CN117925509B - Cell culture substituting serum culture solution and method - Google Patents
Cell culture substituting serum culture solution and method Download PDFInfo
- Publication number
- CN117925509B CN117925509B CN202410341551.8A CN202410341551A CN117925509B CN 117925509 B CN117925509 B CN 117925509B CN 202410341551 A CN202410341551 A CN 202410341551A CN 117925509 B CN117925509 B CN 117925509B
- Authority
- CN
- China
- Prior art keywords
- culture solution
- culture
- microcarrier
- 3dom
- growth factor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000004113 cell culture Methods 0.000 title claims abstract description 27
- 210000002966 serum Anatomy 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims abstract description 9
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 claims abstract description 44
- 239000003102 growth factor Substances 0.000 claims abstract description 31
- 239000000654 additive Substances 0.000 claims abstract description 23
- 239000000186 progesterone Substances 0.000 claims abstract description 22
- 229960003387 progesterone Drugs 0.000 claims abstract description 22
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims abstract description 21
- 230000000996 additive effect Effects 0.000 claims abstract description 10
- 230000010261 cell growth Effects 0.000 claims abstract description 8
- 210000004027 cell Anatomy 0.000 claims description 86
- 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 64
- 239000012752 auxiliary agent Substances 0.000 claims description 47
- 238000012258 culturing Methods 0.000 claims description 38
- 102000004877 Insulin Human genes 0.000 claims description 32
- 108090001061 Insulin Proteins 0.000 claims description 32
- 229940125396 insulin Drugs 0.000 claims description 32
- 239000000203 mixture Substances 0.000 claims description 28
- 230000001464 adherent effect Effects 0.000 claims description 24
- 238000006243 chemical reaction Methods 0.000 claims description 21
- 150000003839 salts Chemical class 0.000 claims description 21
- 229920000881 Modified starch Polymers 0.000 claims description 19
- 150000002736 metal compounds Chemical class 0.000 claims description 18
- 102000009024 Epidermal Growth Factor Human genes 0.000 claims description 17
- 101800003838 Epidermal growth factor Proteins 0.000 claims description 17
- 108010010803 Gelatin Proteins 0.000 claims description 17
- 108010025020 Nerve Growth Factor Proteins 0.000 claims description 17
- 229920001577 copolymer Polymers 0.000 claims description 17
- 229940116977 epidermal growth factor Drugs 0.000 claims description 17
- 229920000159 gelatin Polymers 0.000 claims description 17
- 239000008273 gelatin Substances 0.000 claims description 17
- 235000019322 gelatine Nutrition 0.000 claims description 17
- 235000011852 gelatine desserts Nutrition 0.000 claims description 17
- 229940053128 nerve growth factor Drugs 0.000 claims description 17
- 239000000843 powder Substances 0.000 claims description 17
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 claims description 17
- 102000004338 Transferrin Human genes 0.000 claims description 16
- 108090000901 Transferrin Proteins 0.000 claims description 16
- 239000012581 transferrin Substances 0.000 claims description 16
- 238000010438 heat treatment Methods 0.000 claims description 15
- 239000000463 material Substances 0.000 claims description 15
- 238000003756 stirring Methods 0.000 claims description 15
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 14
- 238000001035 drying Methods 0.000 claims description 14
- 238000000227 grinding Methods 0.000 claims description 14
- 239000002245 particle Substances 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 229920002472 Starch Polymers 0.000 claims description 13
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 12
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 238000001816 cooling Methods 0.000 claims description 12
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 claims description 12
- QNVSXXGDAPORNA-UHFFFAOYSA-N Resveratrol Natural products OC1=CC=CC(C=CC=2C=C(O)C(O)=CC=2)=C1 QNVSXXGDAPORNA-UHFFFAOYSA-N 0.000 claims description 9
- LUKBXSAWLPMMSZ-OWOJBTEDSA-N Trans-resveratrol Chemical compound C1=CC(O)=CC=C1\C=C\C1=CC(O)=CC(O)=C1 LUKBXSAWLPMMSZ-OWOJBTEDSA-N 0.000 claims description 9
- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 0.000 claims description 9
- 230000007910 cell fusion Effects 0.000 claims description 9
- 230000029087 digestion Effects 0.000 claims description 9
- 239000012621 metal-organic framework Substances 0.000 claims description 9
- 235000021283 resveratrol Nutrition 0.000 claims description 9
- 229940016667 resveratrol Drugs 0.000 claims description 9
- 229930002330 retinoic acid Natural products 0.000 claims description 9
- 229960001727 tretinoin Drugs 0.000 claims description 9
- 238000001914 filtration Methods 0.000 claims description 8
- 239000002608 ionic liquid Substances 0.000 claims description 8
- 239000008107 starch Substances 0.000 claims description 8
- 235000019698 starch Nutrition 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 8
- 239000004368 Modified starch Substances 0.000 claims description 7
- 238000004108 freeze drying Methods 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- 235000019426 modified starch Nutrition 0.000 claims description 7
- 239000000047 product Substances 0.000 claims description 7
- 239000012265 solid product Substances 0.000 claims description 7
- 238000001694 spray drying Methods 0.000 claims description 7
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 7
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 6
- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims description 6
- 229910021380 Manganese Chloride Inorganic materials 0.000 claims description 6
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 claims description 6
- 239000001110 calcium chloride Substances 0.000 claims description 6
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 6
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 claims description 6
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 6
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 6
- 239000011565 manganese chloride Substances 0.000 claims description 6
- 235000002867 manganese chloride Nutrition 0.000 claims description 6
- 229940099607 manganese chloride Drugs 0.000 claims description 6
- 239000001103 potassium chloride Substances 0.000 claims description 6
- 235000011164 potassium chloride Nutrition 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 239000011592 zinc chloride Substances 0.000 claims description 6
- 235000005074 zinc chloride Nutrition 0.000 claims description 6
- MXVXFMVCNUZQEW-UHFFFAOYSA-N 1-(2-ethoxyethyl)-2,3-dimethyl-2h-imidazole Chemical compound CCOCCN1C=CN(C)C1C MXVXFMVCNUZQEW-UHFFFAOYSA-N 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 210000000130 stem cell Anatomy 0.000 claims description 5
- GNPAIJQXLFMEHA-UHFFFAOYSA-N 1-(2-ethoxyethyl)-3-methyl-2h-imidazole Chemical compound CCOCCN1CN(C)C=C1 GNPAIJQXLFMEHA-UHFFFAOYSA-N 0.000 claims description 4
- 102000018233 Fibroblast Growth Factor Human genes 0.000 claims description 4
- 108050007372 Fibroblast Growth Factor Proteins 0.000 claims description 4
- 229940126864 fibroblast growth factor Drugs 0.000 claims description 4
- 102000029816 Collagenase Human genes 0.000 claims description 3
- 108060005980 Collagenase Proteins 0.000 claims description 3
- 229960002424 collagenase Drugs 0.000 claims description 3
- WRKJABJOZCTYFH-UHFFFAOYSA-N 1-(ethoxymethyl)-3-methyl-2h-imidazole Chemical compound CCOCN1CN(C)C=C1 WRKJABJOZCTYFH-UHFFFAOYSA-N 0.000 claims description 2
- KAIPKTYOBMEXRR-UHFFFAOYSA-N 1-butyl-3-methyl-2h-imidazole Chemical compound CCCCN1CN(C)C=C1 KAIPKTYOBMEXRR-UHFFFAOYSA-N 0.000 claims description 2
- 239000013207 UiO-66 Substances 0.000 claims description 2
- 210000004923 pancreatic tissue Anatomy 0.000 claims description 2
- 102000015336 Nerve Growth Factor Human genes 0.000 claims 2
- 239000002671 adjuvant Substances 0.000 claims 1
- YOSZVGLIFGVSOL-UHFFFAOYSA-N n,n-diethyl-3-methoxypropan-1-amine Chemical compound CCN(CC)CCCOC YOSZVGLIFGVSOL-UHFFFAOYSA-N 0.000 claims 1
- 239000000243 solution Substances 0.000 description 127
- 239000006228 supernatant Substances 0.000 description 29
- 229910002091 carbon monoxide Inorganic materials 0.000 description 28
- 239000002609 medium Substances 0.000 description 18
- 230000001105 regulatory effect Effects 0.000 description 16
- 102100023995 Beta-nerve growth factor Human genes 0.000 description 15
