vitro of the myogenic cells.
In this example, the smooth muscle cell proliferation medium formulations used in the proliferation stage were each 84vol% F12 basal medium, 15vol% fetal bovine serum, 1vol% penicillin-streptomycin diabody and 5ng/ml basic fibroblast growth factor was added.
In this example, the serum-containing medium (positive control) for inducing matrix protein production in vitro by existing myogenic cells was as follows: the basal formulation was 98vol% DMEM/F12 (1:1) basal medium, 2vol% fetal bovine serum, and 5ng/ml transforming growth factor was added.
The improved culture medium with definite chemical components comprises the following components: the DMEM/F12 basal medium was added with the cell culture cofactors shown in Table 1.
TABLE 1
The cells used in this example were piglet smooth muscle cells, and further, adherent cells.
The culture conditions used in this example were all CO 2 Culturing at 37deg.C in incubator, CO 2 The concentration of (C) was 5% (v/v).
The detection methods employed in this example, unless otherwise indicated, are all experimental methods, detection methods and preparation methods disclosed in the art.
Materials, reagents and the like used in this example are commercially available unless otherwise specified.
The specific treatment method comprises the following steps:
1) Cell inoculation: the piglet smooth muscle cells before P6 after sorting were inoculated into 3.5cm cell culture dishes at a cell density of 100000 cells/dish, and cultured with smooth muscle cell proliferation medium with 1 change of fluid every 2 days.
2) Induction of matrix protein production: after the cells proliferate to the whole culture dish (more than 90% density), the smooth muscle cell proliferation culture medium is sucked, and the culture medium is divided into two groups, and the serum-containing culture medium (positive control) for generating the matrix protein by the existing myogenic cells in vitro induction and the improved culture medium with definite chemical components for generating the matrix protein by the myogenic cells in vitro induction are respectively used for culturing, and liquid is changed for 1 time every 2 days. Cell growth was recorded by observation with an optical microscope.
3) Sirius red staining quantification: sampling on days 0 to 5 of induction matrix protein generation, fixing with 4%vol paraformaldehyde at normal temperature for 30min or at 4 ℃ for 12h, washing with pure water for 2 times, adding 1mL of sirius red dye liquor, dyeing for 1 hour, removing the sirius red dye liquor, washing with pure water for 2 times, and immediately taking a picture by using a high-flux instrument. 1mL of 0.1mM sodium hydroxide solution was added, and the mixture was shaken in a shaker for 5 minutes in a dark environment, and 200ul to 96 well plates were used after the completion of the shaking, and absorbance was measured at 550nm using an ELISA reader. Collagen I is the main component of matrix protein, and sirius red dye is easy to combine with alkaline groups in collagen molecules.
4) The results show that: compared with the existing serum-containing medium for in-vitro induction of matrix protein production by myogenic cells, the improved medium provided by the invention has the advantages that the matrix protein production content and time efficiency of smooth muscle cells can be improved, and more extracellular substances can be observed by open field photographing (figure 1). The quantitative result of sirius red staining shows that after induction, the content of extracellular matrix components of smooth muscle cells is extremely obviously better than that of the existing serum-containing culture medium, specifically, after in vitro induction of smooth muscle cells for 1, 2, 3, 4 and 5 days, the matrix protein generation efficiency is respectively improved by 1.19, 1.34, 1.07 and 1.12 times through the quantitative measurement of sirius red, and the matrix protein generation content of the induced smooth muscle cells is higher than that of the existing serum-containing culture medium for 4 days (figures 2 and 3). Thus, the modified cell culture medium of the present invention, which is clearly defined in terms of chemical composition, has an improved matrix protein production capacity of smooth muscle cells.
Example 2 detection of genes and protein levels associated with the induction of matrix protein production by piglet smooth muscle cells
The myogenic cell myogenic protein and cell specific marker protein alpha-SMA (alpha-smooth muscle actin) are specific products of myogenic cell activation, and the activated myogenic cell secretes a large amount of matrix protein. Collagen I is the major Collagen of tendons, skin, ligaments, cornea and many interstitial connective tissues. Thus, both genes and proteins can be used to characterize the matrix protein production capacity.
1) Gene level detection:
according to the treatment method of example 1, samples were taken on days 0, 3 and 5 of induction in step 2), and the gene expression amounts of Col 1A2 and alpha-SMA were measured on days 0, 3 and 5 of smooth muscle cells induced by the serum-containing medium (positive control) for the production of myogenic cell in vitro matrix protein and the modified medium for the production of myogenic cell in vitro matrix protein provided by the present invention, respectively, by real-time fluorescent quantitative PCR.
The result shows that compared with the existing serum-containing medium (positive control) for generating the myogenic cell in vitro matrix protein, the improved medium with definite chemical components for generating the myogenic cell in vitro matrix protein can remarkably improve the expression level of genes related to smooth muscle matrix protein generation, and specifically, after 3 days of induction, the improved medium remarkably improves the gene expression level of Col 1A 2; after induction for 5 days, the gene expression level of alpha-SMA was significantly increased. Specifically, the matrix protein production-related gene Col 1A2 was increased 1.69-fold on day 3 of induction, and α -SMA was increased 1.22-fold on day 5 of induction (FIG. 4). Thus, the modified medium of the present invention, which is clearly defined in terms of chemical composition, increases the matrix protein production capacity and level of smooth muscle cells.
2) Protein level:
according to the treatment method of example 1, samples were taken on days 0, 3 and 5 of induction in step 2), respectively, serum-containing medium (positive control) for the production of myogenic cell in vitro matrix protein and 2 discs of smooth muscle cells on days 0, 3 and 5 of induction with the modified medium for the production of myogenic cell in vitro matrix protein according to the invention, respectively, 100. Mu.L of RIPA (1 mM PMSF in addition) was added, and the cells were lysed on ice for 30min and collected at-20℃for storage. After 12000g centrifugation for 15 minutes, the supernatant was collected, the BCA kit for the Sieimer's flight was used for measurement and protein concentration, 5X loading buffer was added at 4:1 (V: V), and after mixing, the protein was denatured by heating at 95℃for 5 minutes, and stored at-80 ℃.
SDS-page gel electrophoresis: preparing electrophoresis buffer solution and transfer solution in advance (10% methanol is needed to be added to the transfer solution), under the condition that the electrophoresis buffer solution is over 12% of modified agarose precast gel plates, taking 10 mug modified proteins, respectively adding the modified proteins into the upper sample holes, setting two procedures of electrophoresis at 80V for 30min and electrophoresis at 120V for 60min, and observing whether the protein upper sample solution reaches the bottom of the precast plate.
Transferring: placing PVDF film into methanol for activation for about 10s, placing into transfer printing liquid for standby, placing according to sponge, 2 layers of filter paper, gel, activated PVDF film, 2 layers of filter paper and sponge, clamping by a transfer printing clamp, placing into an electrophoresis tank, adding the prepared transfer printing liquid for 90V, and operating for 90min.
Closing: the PVDF film after transfer was put into a blocking solution (5% skimmed milk powder prepared with TBST), and after blocking for 2 hours in a shaking table at room temperature, the blocking solution was sucked off.
Primary and secondary antibody incubation: collagenI, alpha-SMA, GAPDH primary antibody was diluted according to the specific conditions of the antibody and incubated at 4℃for 14-16h. The primary antibody is recovered after the incubation of the primary antibody is finished, and TBST is washed three times for 5min each time. The diluted secondary antibody was added and incubated for 2h, and after the completion, TBST was washed three times for 5min each time.
Developing: the PVDF film is covered in a dark place by using the developing solution, the developing solution is sucked after incubation for 5min, and the film is photographed under a gel imager. And gray scale analysis was performed using quality One analysis software. The reference protein used in this experiment was GADPH.
The result shows that compared with the existing serum-containing medium (positive control) for generating the myogenic cell in vitro matrix protein, the improved medium with definite chemical components for generating the myogenic cell in vitro matrix protein can improve the expression level of the smooth muscle matrix protein generation related protein, and particularly, the protein expression level of alpha-SMA is obviously improved on the 5 th day of induction; on day 5 of induction, the protein expression level of collageni was increased (no significant difference). Specifically, the expression level of matrix protein production-related protein alpha-SMA was increased by 2.05 times, and the expression level of Collagen I was increased by 1.94 times (FIG. 5). Thus, the modified medium of the present invention, which is clearly defined in terms of chemical composition, increases the matrix protein production capacity and level of smooth muscle cells.