- 230000000052 comparative effect Effects 0.000 description 14
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 13
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 13
- 230000035755 proliferation Effects 0.000 description 13
- AEMRFAOFKBGASW-UHFFFAOYSA-N Glycolic acid Chemical compound OCC(O)=O AEMRFAOFKBGASW-UHFFFAOYSA-N 0.000 description 10
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 10
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 7
- 108010019160 Pancreatin Proteins 0.000 description 7
- 238000005119 centrifugation Methods 0.000 description 7
- 230000001079 digestive effect Effects 0.000 description 7
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 7
- 229940055695 pancreatin Drugs 0.000 description 7
- 229920002261 Corn starch Polymers 0.000 description 6
- 230000004663 cell proliferation Effects 0.000 description 6
- 239000008120 corn starch Substances 0.000 description 6
- 206010012601 diabetes mellitus Diseases 0.000 description 6
- -1 imine salt compound Chemical class 0.000 description 6
- 239000004310 lactic acid Substances 0.000 description 5
- 235000014655 lactic acid Nutrition 0.000 description 5
- 229920001592 potato starch Polymers 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 229940100445 wheat starch Drugs 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 239000007640 basal medium Substances 0.000 description 3
- 210000000496 pancreas Anatomy 0.000 description 3
- 239000013154 zeolitic imidazolate framework-8 Substances 0.000 description 3
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000012894 fetal calf serum Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 150000003212 purines Chemical class 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 210000003079 salivary gland Anatomy 0.000 description 2
- 239000004017 serum-free culture medium Substances 0.000 description 2
- PFNFFQXMRSDOHW-UHFFFAOYSA-N spermine Chemical compound NCCCNCCCCNCCCN PFNFFQXMRSDOHW-UHFFFAOYSA-N 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 208000008960 Diabetic foot Diseases 0.000 description 1
- 206010018429 Glucose tolerance impaired Diseases 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- ZOKXTWBITQBERF-UHFFFAOYSA-N Molybdenum Chemical compound [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 description 1
- 102100037209 Peroxisomal N(1)-acetyl-spermine/spermidine oxidase Human genes 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 208000001280 Prediabetic State Diseases 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001805 chlorine compounds Chemical class 0.000 description 1
- 229910052804 chromium Inorganic materials 0.000 description 1
- 239000011651 chromium Substances 0.000 description 1
- 208000020832 chronic kidney disease Diseases 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 238000011841 epidemiological investigation Methods 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 230000002641 glycemic effect Effects 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 210000004153 islets of langerhan Anatomy 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 229910052750 molybdenum Inorganic materials 0.000 description 1
- 239000011733 molybdenum Substances 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 108010089000 polyamine oxidase Proteins 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 201000009104 prediabetes syndrome Diseases 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229940063675 spermine Drugs 0.000 description 1
- 238000011476 stem cell transplantation Methods 0.000 description 1
- 229910052718 tin Inorganic materials 0.000 description 1
- 229910021654 trace metal Inorganic materials 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 229910052720 vanadium Inorganic materials 0.000 description 1
- GPPXJZIENCGNKB-UHFFFAOYSA-N vanadium Chemical compound [V]#[V] GPPXJZIENCGNKB-UHFFFAOYSA-N 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of cell culture, and particularly relates to a serum replacement culture solution for cell culture and a method. The invention discloses a culture solution for replacing serum in cell culture, which consists of a basic culture solution and an exogenous additive, wherein the basic culture solution is a DMEM culture solution or a F12 culture solution or a mixed culture solution of the DMEM culture solution and the F12 culture solution according to the volume ratio of 1:2; the exogenous additives are necessary supplementary factors, cell growth factors, microcarriers, microcarrier auxiliaries, progesterone and pH regulator. The culture solution has simple components and can realize efficient culture of cells.
Description
Technical Field
The invention belongs to the technical field of cell culture, and particularly relates to a serum replacement culture solution for cell culture and a method.
Background
Diabetes and complications have become public health problems that seriously jeopardize the health of people in China. At present, epidemiological investigation shows that the prevalence of diabetes in China is up to 12.8%, and complications such as cardiovascular and cerebrovascular diseases, chronic kidney diseases, eyeground pathological changes, diabetic feet and the like caused by poor blood sugar control are main causes of death or disability of diabetics, and bring heavy burden to national health and social economy. Although the current treatment for diabetes has a lot of choices, mainly for glycemic control in the diabetic stage, there are few preventive measures from euglycemia to pre-diabetes and the progression of diabetes. At present, islet stem cell transplantation is an ideal method for radically treating diabetes. But the addition of heterologous serum to the medium used for large-scale in vitro culture of islet stem cells greatly increases the risk of rejection in humans during transplantation.
Serum-free medium refers to a basal medium in which the heterologous serum is replaced by the addition of additives, thereby reducing the effect of the heterologous serum on the cells. Since conventional mammalian cell culture solutions require the use of DMEM (Dulbecco's Modified Eagle Med ium) Medium, MEM (Minimum Eagle's Medium) Medium, or the like as a basal Medium, about 10% fetal bovine serum or calf serum must be added to the basal Medium. For artificially cultured cells, the primary role of serum includes providing basic nutrients; providing hormones and various growth factors; providing a binding protein; providing a pro-contact and extension factor to protect the cell wall from mechanical damage; certain protective effects are exerted on the cells in culture. However, there are problems in using serum as an additive, such as high serum consumption and high cost, and in containing substances which are toxic to cells, such as polyamine oxidase, which can react to produce a toxic action of spermine, etc. In addition, the biological safety of the serum is difficult to ensure because of the influence of factors such as sources, processing technology and the like, the quality is unstable, and the cell growth and the quality of the final product are finally influenced.
There is also a considerable amount of literature on serum-free culture solutions, for example, patent 200710057738.1 discloses a serum-free or low serum concentration cell culture method comprising the steps of: the key point of the patent is that the serum with higher concentration is replaced by the stress pressure, and the cells grow under the conditions of no serum or low serum and certain stress. For another example, patent 20110303107. X discloses a serum-free culture solution for culturing salivary gland epithelial cells and salivary gland stem cells of mammals, wherein the culture solution is prepared by taking MCDB153HAA as a basic culture medium and adding amino acids, vitamins, salts, lipids, trace elements, buffer solution, hormonal compounds, transferrin, antioxidants, serum albumin, saccharides, purines, pyrimidine base substances and pH value indicators. The existing serum-free culture solution mainly replaces the effect of serum by adding substances with similar nutritional compositions as serum, such as amino acids, vitamins, purines and the like, but the serum-free culture solution has unstable factors and also has different composition components and no wide adaptability because the serum-free culture medium has specificity, namely, the composition components of the culture medium are different for different cells.
Therefore, development of a serum-free culture medium for islet-like cells is also a current direction of research.