EXAMPLE 3 investigation of the concentration ranges of the modified Medium
In order to investigate the applicable concentration ranges of the modified medium for in vitro matrix protein production of myogenic cells of the present invention, which are clear in chemical composition, for matrix protein production of smooth muscle cells, the concentrations of the components used were simultaneously adjusted based on the concentrations of the components in table 1, and the specific group settings are as shown in table 2.
TABLE 2
1) Cell inoculation: the piglet smooth muscle cells before P6 after sorting were inoculated into 24-well plates at a cell density of 12000 cells/well, cultured with smooth muscle cell proliferation medium, and changed 1 time every 2 days.
2) Induction of matrix protein production: after the cells proliferated to confluence (at a density of 90% or more) in the whole dish, the smooth muscle cell proliferation medium was aspirated and divided into 8 groups, and the cells were induced with medium for inducing the production of matrix protein at different formulation concentrations for each group in table 2, and the liquid was changed 1 time every 2 days. Cell growth was recorded by observation with an optical microscope.
3) Sirius red staining quantification: taking the cells induced for 5 days, fixing with 4%vol paraformaldehyde for 30min at normal temperature or fixing for 12h at 4 ℃, washing with pure water for 2 times, adding 0.5mL of sirius red dye liquor, dyeing for 1 hour, removing the sirius red dye liquor, washing with pure water for 2 times, and immediately taking a picture by using a high-flux instrument. 0.3ml of 0.1mM sodium hydroxide solution was added and the mixture was shaken in a shaker for 5 minutes in a dark environment, 200. Mu.l was taken to 96 well plates after the completion and absorbance was measured at 550nm using an enzyme-labeled instrument.
The results showed that the modified medium of the present invention showed significantly greater or equal collagen content produced by smooth muscle cells than the serum-containing medium control group (FIGS. 6, 7, 8) at a cell supplement factor concentration of not less than 1/2 as described in Table 1 (groups 1-3) compared to the serum-containing medium (positive control) in which the production of matrix proteins was induced by myogenic cells in vitro. It was thus concluded that the concentration of each cell supplement factor added to the medium for inducing matrix protein production in vitro of myogenic cells according to the present invention is in the range of 1/2 to 2 times the concentration described in table 1, and that the effect of inducing smooth muscle cell matrix protein production can be the same as or better than that of the existing serum-containing medium (positive control) for inducing matrix protein production in vitro of myogenic cells.
Example 5 piglet fibroblast-induced matrix protein production and quantification of sirius red staining
The experiment of the embodiment is divided into two groups, namely a serum-containing culture medium for generating matrix proteins by conventional myogenic cells in vitro induction and an improved culture medium treatment group with definite chemical components for generating matrix proteins by myogenic cells in vitro induction.
In this example, the fibroblast proliferation medium formulation used in the proliferation stage was 89vol% DMEM/F12 (1:1) basal medium, 10vol% fetal bovine serum, 1vol% penicillin-streptomycin diabody, and 5ng/ml basic fibroblast growth factor was added.
In this example, the conventional serum-containing medium method (positive control) for in vitro matrix protein production of myogenic cells is as follows: the basal formulation was 98vol% DMEM/F12 (1:1) basal medium, 2vol% fetal bovine serum and 5ng/ml transforming growth factor was added.
The improved culture medium with definite chemical components comprises the following components: the DMEM/F12 basal medium was added with cell culture cofactors shown in Table 3.
TABLE 3 Table 3
The cells adopted in the embodiment are piglet ear fibroblasts, and are further separated from the piglet body, and the cells with the purity higher than 90% are identified through the immunization of Vimentin, TE-7 and TCF-4 protein antibodies.
The culture conditions used in this example were all CO 2 Culturing at 37deg.C in incubator, CO 2 The concentration of (C) was 5% (v/v).
Materials, reagents and the like used in this example are commercially available unless otherwise specified.
The specific treatment method comprises the following steps:
1) Cell inoculation: piglet fibroblasts were inoculated into 3.5cm cell culture dishes at a cell density of 100000 per dish and cultured with fibroblast proliferation medium with 1 exchange every 2 days.
2) Induction of matrix protein production: after the cells proliferate to the whole culture dish (more than 90% density), the fibroblast proliferation culture medium is sucked and divided into two groups, and the culture is carried out by using a serum-containing culture medium (positive control) for inducing the generation of matrix proteins in vitro by conventional myogenic cells and a modified culture medium with definite chemical components for inducing the generation of matrix proteins in vitro by the myogenic cells, wherein the culture medium is used for culturing, and liquid is changed 1 time every 2 days. Cell growth was recorded by observation with an optical microscope.
3) Sirius red staining quantification: sampling on days 0 to 5 of induction matrix protein generation, fixing with 4%vol paraformaldehyde at normal temperature for 30min or at 4 ℃ for 12h, washing with pure water for 2 times, adding 1mL of sirius red dye liquor, dyeing for 1 hour, removing the sirius red dye liquor, washing with pure water for 2 times, and immediately taking a picture by using a high-flux instrument. 1mL of 0.1mM sodium hydroxide solution was added, and the mixture was shaken in a shaker for 5 minutes in a dark environment, and 200ul to 96 well plates were used after the completion of the shaking, and absorbance was measured at a wavelength of 550nm using an ELISA reader.
4) The results show that: compared with the serum-containing medium for generating the matrix protein by the conventional myogenic cell in-vitro induction, the improved medium with definite chemical components for generating the matrix protein by the myogenic cell in-vitro induction can improve the matrix protein generation content and time efficiency of the fibroblast, and can observe more extracellular substances by bright field photographing (figure 9). The sirius red staining quantification results showed that the extracellular matrix component content of fibroblasts was significantly better or equal to that of the conventional serum-containing medium on days 1, 2, and 3 after induction, and that the amount of matrix protein production was higher on day 2 than on day 4 after induction of the conventional serum-containing medium (fig. 10, 11). Thus, the improved culture medium with definite chemical composition of the invention improves the matrix protein production capacity of the fibroblast.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (12)
1. An improved differentiation medium with definite chemical composition for inducing extracellular matrix secretion in vitro, which is characterized in that the improved differentiation medium comprises a basal differentiation medium and cell culture supplement factors, and the improved differentiation medium does not contain serum.
Streptomycin diabody.
2. The improved differentiation medium according to claim 1, wherein said basal differentiation medium is selected from one of DMEM medium, MEM medium, DMEM/F12 medium, F10 medium, F12 medium.
3. The improved differentiation medium according to claim 1, wherein said cell culture supplement factor comprises one or more of hormonal compounds, proteinaceous substances, lipids, small molecule compounds.
4. The improved differentiation medium according to claim 3, wherein said hormonal compound is insulin;
the protein substance is selected from one or more of transferrin, albumin, insulin-like growth factor, interleukin and transforming growth factor;
the lipid is selected from one or more of Tween 80, arachidonic acid, cholesterol, vitamin E acetate, linoleic acid, linolenic acid, tetradecanoic acid, oleic acid, palmitic acid, palmitoleic acid, pramipexole F68 and octadecanoic acid;
the small molecule compound is selected from one or a combination of more of sodium selenite, ethanolamine and L-ascorbic acid.
5. The modified differentiation medium according to claim 3, wherein the total concentration range of the cell culture supplement factor addition in the modified differentiation medium is not less than 5.06mg/mL;
preferably, the total concentration of the cell culture supplement factor is added in the range of 5.06mg/mL to 20.25mg/mL.
6. The improved differentiation medium according to claim 3, wherein the concentration of any one of the cell culture supplement factors is not less than 1ng/mL;
preferably, the concentration of any cell culture supplement factor ranges from 1ng/mL to 20mg/mL;
7. use of the modified differentiation medium of claim 1 for inducing myogenic cells to produce extracellular matrix secretion in vitro.