Disclosure of Invention
The invention aims at overcoming the defects in the prior art and provides a cell culture replacement serum culture solution and a method. The culture solution provided by the invention can be used for culturing islet cells, so that the proliferation capability of the cells can be effectively improved, the number of islet stem cells with a differentiation function can be increased, and serum is not added into the culture solution, so that the influence of heterologous serum on the cells is avoided.
The aim and the technical problems of the invention are realized by adopting the following technical proposal.
The invention provides a culture solution for replacing serum in cell culture, which consists of a basic culture solution and an exogenous additive, wherein the basic culture solution is a DMEM culture solution or an F12 culture solution or a mixed culture solution of the DMEM culture solution and the F12 culture solution according to a volume ratio of 1:2; the exogenous additives are necessary supplementary factors, cell growth factors, microcarriers, microcarrier auxiliary agents, progesterone and pH regulators; wherein,
The preparation method of the microcarrier comprises the following steps: mixing gelatin and polylactic acid-glycolic acid copolymer according to the mass ratio of 1:2-5 to obtain a mixture, dissolving the mixture in tetrahydrofuran with the volume fraction of 40-50%, heating the whole reaction system to 60-85 ℃, preserving heat for 60-100 min, cooling to 40-50 ℃, adding a three-dimensional ordered macroporous metal-organic frame material, preserving heat for 3-8 h, cooling to room temperature, filtering, sequentially freeze-drying the obtained solid product at-50 to-30 ℃, drying in a vacuum constant temperature drying oven at 40-50 ℃, and finally grinding the product until particles with the particle diameter of 100-500 mu m are microcarriers;
The preparation method of the microcarrier auxiliary agent comprises the following steps: firstly, starch and water are mixed according to the mass volume ratio of 1mg: placing the 1-3 mL of the mixture into a reaction kettle, heating, stirring and dissolving the mixture, sequentially adding the ionic liquid and the metal compound while stirring when the temperature reaches the gelatinization temperature, heating the system to 40-60 ℃ after all the adding is completed, continuously stirring the mixture for 8-10 hours, finally performing spray drying and grinding to obtain modified starch powder, ultrasonically dispersing the obtained modified starch powder into water, and adjusting the pH value of the solution to be 7 to obtain a modified starch solution with the concentration of 30-50wt%, namely the microcarrier auxiliary agent;
The addition amount of the three-dimensional ordered macroporous metal-organic framework material in the reaction process is 0.5-1 times of the total mass of the gelatin and the polylactic acid-glycolic acid copolymer;
in the reaction process, the ionic liquid, the metal compound and the starch are mixed according to the ratio of 0.03-0.06 mL: 0.4-0.8 mg:1 g.
Further, the three-dimensional ordered macroporous metal-organic framework material is selected from one or a mixture of more than two of 3DOM-PCN-601、3DOM-PCN-14、3DOM-ZIF-8、3DOM-ZIF-67、3DOM-ZIF-68、3DOM-ZIF-69、3DOM-ZIF-70、3DOM-ZIF-78、3DOM-ZIF-81、3DOM-ZIF-82、3DOM-ZIF-95、3DOM-ZIF-100、3DOM-UIO-66.
Further, the ionic liquid is selected from any one of 1, 2-dimethyl-3-ethoxyethylimidazole bistrifluoromethylsulfonylimine salt, 1-methyl-3-ethoxyethylimidazole bistrifluoromethylsulfonylimine salt, N-butyl-N-methylpyrrolidine bistrifluoromethylsulfonylimine salt, 1-methyl-3-ethoxymethylimidazole bistrifluoromethylsulfonylimine salt, N-ethoxymethyl-N-methylpyrrolidine bistrifluoromethylsulfonylimine salt, 1-methyl-3-butylimidazole bistrifluoromethylsulfonylimine salt, N-ethoxyethyl-N-methylpyrrolidine bistrifluoromethylsulfonylimine salt, N-diethyl-2-methoxyethyl-N-methylamine bistrifluoromethylsulfonylimine salt.
Further, the metal compound is selected from the group consisting of chlorides of any one or more of the following metal elements: zinc, copper, sodium, potassium, calcium, magnesium, iron, manganese, chromium, molybdenum, cobalt, nickel, vanadium, tin.
Further, the concentration of the microcarrier is 1-3 g/L; the concentration of the microcarrier auxiliary agent is 10-50 mL/L.
Further, the necessary supplementary factors are insulin with the concentration of 5-30mg/L and transferrin with the concentration of 10-20 mg/L; the growth factors comprise an epidermal growth factor, a nerve growth factor, an insulin growth factor and an alkaline fibroblast growth factor, wherein the concentration of the epidermal growth factor is 15-25 mg/L, the concentration of the nerve growth factor is 20-30 mg/L, the concentration of the insulin growth factor is 15-25 mg/L and the concentration of the alkaline fibroblast growth factor is 10-30 mg/L; the concentration of the progesterone is 3-5 mmol/L; and the pH regulator regulates the pH of the culture solution to 6.9-7.3.
The aim and the technical problems of the invention are also realized by adopting the following technical proposal.
The invention also provides a culture method of islet stem cells, which adopts the culture solution of the cell culture replacement serum for culture, and comprises the following steps:
S1: adding growth factors, cell growth factors and progesterone which are required to be supplemented into the basic culture solution, and adjusting the pH to 6.9-7.3 to obtain a culture solution A;
Re-suspending pancreatic cells to a density of1×10 5 to 1×10 6 cells/mL with culture solution a, culturing for 1h, and transferring non-adherent cells;
S2: adding growth factors, cell growth factors, progesterone and microcarriers which are required to be supplemented into a basic culture solution, and adjusting the pH to 6.9-7.3 to obtain a culture solution B;
Culturing the non-adherent cells with a culture solution B for 18 hours, and then discarding the non-adherent cells and the culture solution B to obtain adherent cells;
S3: culturing the adherent cells in a culture solution B until the cell fusion degree is 70% -90%, adding microcarrier auxiliary agent into the culture solution B, uniformly stirring, and performing subculture for 8 times.
Further, in the step S3, retinoic acid and resveratrol with the concentration of 1-10 mmol/L are added within 6-8 hours after the microcarrier auxiliary agent is added.
Further, the preparation method of the pancreatic cells comprises the following steps: pancreatic cells were prepared by digestion of pancreatic tissue with collagenase type four and washing with PBS.
By means of the technical scheme, the invention has at least the following advantages: the culture solution provided by the invention is a serum-free culture solution, namely, the traditional fetal calf serum or calf serum is abandoned, and instead, an exogenous additive is added, the exogenous additive disclosed by the invention comprises a plurality of essential components, a microcarrier and a microcarrier auxiliary agent are additionally added, the microcarrier is added to play a role in promoting cell adhesion, islet-like cells and other pancreatic cells can be well separated, and the synergistic effect of the microcarrier and the microcarrier auxiliary agent can promote cell proliferation and increase the subculture times.
The microcarrier is a micron-sized carrier substance with a three-dimensional ordered macroporous metal-organic frame material as a matrix core and polylactic acid-glycolic acid copolymer and gelatin adhered to the outer surface, the microcarrier uses a blend formed by the polylactic acid-glycolic acid copolymer and gelatin as a raw material, the blend is coated and adhered on the surface of the three-dimensional ordered macroporous metal-organic frame material through the adhesive effect of the polymer by full contact, and the carrier can slowly release groups of the polylactic acid-glycolic acid copolymer and the gelatin to provide nutrition for cell culture. The microcarrier auxiliary agent is modified starch obtained by modifying starch serving as a raw material through an ionic liquid and a metal compound, the starch can provide various nutrients such as needed sugar for cell culture, the metal compound is a compound formed by trace metal elements and provides trace elements for culture solution, and the ionic liquid is an imine salt compound and can change the three-dimensional structure of the starch so that the starch is easier to decompose into sugar substances.
The foregoing description is only an overview of the present invention, and is intended to provide a more thorough understanding of the present invention, and is to be accorded the full scope of the present invention.
Drawings
FIG. 1 is a graph showing proliferation of cell culture in example 1;
FIG. 2 is a graph showing the proliferation of cell culture in comparative example 1;
FIG. 3 is a graph showing proliferation of cell culture in comparative example 2;
FIG. 4 is a graph showing proliferation of cell culture in comparative example 3;
FIG. 5 is a graph showing the proliferation of cell culture in comparative example 4.
Detailed Description
In order to make the technical means, the creation features, the achievement of the purposes and the effects of the present invention easy to understand, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Unless otherwise specified, the experimental methods in the examples of the present invention are all conventional methods.
Materials, reagents, and the like used in the examples of the present invention are commercially available unless otherwise specified.
The pancreatic cells in the examples of the present invention were prepared as follows, unless otherwise specified: and (3) picking 4-7 weeks old SD rat pancreas, cleaning the picked pancreas with PBS for 2 times, shearing the pancreas into 3mm size, adding 1g/L four-type collagenase according to 5 times volume, shaking and digesting for 50 minutes at 37 ℃, adding an equivalent amount of PBS to dilute the digested liquid, and filtering the digested liquid with a 200-mesh filter screen to remove impurities. 2000g of the filtrate was centrifuged for 5 minutes, and the supernatant was removed to obtain pancreatic cells.
Example 1:
Preparing a microcarrier: mixing gelatin and polylactic acid-glycolic acid copolymer according to the mass ratio of 1:3.5 to obtain a mixture, dissolving the mixture in tetrahydrofuran with the volume fraction of 45%, heating the whole reaction system to 73 ℃, preserving heat for 80min, then cooling to 45 ℃, adding a three-dimensional ordered macroporous metal-organic framework material 3DOM-ZIF-8 which is 0.75 times of the total mass of the gelatin and the polylactic acid-glycolic acid copolymer (formed by copolymerizing 70% lactic acid and 30% glycolic acid), preserving heat for 5.5h, cooling the system to room temperature, filtering, sequentially freeze-drying the obtained solid product at-40 ℃, drying in a vacuum constant-temperature drying oven at 45 ℃, and finally grinding the product to particles with the particle diameter of 100-500 mu m to obtain the microcarrier;
Preparing a microcarrier auxiliary agent: firstly, corn starch and water are mixed according to the mass volume ratio of 1mg:2mL of the modified corn starch powder is added with 1, 2-dimethyl-3-ethoxyethylimidazole bistrifluoromethylsulfonylimine salt and a metal compound (comprising zinc chloride, copper chloride, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, ferric chloride and manganese chloride according to the mass ratio of 1:1:1:1:1:1:1:1) in sequence while stirring when the temperature reaches the gelatinization temperature, and the system is heated to 50 ℃ after the whole addition is finished, and then is continuously stirred for 9 hours, finally, spray drying and grinding are carried out to obtain the modified corn starch powder, the obtained modified corn starch powder is dispersed in water in an ultrasonic manner, and the pH=7 of the solution is regulated to obtain a modified corn starch solution with the concentration of 40wt%, namely the microcarrier auxiliary agent; during the reaction, 1, 2-dimethyl-3-ethoxyethylimidazole bis (trifluoromethanesulfonyl) imide salt, metal compound and corn starch are mixed according to 0.045mL:0.6mg:1 g.
Preparing a culture solution A: the basic culture solution is DMEM culture solution; the exogenous additives are 12.5mg/L of insulin, 15mg/L of transferrin, 20mg/L of epidermal growth factor, 25mg/L of nerve growth factor, 20mg/L of insulin growth factor, 20mg/L of basic fibroblast growth factor and 4mmol/L of progesterone, and the pH value of the solution is regulated to 6.9-7.3.
Preparing a culture solution B: the basic culture solution is DMEM culture solution; the exogenous additive is 12.5mg/L of insulin, 15mg/L of transferrin, 20mg/L of epidermal growth factor, 25mg/L of nerve growth factor, 20mg/L of insulin growth factor, 20mg/L of basic fibroblast growth factor, 4mmol/L of progesterone, 2g/L of microcarrier (30 mL/L of microcarrier auxiliary agent) and the pH value of the solution is regulated to 6.9-7.3.
The cell culture method of this example is as follows: pancreatic cells were resuspended in medium A to a density of 1X 10 5 to 1X 10 6 cells/mL and inoculated into culture flasks. Culturing the culture flask in a culture flask containing 5% CO 2 at 37 ℃ for 1 hour, sucking non-adherent cells, transferring the culture flask to a culture flask containing the culture solution B (without microcarrier auxiliary agent) and culturing the culture flask for 18 hours at 37 ℃ with 5% CO 2, discarding the non-adherent cells and supernatant, adding the culture solution B (without microcarrier auxiliary agent) equal to the discarded supernatant at 37 ℃ with 5% CO 2, and continuously culturing the culture flask once every 3 days. The equal cell fusion degree reaches 80%, the supernatant is discarded, PBS is used for washing for 2 times, pancreatin with 0.4% EDTA concentration of 0.25% is added for digesting cells for 1 minute, 3 times of the volume of the digestive juice is added into culture solution B (containing microcarrier auxiliary agent) to stop digestion, the supernatant is removed after centrifugation, cells are resuspended according to the density of 1X 10 5/mL-1X 10 6/mL by using the culture solution B (containing microcarrier auxiliary agent), 5.5 mmol/L of retinoic acid and resveratrol are added simultaneously, and the cells are transferred into a new culture flask for culturing at 37 ℃ and 5% CO 2, and the culture is marked as P1 generation. Subculturing to P8, proliferation of cells in the medium was recorded as shown in Table 1 and FIG. 1.
Example 2:
preparing a microcarrier: mixing gelatin and polylactic acid-glycolic acid copolymer according to the mass ratio of 1:2 to obtain a mixture, dissolving the mixture in 50% tetrahydrofuran, heating the whole reaction system to 60 ℃, preserving heat for 100min, then reducing the temperature to 40 ℃, adding three-dimensional ordered macroporous metal-organic framework material 3DOM-PCN-14 which is 1 time of the total mass of the gelatin and the polylactic acid-glycolic acid copolymer (formed by copolymerizing 70% lactic acid and 30% glycolic acid), preserving heat for 8h, finally cooling the system to room temperature, filtering, sequentially freeze-drying the obtained solid product at-30 ℃, drying in a vacuum constant temperature drying oven at 50 ℃, and finally grinding the product to particles with the particle size of 100-500 mu m to obtain the microcarrier;
Preparing a microcarrier auxiliary agent: wheat starch and water are firstly mixed according to the mass volume ratio of 1mg: putting the 1mL weight ratio into a reaction kettle, heating, stirring and dissolving, when the temperature reaches the gelatinization temperature, sequentially adding 1-methyl-3-ethoxyethylimidazole bistrifluoromethylsulfonyl imide salt and a metal compound (composed of zinc chloride, copper chloride, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, ferric chloride and manganese chloride according to the mass ratio of 1:1:1:1:1:1:1:1:1), heating the system to 40 ℃ after all the components are added, continuously stirring for 10 hours, finally performing spray drying and grinding to obtain modified wheat starch powder, ultrasonically dispersing the obtained modified wheat starch powder into water, and adjusting the pH=7 of the solution to obtain a modified wheat starch solution with the concentration of 50wt%, namely a microcarrier auxiliary agent; during the reaction, 1-methyl-3-ethoxyethylimidazole bistrifluoromethylsulfonylimine salt, metal compound and wheat starch are mixed according to 0.03mL:0.8mg:1 g.
Preparing a culture solution A: the basic culture solution is DMEM culture solution; the exogenous additives are 5-30mg/L of insulin, 20mg/L of transferrin, 15mg/L of epidermal growth factor, 20mg/L of nerve growth factor, 25mg/L of insulin growth factor, 10mg/L of basic fibroblast growth factor and 5mmol/L of progesterone, and the pH value of the solution is regulated to 6.9-7.3.
Preparing a culture solution B: the basic culture solution is DMEM culture solution; the exogenous additive is 5-30mg/L of insulin, 20mg/L of transferrin, 15mg/L of epidermal growth factor, 20mg/L of nerve growth factor, 25mg/L of insulin growth factor, 10mg/L of basic fibroblast growth factor, 5mmol/L of progesterone, 1g/L of microcarrier (10 mL/L of microcarrier auxiliary agent) and the pH value of the solution is regulated to 6.9-7.3.
The cell culture method of this example is as follows: pancreatic cells were resuspended in medium A to a density of 1X 10 5 to 1X 10 6 cells/mL and inoculated into culture flasks. Culturing the culture flask in a culture flask containing 5% CO 2 at 37 ℃ for 1 hour, sucking non-adherent cells, transferring the culture flask to a culture flask containing the culture solution B (without microcarrier auxiliary agent) and culturing the culture flask for 18 hours at 37 ℃ with 5% CO 2, discarding the non-adherent cells and supernatant, adding the culture solution B (without microcarrier auxiliary agent) equal to the discarded supernatant at 37 ℃ with 5% CO 2, and continuously culturing the culture flask once every 3 days. The equal cell fusion degree reaches 80%, the supernatant is discarded, PBS is used for washing for 2 times, pancreatin with 0.4% EDTA concentration of 0.25% is added for digesting cells for 1 minute, 3 times of the volume of the digestive juice is added into culture solution B (containing microcarrier auxiliary agent) to stop digestion, the supernatant is removed after centrifugation, cells are resuspended according to the density of 1X 10 5/mL-1X 10 6/mL by using the culture solution B (containing microcarrier auxiliary agent), 1 mmol/L of retinoic acid and resveratrol are added simultaneously, and the cells are transferred into a new culture flask for culturing at 37 ℃ and 5% CO 2, and the culture is marked as generation P1. Subculturing to P8, proliferation of cells in the medium was recorded as shown in Table 1.
Example 3:
Preparing a microcarrier: mixing gelatin and polylactic acid-glycolic acid copolymer according to the mass ratio of 1:5 to obtain a mixture, dissolving the mixture in tetrahydrofuran with the volume fraction of 40%, heating the whole reaction system to 85 ℃, preserving heat for 60min, then reducing the temperature to 50 ℃, adding three-dimensional ordered macroporous metal-organic framework material 3DOM-PCN-601 which is 0.5 times of the total mass of the gelatin and the polylactic acid-glycolic acid copolymer (formed by copolymerizing 70% lactic acid and 30% glycolic acid), preserving heat for 3h, finally cooling the system to room temperature, filtering, sequentially freeze-drying the obtained solid product at-50 ℃, drying in a vacuum constant temperature drying oven at 40 ℃, and finally grinding the product to particles with the particle size of 100-500 mu m to obtain the microcarrier;
Preparing a microcarrier auxiliary agent: firstly, potato starch and water are mixed according to the mass volume ratio of 1mg: putting the 3mL weight ratio into a reaction kettle, heating, stirring and dissolving, when the temperature reaches the gelatinization temperature, sequentially adding N-butyl-N-methylpyrrolidine bistrifluoromethylsulfonyl imide salt and a metal compound (comprising zinc chloride, copper chloride, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, ferric chloride and manganese chloride according to the mass ratio of 1:1:1:1:1:1:1:1), heating the system to 60 ℃ after all the components are added, continuously stirring for 8 hours, finally performing spray drying and grinding to obtain modified potato starch powder, ultrasonically dispersing the obtained modified potato starch powder into water, and adjusting the pH=7 of the solution to obtain a modified potato starch solution with the concentration of 30wt%, namely the microcarrier auxiliary agent; during the reaction, the N-butyl-N-methylpyrrolidine bis (trifluoromethanesulfonyl) imide salt, the metal compound and potato starch are mixed according to 0.06mL:0.4mg:1 g.
Preparing a culture solution A: the basic culture solution is DMEM culture solution; the exogenous additives are 5-30mg/L of insulin, 10mg/L of transferrin, 25mg/L of epidermal growth factor, 20mg/L of nerve growth factor, 15mg/L of insulin growth factor, 30mg/L of basic fibroblast growth factor and 3mmol/L of progesterone, and the pH value of the solution is regulated to 6.9-7.3.
Preparing a culture solution B: the basic culture solution is DMEM culture solution; the exogenous additive is 5-30mg/L of insulin, 10mg/L of transferrin, 25mg/L of epidermal growth factor, 20mg/L of nerve growth factor, 15mg/L of insulin growth factor, 30mg/L of basic fibroblast growth factor, 3mmol/L of progesterone, 3g/L of microcarrier (50 mL/L of microcarrier auxiliary agent) and the pH value of the solution is regulated to 6.9-7.3.
The cell culture method of this example is as follows: pancreatic cells were resuspended in medium A to a density of 1X 10 5 to 1X 10 6 cells/mL and inoculated into culture flasks. Culturing the culture flask in a culture flask containing 5% CO 2 at 37 ℃ for 1 hour, sucking non-adherent cells, transferring the culture flask to a culture flask containing the culture solution B (without microcarrier auxiliary agent) and culturing the culture flask for 18 hours at 37 ℃ with 5% CO 2, discarding the non-adherent cells and supernatant, adding the culture solution B (without microcarrier auxiliary agent) equal to the discarded supernatant at 37 ℃ with 5% CO 2, and continuously culturing the culture flask once every 3 days. The equal cell fusion degree reaches 80%, the supernatant is discarded, PBS is used for washing for 2 times, pancreatin with 0.4% EDTA concentration of 0.25% is added for digesting cells for 1 minute, 3 times of the volume of the digestive juice is added into culture solution B (containing microcarrier auxiliary agent) to stop digestion, the supernatant is removed after centrifugation, cells are resuspended according to the density of 1X 10 5/mL-1X 10 6/mL by using the culture solution B (containing microcarrier auxiliary agent), 10 mmol/L of retinoic acid and resveratrol are added simultaneously, and the cells are transferred into a new culture flask for culturing at 37 ℃ and 5% CO 2, and the culture is marked as generation P1. Subculturing to P8, proliferation of cells in the medium was recorded as shown in Table 1.
Comparative example 1:
Preparing a microcarrier: mixing gelatin and polylactic acid-glycolic acid copolymer according to the mass ratio of 1:3.5 to obtain a mixture, dissolving the mixture in tetrahydrofuran with the volume fraction of 45%, heating the whole reaction system to 73 ℃, preserving heat for 80min, then cooling to 45 ℃, adding a three-dimensional ordered macroporous metal-organic framework material 3DOM-ZIF-8 which is 0.75 times of the total mass of the gelatin and the polylactic acid-glycolic acid copolymer (formed by copolymerizing 70% lactic acid and 30% glycolic acid), preserving heat for 5.5h, cooling the system to room temperature, filtering, sequentially freeze-drying the obtained solid product at-40 ℃, drying in a vacuum constant-temperature drying oven at 45 ℃, and finally grinding the product to particles with the particle diameter of 100-500 mu m to obtain the microcarrier;
Preparing a culture solution A: the basic culture solution is DMEM culture solution; the exogenous additives are 12.5mg/L of insulin, 15mg/L of transferrin, 20mg/L of epidermal growth factor, 25mg/L of nerve growth factor, 20mg/L of insulin growth factor, 20mg/L of basic fibroblast growth factor and 4mmol/L of progesterone, and the pH value of the solution is regulated to 6.9-7.3.
Preparing a culture solution B: the basic culture solution is DMEM culture solution; the exogenous additives are 12.5mg/L of insulin, 15mg/L of transferrin, 20mg/L of epidermal growth factor, 25mg/L of nerve growth factor, 20mg/L of insulin growth factor, 20mg/L of basic fibroblast growth factor, 4mmol/L of progesterone and 2g/L of microcarrier, and the pH value of the solution is regulated to 6.9-7.3.
The cell culture method of this example is as follows: pancreatic cells were resuspended in medium A to a density of 1X 10 5 to 1X 10 6 cells/mL and inoculated into culture flasks. Culturing the culture flask in a culture flask containing 5% CO 2 at 37deg.C for 1 hr, sucking non-adherent cells, transferring into a culture flask containing culture solution B, culturing at 37deg.C for 5% CO 2 for 18 hr, discarding non-adherent cells and supernatant, adding the culture solution B equal to the supernatant, and culturing at 37deg.C for 5% CO 2. The equal cell fusion degree reaches 80%, the supernatant is discarded, PBS is used for washing for 2 times, pancreatin with 0.4% EDTA concentration of 0.25% is added for digesting cells for 1 minute, 3 times of the volume of the digestive juice is added to culture solution B for stopping digestion, the supernatant is removed after centrifugation, the cells are resuspended according to the density of 1X 10 5/mL-1X 10 6/mL by using the culture solution B, 5.5 mmol/L of retinoic acid and resveratrol are added simultaneously, and the cells are transferred into a new culture flask for culturing at 37 ℃ and 5% CO 2, and the culture is marked as P1 generation. Subculturing to P8, proliferation of cells in the medium was recorded as shown in Table 1 and FIG. 2.
Comparative example 2:
Preparing a microcarrier auxiliary agent: firstly, corn starch and water are mixed according to the mass volume ratio of 1mg:2mL of the modified corn starch powder is added with 1, 2-dimethyl-3-ethoxyethylimidazole bistrifluoromethylsulfonylimine salt and a metal compound (comprising zinc chloride, copper chloride, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, ferric chloride and manganese chloride according to the mass ratio of 1:1:1:1:1:1:1:1) in sequence while stirring when the temperature reaches the gelatinization temperature, and the system is heated to 50 ℃ after the whole addition is finished, and then is continuously stirred for 9 hours, finally, spray drying and grinding are carried out to obtain the modified corn starch powder, the obtained modified corn starch powder is dispersed in water in an ultrasonic manner, and the pH=7 of the solution is regulated to obtain a modified corn starch solution with the concentration of 40wt%, namely the microcarrier auxiliary agent; during the reaction, 1, 2-dimethyl-3-ethoxyethylimidazole bis (trifluoromethanesulfonyl) imide salt, metal compound and corn starch are mixed according to 0.045mL:0.6mg:1 g.
Preparing a culture solution A: the basic culture solution is DMEM culture solution; the exogenous additives are 12.5mg/L of insulin, 15mg/L of transferrin, 20mg/L of epidermal growth factor, 25mg/L of nerve growth factor, 20mg/L of insulin growth factor, 20mg/L of basic fibroblast growth factor and 4mmol/L of progesterone, and the pH value of the solution is regulated to 6.9-7.3.
Preparing a culture solution B: the basic culture solution is DMEM culture solution; the exogenous additives are 12.5mg/L of insulin, 15mg/L of transferrin, 20mg/L of epidermal growth factor, 25mg/L of nerve growth factor, 20mg/L of insulin growth factor, 20mg/L of basic fibroblast growth factor, 4mmol/L of progesterone and 30mL/L of microcarrier auxiliary agent, and the pH value of the solution is regulated to 6.9-7.3.
The cell culture method of this example is as follows: pancreatic cells were resuspended in medium A to a density of 1X 10 5 to 1X 10 6 cells/mL and inoculated into culture flasks. Culturing the culture flask in a culture flask containing 5% CO 2 at 37deg.C for 1 hr, sucking non-adherent cells, transferring into a culture flask containing culture solution B, culturing at 37deg.C for 5% CO 2 for 18 hr, discarding non-adherent cells and supernatant, adding the culture solution B equal to the supernatant, and culturing at 37deg.C for 5% CO 2. The equal cell fusion degree reaches 80%, the supernatant is discarded, PBS is used for washing for 2 times, pancreatin with 0.4% EDTA concentration of 0.25% is added for digesting cells for 1 minute, 3 times of the volume of the digestive juice is added to culture solution B for stopping digestion, the supernatant is removed after centrifugation, the cells are resuspended according to the density of 1X 10 5/mL-1X 10 6/mL by using the culture solution B, 5.5 mmol/L of retinoic acid and resveratrol are added simultaneously, and the cells are transferred into a new culture flask for culturing at 37 ℃ and 5% CO 2, and the culture is marked as P1 generation. Subculturing to P8, proliferation of cells in the medium was recorded as shown in Table 1 and FIG. 3.
Comparative example 3:
Preparing a culture solution A: the basic culture solution is DMEM culture solution; the exogenous additives are 12.5mg/L of insulin, 15mg/L of transferrin, 20mg/L of epidermal growth factor, 25mg/L of nerve growth factor, 20mg/L of insulin growth factor, 20mg/L of basic fibroblast growth factor and 4mmol/L of progesterone, and the pH value of the solution is regulated to 6.9-7.3.
The cell culture method of this example is as follows: pancreatic cells were resuspended in medium A to a density of 1X 10 5 to 1X 10 6 cells/mL and inoculated into culture flasks. Culturing the culture flask in a culture flask containing 5% CO 2 at 37deg.C for 1 hr, sucking non-adherent cells, transferring into a culture flask containing culture solution A, culturing at 37deg.C for 5% CO 2 for 18 hr, discarding non-adherent cells and supernatant, adding the culture solution A equivalent to the discarded supernatant, and culturing at 37deg.C with 5% CO 2. The equal cell fusion degree reaches 80%, the supernatant is discarded, PBS is used for washing for 2 times, pancreatin with 0.4% EDTA concentration of 0.25% is added for digesting cells for 1 minute, 3 times of the volume of the digestive juice is added to culture solution A to stop digestion, the supernatant is removed after centrifugation, the cells are resuspended according to the density of 1X 10 5/mL-1X 10 6/mL by using the culture solution A, 5.5 mmol/L of retinoic acid and resveratrol are added simultaneously, and the cells are transferred into a new culture flask for culturing at 37 ℃ and 5% CO 2, and the culture is recorded as P1 generation. Subculturing to P8, proliferation of cells in the medium was recorded as shown in Table 1 and FIG. 4.
Comparative example 4:
Preparing a microcarrier: mixing gelatin and polylactic acid-glycolic acid copolymer according to the mass ratio of 1:3.5 to obtain a mixture, dissolving the mixture in tetrahydrofuran with the volume fraction of 45%, heating the whole reaction system to 73 ℃, preserving heat for 80min, then cooling to 45 ℃, adding a three-dimensional ordered macroporous metal-organic framework material 3DOM-ZIF-8 which is 0.75 times of the total mass of the gelatin and the polylactic acid-glycolic acid copolymer (formed by copolymerizing 70% lactic acid and 30% glycolic acid), preserving heat for 5.5h, cooling the system to room temperature, filtering, sequentially freeze-drying the obtained solid product at-40 ℃, drying in a vacuum constant-temperature drying oven at 45 ℃, and finally grinding the product to particles with the particle diameter of 100-500 mu m to obtain the microcarrier;
Preparing a microcarrier auxiliary agent: firstly, corn starch and water are mixed according to the mass volume ratio of 1mg:2mL of the modified corn starch powder is put into a reaction kettle, heated, stirred and dissolved, when the temperature reaches the gelatinization temperature, 1, 2-dimethyl-3-ethoxyethylimidazole bistrifluoromethylsulfonylimine salt and a metal compound (composed of zinc chloride, copper chloride, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, ferric chloride and manganese chloride according to the mass ratio of 1:1:1:1:1:1:1:1) are sequentially added while stirring, after all the materials are added, the system is heated to 50 ℃ and then is continuously stirred for 9 hours, finally, spray drying and grinding are carried out to obtain modified corn starch powder, the obtained modified corn starch powder is dispersed in water in an ultrasonic manner, and the pH=7 of the solution is regulated to obtain a modified corn starch solution with the concentration of 40wt%, namely the microcarrier auxiliary agent; during the reaction, 1, 2-dimethyl-3-ethoxyethylimidazole bis (trifluoromethanesulfonyl) imide salt, metal compound and corn starch are mixed according to 0.045mL:0.6mg:1 g.
Preparing a culture solution A: the basic culture solution is DMEM culture solution; the exogenous additives are 12.5mg/L of insulin, 15mg/L of transferrin, 20mg/L of epidermal growth factor, 25mg/L of nerve growth factor, 20mg/L of insulin growth factor, 20mg/L of basic fibroblast growth factor and 4mmol/L of progesterone, and the pH value of the solution is regulated to 6.9-7.3.
Preparing a culture solution B: the basic culture solution is DMEM culture solution; the exogenous additive is 12.5mg/L of insulin, 15mg/L of transferrin, 20mg/L of epidermal growth factor, 25mg/L of nerve growth factor, 20mg/L of insulin growth factor, 20mg/L of basic fibroblast growth factor, 4mmol/L of progesterone, 2g/L of microcarrier (30 mL/L of microcarrier auxiliary agent) and the pH value of the solution is regulated to 6.9-7.3.
The cell culture method of this example is as follows: pancreatic cells were resuspended in medium A to a density of 1X 10 5 to 1X 10 6 cells/mL and inoculated into culture flasks. Culturing the culture flask in a culture flask containing 5% CO 2 at 37 ℃ for 1 hour, sucking non-adherent cells, transferring the culture flask to a culture flask containing the culture solution B (without microcarrier auxiliary agent) and culturing the culture flask for 18 hours at 37 ℃ with 5% CO 2, discarding the non-adherent cells and supernatant, adding the culture solution B (without microcarrier auxiliary agent) equal to the discarded supernatant at 37 ℃ with 5% CO 2, and continuously culturing the culture flask once every 3 days. The equal cell fusion degree reaches 80%, the supernatant is discarded, PBS is used for cleaning for 2 times, pancreatin with 0.4% EDTA concentration of 0.25% is added for digesting cells for 1 minute, 3 times of the volume of the digestive juice is added into culture solution B (containing microcarrier auxiliary agent) to stop digestion, after centrifugation, the supernatant is removed, cells are resuspended according to the density of 1X 10 5/mL-1X 10 6/mL by using the culture solution B (containing microcarrier auxiliary agent), and the cells are transferred into a new culture flask for culturing at 37 ℃ and 5% CO 2, and the culture is marked as generation P1. Subculturing to P8, proliferation of cells in the medium was recorded as shown in Table 1 and FIG. 5.
TABLE 1 summary of cell proliferation
As can be seen from Table 1 and FIGS. 1 to 5, the culture solutions of examples 1 to 3 of the present invention can significantly improve the cell proliferation ability as compared with comparative examples 1 to 4. Comparative example 4, in which retinoic acid and resveratrol were absent, was able to slightly reduce the number of cell proliferation, but had significantly less effect than comparative example 1 without microcarrier aid, comparative example 2 without microcarrier and comparative example 3 without microcarrier aid at the same time as microcarrier. Meanwhile, compared with the example, the comparative example 3 without microcarrier auxiliary agent and microcarrier has obviously reduced cell proliferation capacity, which shows that the microcarrier and microcarrier auxiliary agent have synergistic effect and obviously improve cell proliferation capacity.
While the invention has been described with respect to preferred embodiments, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention, and that any such changes and modifications as described in the above embodiments are intended to be within the scope of the invention.
Claims (5)
1. The culture solution for replacing serum in cell culture consists of a basic culture solution and an exogenous additive, and is characterized in that the basic culture solution is a DMEM culture solution or a F12 culture solution or a mixed culture solution of the DMEM culture solution and the F12 culture solution according to a volume ratio of 1:2; the exogenous additives are necessary supplementary factors, cell growth factors, microcarriers, microcarrier auxiliary agents, progesterone and pH regulators; wherein,
The preparation method of the microcarrier comprises the following steps: mixing gelatin and polylactic acid-glycolic acid copolymer according to the mass ratio of 1:2-5 to obtain a mixture, dissolving the mixture in tetrahydrofuran with the volume fraction of 40-50%, heating the whole reaction system to 60-85 ℃, preserving heat for 60-100 min, cooling to 40-50 ℃, adding a three-dimensional ordered macroporous metal-organic frame material, preserving heat for 3-8 h, cooling to room temperature, filtering, sequentially freeze-drying the obtained solid product at-50 to-30 ℃, drying in a vacuum constant temperature drying oven at 40-50 ℃, and finally grinding the product until particles with the particle diameter of 100-500 mu m are microcarriers;
The preparation method of the microcarrier auxiliary agent comprises the following steps: firstly, starch and water are mixed according to the mass volume ratio of 1mg: placing the 1-3 mL of the mixture into a reaction kettle, heating, stirring and dissolving the mixture, sequentially adding the ionic liquid and the metal compound while stirring when the temperature reaches the gelatinization temperature, heating the system to 40-60 ℃ after all the adding is completed, continuously stirring the mixture for 8-10 hours, finally performing spray drying and grinding to obtain modified starch powder, ultrasonically dispersing the obtained modified starch powder into water, and adjusting the pH value of the solution to be 7 to obtain a modified starch solution with the concentration of 30-50wt%, namely the microcarrier auxiliary agent;
The addition amount of the three-dimensional ordered macroporous metal-organic framework material in the reaction process is 0.5-1 times of the total mass of the gelatin and the polylactic acid-glycolic acid copolymer;
In the reaction process, the ionic liquid, the metal compound and the starch are mixed according to the ratio of 0.03-0.06 mL: 0.4-0.8 mg:1g of the mixture is added;
The ionic liquid is selected from any one of 1, 2-dimethyl-3-ethoxyethylimidazole bistrifluoromethylsulfonylimine salt, 1-methyl-3-ethoxyethylimidazole bistrifluoromethylsulfonylimine salt, N-butyl-N-methylpyrrolidine bistrifluoromethylsulfonylimine salt, 1-methyl-3-ethoxymethylimidazole bistrifluoromethylsulfonylimine salt, N-ethoxymethyl-N-methylpyrrolidine bistrifluoromethylsulfonylimine salt, 1-methyl-3-butylimidazole bistrifluoromethylsulfonylimine salt, N-ethoxyethyl-N-methylpyrrolidine bistrifluoromethylsulfonylimine salt and N, N-diethyl-2-methoxyethyl-N-methylamine bistrifluoromethylsulfonylimine salt;
The metal compound consists of zinc chloride, copper chloride, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, ferric chloride and manganese chloride according to the mass ratio of 1:1:1:1:1:1:1:1;
the concentration of the microcarrier is 1-3 g/L; the concentration of the microcarrier auxiliary agent is 10-50 mL/L;
The necessary supplementary factors are insulin with the concentration of 5-30mg/L and transferrin with the concentration of 10-20 mg/L; the growth factors comprise an epidermal growth factor, a nerve growth factor, an insulin growth factor and an alkaline fibroblast growth factor, wherein the concentration of the epidermal growth factor is 15-25 mg/L, the concentration of the nerve growth factor is 20-30 mg/L, the concentration of the insulin growth factor is 15-25 mg/L and the concentration of the alkaline fibroblast growth factor is 10-30 mg/L; the concentration of the progesterone is 3-5 mmol/L; and the pH regulator regulates the pH of the culture solution to 6.9-7.3.
2. The culture broth of claim 1, wherein the three-dimensional ordered macroporous metal-organic framework material is selected from one or a mixture of two or more of 3DOM-PCN-601、3DOM-PCN-14、3DOM-ZIF-8、3DOM-ZIF-67、3DOM-ZIF-68、3DOM-ZIF-69、3DOM-ZIF-70、3DOM-ZIF-78、3DOM-ZIF-81、3DOM-ZIF-82、3DOM-ZIF-95、3DOM-ZIF-100、3DOM-UIO-66.
3. A method for culturing islet stem cells, characterized by culturing with the culture solution according to claim 1 or 2, comprising the steps of:
S1: adding growth factors, cell growth factors and progesterone which are required to be supplemented into the basic culture solution, and adjusting the pH to 6.9-7.3 to obtain a culture solution A;
Re-suspending pancreatic cells to a density of1×10 5 to 1×10 6 cells/mL with culture solution a, culturing for 1h, and transferring non-adherent cells;
S2: adding growth factors, cell growth factors, progesterone and microcarriers which are required to be supplemented into a basic culture solution, and adjusting the pH to 6.9-7.3 to obtain a culture solution B;
Culturing the non-adherent cells with a culture solution B for 18 hours, and then discarding the non-adherent cells and the culture solution B to obtain adherent cells;
S3: culturing the adherent cells in a culture solution B until the cell fusion degree is 70% -90%, adding microcarrier auxiliary agent into the culture solution B, uniformly stirring, and performing subculture for 8 times.
4. The method according to claim 3, wherein in step S3, retinoic acid and resveratrol with concentrations of 1-10 mmol/L are added within 6-8 hours after adding the microcarrier adjuvant.
5. The method of claim 3, wherein the pancreatic cells are prepared by: pancreatic cells were prepared by digestion of pancreatic tissue with collagenase type four and washing with PBS.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410341551.8A CN117925509B (en) | 2024-03-25 | 2024-03-25 | Cell culture substituting serum culture solution and method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410341551.8A CN117925509B (en) | 2024-03-25 | 2024-03-25 | Cell culture substituting serum culture solution and method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117925509A CN117925509A (en) | 2024-04-26 |
| CN117925509B true CN117925509B (en) | 2024-06-11 |
Family
ID=90768759
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410341551.8A Active CN117925509B (en) | 2024-03-25 | 2024-03-25 | Cell culture substituting serum culture solution and method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117925509B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR19990042747A (en) * | 1997-11-28 | 1999-06-15 | 조생현 | Serum-free medium for cell culture |
| WO2004055174A1 (en) * | 2002-12-13 | 2004-07-01 | Celogos | Culture medium composition, culture method, and myoblasts obtained, and their uses |
| CN101121926A (en) * | 2007-07-02 | 2008-02-13 | 西北农林科技大学 | Limbus corneae stem cell serum-free culture medium |
| CN104928233A (en) * | 2015-07-02 | 2015-09-23 | 广州赛莱拉干细胞科技股份有限公司 | Culture solution and method of islet-like cells |
| CN107129966A (en) * | 2017-06-18 | 2017-09-05 | 广东博溪生物科技有限公司 | A kind of corneal epithelial cell nutrient solution containing serum |
| CN115942952A (en) * | 2020-08-21 | 2023-04-07 | 迈阿密大学 | Therapeutic compositions and methods using microvesicles from bone marrow-derived mesenchymal stem cells |
-
2024
- 2024-03-25 CN CN202410341551.8A patent/CN117925509B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR19990042747A (en) * | 1997-11-28 | 1999-06-15 | 조생현 | Serum-free medium for cell culture |
| WO2004055174A1 (en) * | 2002-12-13 | 2004-07-01 | Celogos | Culture medium composition, culture method, and myoblasts obtained, and their uses |
| CN101121926A (en) * | 2007-07-02 | 2008-02-13 | 西北农林科技大学 | Limbus corneae stem cell serum-free culture medium |
| CN104928233A (en) * | 2015-07-02 | 2015-09-23 | 广州赛莱拉干细胞科技股份有限公司 | Culture solution and method of islet-like cells |
| CN107129966A (en) * | 2017-06-18 | 2017-09-05 | 广东博溪生物科技有限公司 | A kind of corneal epithelial cell nutrient solution containing serum |
| CN115942952A (en) * | 2020-08-21 | 2023-04-07 | 迈阿密大学 | Therapeutic compositions and methods using microvesicles from bone marrow-derived mesenchymal stem cells |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117925509A (en) | 2024-04-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106119186B (en) | It is a kind of for the serum free medium and preparation method thereof for cultivating mdck cell that suspends entirely | |
| CN108220230B (en) | Method for separating and culturing human adipose-derived stem cells | |
| CN108949678A (en) | A kind of stem cell media and cultural method | |
| CN111454877B (en) | CHO cell culture method | |
| CN111206017A (en) | Serum-free culture medium for stem cells and application thereof | |
| Hewlett | Strategies for optimising serum-free media | |
| CN111440764B (en) | Serum-free culture medium of mesenchymal stem cells and clinical-grade large-scale culture method of mesenchymal stem cells | |
| CN117925509B (en) | Cell culture substituting serum culture solution and method | |
| CN103484426B (en) | Non-animal source low-protein culture medium | |
| EP1438395A1 (en) | Growing xenotransplant material in culture | |
| CN103421736B (en) | Medium additive replacing animal serum in CHO cell culture and preparation method thereof | |
| CN109439628B (en) | Primary culture method of limbal stem cells | |
| CA3225535A1 (en) | Cell culture medium and supplements for corneal and skin cell culture | |
| CN110564688B (en) | Method for culturing limbal stromal stem cells without serum and inducing balling and differentiation in vitro | |
| CN111944763B (en) | CAR-T cell culture medium and culture method | |
| CN110923205A (en) | Lymphatic endothelial cell culture medium and preparation method and application thereof | |
| CN114214266A (en) | Gel composition, biological scaffold gel, and preparation method and application thereof | |
| CN111849863A (en) | Culture medium additive supporting CHO cell to efficiently produce monoclonal antibody and preparation method and application thereof | |
| CN112342187A (en) | Chondrocyte culture medium and preparation method thereof | |
| CN113106054A (en) | Inducer for inducing and differentiating mesenchymal stem cells into islet cells | |
| CN110669728A (en) | Crocodile adipose-derived stem cell separation culture method and serum-free culture medium | |
| CN109880796B (en) | Cell culture method for rapidly obtaining primary cells in vitro | |
| CN113293126B (en) | In-vitro construction method of human vaginal mucosa model | |
| CN115896008A (en) | Low-serum culture medium and application thereof in preparation of cell culture meat of large yellow croaker | |
| CN115109745B (en) | Efficient separation and primary culture method for liver cells of pelteobagrus fulvidraco |
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 | ||
| GR01 | Patent grant |