8. The use according to claim 7, wherein the myogenic cells are smooth muscle cells or fibroblasts.
9. The use according to claim 7, wherein the modified differentiation medium is capable of increasing the extracellular matrix secretion in the in vitro induction of extracellular matrix secretion.
10. The use according to claim 7, wherein the modified differentiation medium is capable of increasing the gene expression level of the extracellular matrix secretion-related genes Col 1A2, α -SMA and increasing the protein expression level of the extracellular matrix secretion-related proteins Collagen i, α -SMA.
11. Use of the modified differentiation medium according to claim 1 for the preparation of a cell culture meat, characterized in that the modified differentiation medium according to claim 1 is used for in vitro extracellular matrix secretion induction of cells in a cell culture meat gel.
12. A cell culture meat obtained by inducing secretion of extracellular matrix using the modified differentiation medium according to claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310717527.5A CN116769702A (en) | 2023-06-16 | 2023-06-16 | Improved culture medium with definite chemical components for inducing in-vitro matrix protein generation and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310717527.5A CN116769702A (en) | 2023-06-16 | 2023-06-16 | Improved culture medium with definite chemical components for inducing in-vitro matrix protein generation and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116769702A true CN116769702A (en) | 2023-09-19 |
Family
ID=87992398
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310717527.5A Pending CN116769702A (en) | 2023-06-16 | 2023-06-16 | Improved culture medium with definite chemical components for inducing in-vitro matrix protein generation and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116769702A (en) |
-
2023
- 2023-06-16 CN CN202310717527.5A patent/CN116769702A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| RU2272839C2 (en) | Serum-free medium for cultured cells used for reconstruction of osseous and cartilage segments (variants) | |
| CN112708591B (en) | Culture medium with definite chemical components for in-vitro differentiation of muscle stem cells | |
| US6709864B1 (en) | Adipogenic differentiation of human mesenchymal stem cells | |
| CN111394299A (en) | In-vitro construction method and application of liver organoid | |
| Huss et al. | Mammary epithelial cell and adipocyte co-culture in a 3-D matrix: the first step towards tissue-engineered human breast tissue | |
| Mujaj et al. | Serum-free primary human fibroblast and keratinocyte coculture | |
| CN112608894A (en) | Mesenchymal stem cell culture medium | |
| KR102684266B1 (en) | Use of pharmaceutical compositions in the regulation of fibroblast growth | |
| CN115927172A (en) | Improved culture medium with specific chemical components for inducing in vitro adipogenic differentiation and application thereof | |
| CN114606186B (en) | Method for improving proliferation of umbilical cord mesenchymal stem cells | |
| CN109852581A (en) | A kind of method for promoting stem cell to break up at rouge and its agents useful for same or kit | |
| CN114058570A (en) | Culture medium for inducing differentiation of dental pulp stem cells into chondrocytes and application of culture medium | |
| CN104988111A (en) | Inducing liquid for converting UC-MSC into islet cells and application thereof | |
| CN113621554A (en) | Simple preparation process and preservation of epidermal tissue by using same serum-free culture medium | |
| CN116769702A (en) | Improved culture medium with definite chemical components for inducing in-vitro matrix protein generation and application thereof | |
| CN114752554B (en) | Serum-free myogenic differentiation medium containing natural compounds and application thereof | |
| CN114736851B (en) | Method for preparing vegetable-based fat-cultured meat | |
| CN109182256A (en) | A kind of spotted maigre Preadipocyte In Vitro be separately cultured and its abductive approach | |
| CN102796698A (en) | Method for inducing differentiation of placenta-derived mesenchymal stem cells into islet-like cells | |
| CN112941015B (en) | Additive and method for preparing keratinocytes based on differentiation of pluripotent stem cells | |
| CN109266600A (en) | A kind of fibroblastic culture medium and fibroblastic method is cultivated using its | |
| CN113046300B (en) | Culture method for preparing keratinocytes based on differentiation of pluripotent stem cells | |
| CN112680410B (en) | Method for inducing pluripotent stem cells to culture heart fibroblasts in differentiation mode and culture solution thereof | |
| CN113817665A (en) | Isolation culture and subculture method of mouse breast cancer cells | |
| CN105039239A (en) | Cell transformation induction liquid and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |