CN109880793A - A kind of youth's porcine islet culture medium and its application method - Google Patents
A kind of youth's porcine islet culture medium and its application method Download PDFInfo
- Publication number
- CN109880793A CN109880793A CN201910194216.9A CN201910194216A CN109880793A CN 109880793 A CN109880793 A CN 109880793A CN 201910194216 A CN201910194216 A CN 201910194216A CN 109880793 A CN109880793 A CN 109880793A
- Authority
- CN
- China
- Prior art keywords
- culture
- culture medium
- islet
- cell
- medium
- 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.)
- Granted
Links
- 239000001963 growth medium Substances 0.000 title claims abstract description 65
- 238000000034 method Methods 0.000 title abstract description 13
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims abstract description 26
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 claims abstract description 24
- 229920000669 heparin Polymers 0.000 claims abstract description 15
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims abstract description 14
- 108010024636 Glutathione Proteins 0.000 claims abstract description 13
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 claims abstract description 13
- 229960003180 glutathione Drugs 0.000 claims abstract description 13
- 239000011570 nicotinamide Substances 0.000 claims abstract description 13
- 229960003966 nicotinamide Drugs 0.000 claims abstract description 13
- 235000005152 nicotinamide Nutrition 0.000 claims abstract description 13
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims abstract description 12
- BHHGXPLMPWCGHP-UHFFFAOYSA-N Phenethylamine Chemical compound NCCC1=CC=CC=C1 BHHGXPLMPWCGHP-UHFFFAOYSA-N 0.000 claims abstract description 12
- 229930003427 Vitamin E Natural products 0.000 claims abstract description 12
- GDXWHFPKFUYWBE-UHFFFAOYSA-N [F].Cl Chemical compound [F].Cl GDXWHFPKFUYWBE-UHFFFAOYSA-N 0.000 claims abstract description 12
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 claims abstract description 12
- 229960002897 heparin Drugs 0.000 claims abstract description 12
- 235000019165 vitamin E Nutrition 0.000 claims abstract description 12
- 229940046009 vitamin E Drugs 0.000 claims abstract description 12
- 239000011709 vitamin E Substances 0.000 claims abstract description 12
- HTQBXNHDCUEHJF-XWLPCZSASA-N Exenatide Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(=O)NCC(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CO)C(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 HTQBXNHDCUEHJF-XWLPCZSASA-N 0.000 claims abstract description 11
- 108010011459 Exenatide Proteins 0.000 claims abstract description 11
- 229960001519 exenatide Drugs 0.000 claims abstract description 11
- 239000007640 basal medium Substances 0.000 claims abstract description 3
- 241000282898 Sus scrofa Species 0.000 claims description 25
- 239000002609 medium Substances 0.000 claims description 14
- 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 12
- 210000002966 serum Anatomy 0.000 claims description 10
- 102000004877 Insulin Human genes 0.000 claims description 6
- 108090001061 Insulin Proteins 0.000 claims description 6
- 239000002054 inoculum Substances 0.000 claims description 6
- 229940125396 insulin Drugs 0.000 claims description 6
- 102000002070 Transferrins Human genes 0.000 claims description 2
- 108010015865 Transferrins Proteins 0.000 claims description 2
- 230000004899 motility Effects 0.000 abstract description 12
- 210000004027 cell Anatomy 0.000 description 47
- 210000004153 islets of langerhan Anatomy 0.000 description 45
- 210000000496 pancreas Anatomy 0.000 description 22
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 15
- 230000006907 apoptotic process Effects 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 239000011248 coating agent Substances 0.000 description 6
- 238000000576 coating method Methods 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 230000001427 coherent effect Effects 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 230000003914 insulin secretion Effects 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- UOFGSWVZMUXXIY-UHFFFAOYSA-N 1,5-Diphenyl-3-thiocarbazone Chemical compound C=1C=CC=CC=1N=NC(=S)NNC1=CC=CC=C1 UOFGSWVZMUXXIY-UHFFFAOYSA-N 0.000 description 4
- DPKHZNPWBDQZCN-UHFFFAOYSA-N acridine orange free base Chemical compound C1=CC(N(C)C)=CC2=NC3=CC(N(C)C)=CC=C3C=C21 DPKHZNPWBDQZCN-UHFFFAOYSA-N 0.000 description 4
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 4
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 4
- 229960005542 ethidium bromide Drugs 0.000 description 4
- 238000002054 transplantation Methods 0.000 description 4
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 description 3
- 229960001008 heparin sodium Drugs 0.000 description 3
- 239000007928 intraperitoneal injection Substances 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical group OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 206010021143 Hypoxia Diseases 0.000 description 2
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000000306 component Substances 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 230000007954 hypoxia Effects 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000035800 maturation Effects 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- GDSOZVZXVXTJMI-SNAWJCMRSA-N (e)-1-methylbut-1-ene-1,2,4-tricarboxylic acid Chemical compound OC(=O)C(/C)=C(C(O)=O)\CCC(O)=O GDSOZVZXVXTJMI-SNAWJCMRSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 239000012981 Hank's balanced salt solution Substances 0.000 description 1
- 208000013016 Hypoglycemia Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 1
- 101000718529 Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) Alpha-galactosidase Proteins 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical class [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 229960004308 acetylcysteine Drugs 0.000 description 1
- YAJCHEVQCOHZDC-QMMNLEPNSA-N actrapid Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3N=CNC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@H](C)O)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@H](C)CC)[C@H](C)CC)[C@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C(N)=O)C1=CNC=N1 YAJCHEVQCOHZDC-QMMNLEPNSA-N 0.000 description 1
- NNISLDGFPWIBDF-MPRBLYSKSA-N alpha-D-Gal-(1->3)-beta-D-Gal-(1->4)-D-GlcNAc Chemical compound O[C@@H]1[C@@H](NC(=O)C)C(O)O[C@H](CO)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@@H](CO)O1 NNISLDGFPWIBDF-MPRBLYSKSA-N 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 210000000270 basal cell Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- LEMUFSYUPGXXCM-JNEQYSBXSA-N caninsulin Chemical compound [Zn].C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC3N=CN=C3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)O)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1C=NC=N1 LEMUFSYUPGXXCM-JNEQYSBXSA-N 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000011748 cell maturation Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000005713 exacerbation Effects 0.000 description 1
- 210000003499 exocrine gland Anatomy 0.000 description 1
- 230000002218 hypoglycaemic effect Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 230000001861 immunosuppressant effect Effects 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 150000002978 peroxides Chemical class 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229940119528 pork insulin Drugs 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000002689 xenotransplantation Methods 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of young porcine islet culture medium and its application methods.The culture medium is the addition in basal medium (M199): glutathione 1-50mM, glutamine 1-50mM, ITS 0.01-1%, niacinamide 1-50mM, 0.1-20 μM of watermiscible vitamin E, heparin 1-100U/L, 4- (2- aminoethyl) benzene sulfonyl fluorine hydrochloride 0.01-0.5mM, 0.01-0.1 μM of Exenatide.Young porcine islet motility rate and its maturing rate can be significantly improved compared to ordinary culture medium (1640, F10, EGM2).
Description
Technical field
The invention belongs to animal insulin technical field of cell culture, and in particular to it is a kind of youth porcine islet culture medium and
Its application method.
Background technique
Type-1 diabetes mellitus is that most threatening one of chronic metabolic diseases are generally acknowledged in the whole world, and feature, which is that selectivity is immune, to be situated between
Destruction beta Cell of islet is led, patient is made to need exogenous insulin to treat all the life.The terminal stage of a disease breaks out various complication, will lead
Cause the great exacerbation and high mortality of minimal invasive treatment and financial burden.After edmonton protocol successful implementation, pancreatic islets transplantation
By highly visible.After summarizing the pancreatic islets transplantation research achievement basis in the whole world, promote transplantation method and immunosuppressant scheme
It improves, brings hope for type-1 diabetes mellitus patient.Human pancreatic's islet allografts have proven to be a kind of feasible
The method for treating type-1 diabetes mellitus.It can improve and control patient blood glucose after transplanting, reduce the demand to insulin, and reduce
Hypoglycemia occurrence probability.But the difficulty in people's pancreas islet source, isolated pancreatic islet purification technique it is not perfect, there are the wind of transmission
Danger, so that people's pancreatic islets transplantation has significant limitation.
Pork insulin and actrapid monotard are quite similar, and blood glucose set point is close, very as human pancreatic islet substitute
It is suitble to.Young pig pancreas islet compares that adult pig immunogenicity is low, and β cell has certain division potential；Although young pig pancreas is not complete
Complete reaches maturity, but compares with neonatal pig, and the pancreas volume of young pig and weight etc. are all bigger, the α-Gal antigen of pancreas islet
Expression is weak, and the maturity of pancreas islet is also relatively high, therefore can obtain the islet cells of a large amount of maturations.From Physiology and biochemistry spy
The substitute of people's pancreas islet is more suitable for in property.
Porcine islet culture now is all made of CMRL1066, and the cell culture mediums such as RPMI are cultivated 3-5 days, maintains pancreas islet
Growth or induce its directed differentiation.But these application methods and culture medium are constantly present many defects, such as: pancreas after culture
Island cell quantity is unstable, the different (2000IEQ/g- of islet cells amount for the active function of obtaining after purification
4000IEQ/g), there is also difference (70-90%) for the islet cells purity of culture；Islet cells is because of rapid apoptosis in incubation
And cell is largely lost, especially β cell.The length of incubation is also to be difficult to select, and the short β cell of incubation time cannot develop
Maturation, long a large amount of islet cells quantity due to apoptosis that has again of incubation time are reduced, and since the apoptosis of β cell makes same number
The reduction of amount of insulin secretion in islet cells under amount (insulin/DNA value is < 49.00mIU/g).In addition, with culture
The extension of time, islet cells group is more easily broken, so that forming smaller islet cells group (diameter < 50um) is easier to apoptosis,
It is difficult to continue to cultivate.Even to this day active and constant rate of production the young pig pancreas islet for not also being capable of providing high-purity is thin
Born of the same parents' cultural method and culture medium, so how to establish the medium component of optimization and training method becomes young pig pancreas islet
The cell culture most pressing problem.
Summary of the invention
The purpose of the present invention is provide a kind of efficient and stable young porcine islet group culture for clinical xenotransplantation
System.
Young pig pancreas is more mature compared to neonatal pig pancreas, has more mature exocrine gland, so as to cause being separately cultured
Digestive ferment abundant is secreted in the process, and islet cells is caused to damage；Therefore addition proteases inhibitors are needed during culture, are prevented
Only islet cells group is digested the destruction such as enzyme.Although young porcine islet group is compared with live pig pancreatic island cell group, yield
And function is more advantageous, but since young pig pancreas islet group itself is more mature, volume is relatively large, hypoxia endurance is wanted
Than live pig pancreatic island cell, group is poor, therefore additional addition antioxidant is considered in later period culture, is generated with removing due to anoxic
Peroxide, superoxides are damaged caused by cell, to improve hypoxia-bearing capability, reduce the cellular damage because of caused by anoxic.
Lasting culture discovery F10 (containing niacinamide, glutamine etc.), EGM-2 are (containing Sodium Pyruvate, niacinamide
Deng) and RPMI1640 culture medium cultivate young porcine islet respectively, effect is not up to expected.Cultivate 3 days islet cells
Group can not form a film, and unicellular more, can not cultivate to its β cell maturation of the 5th angel (see Fig. 1).
Culture medium of the invention is in basal medium M199 containing glutathione 1-50mM, glutamine 1-50mM, pancreas
Island element transferrins (ITS) 0.01-1%, niacinamide 1-50mM, 0.1-20 μM of watermiscible vitamin E, heparin 50-100U/
L, 4- (2- aminoethyl) benzene sulfonyl fluorine hydrochloride 0.01-0.5mM, 0.01-0.1 μM of Exenatide.
It is preferred that: it is to contain glutathione 1-25mM, glutamine 1-25mM, ITS 0.01-0.5% in minimal medium,
Niacinamide 1-25mM, 0.1-10 μM of watermiscible vitamin E, heparin 50-100U/L, 4- (2- aminoethyl) benzene sulfonyl fluorine hydrochloric acid
Salt 0.01-0.25mM, 0.05-0.1 μM of Exenatide.
It is further preferred: to be to contain glutathione 2mM, glutamine 2mM, ITS 0.06%, Ni Ke in minimal medium
Amide 2mM, 0.1 μM of watermiscible vitamin E, heparin 100U/L, 4- (2- aminoethyl) benzene sulfonyl fluorine hydrochloride 0.2mM, Ai Saina
0.1 μM of peptide.
Further, Swine serum, the preferably Swine serum of 10% volume are also contained in minimal medium M199.
The application method of the young porcine islet culture medium: the inoculation of young porcine islet in the medium is dense
Degree is 5000-10000IEQ/25-30mL, in 37 DEG C, 5%CO2, culture in 95% air jet flow case.Cell is first when culture
Its replacement culture medium and culture dish, hereafter every other day replacement is primary, cultivates to 5-7 days.
Beneficial effects of the present invention (following data derives from many experimental results, and has statistical significance):
Same pancreas islet equivalent (1 × 104IEQ it) cultivates 5 days, the active islet cells motility rate of culture medium culture of the present invention reaches
93.48 ± 1.25%, it is higher than the islet cells motility rate 70.03 ± 2.36% of general culture (1640 culture medium), and the present invention trains
The islet cells group particle diameter for supporting base culture 5 days is greater than 100 μm mostly, and cell mass coating is complete, and it is (straight to be crushed islet cells group
Diameter < 50 μm) less than the islet cells of general culture medium (1640 culture medium) culture 5 days, it is more suitable for transplanting.It cultivates 5 days and obtains
Islet cells there is sensitive responsibility to glucose stimulation, SI index is the 2 of general culture medium (1640 culture medium)
Times or so, and β cell proportion is 80.13 ± 1.69% in the islet cells of culture 5 days, culture of the present invention 7 days β cell ratios
Example is 85.96 ± 1.56%, and the relatively ratio of generally culture (1640 culture medium) is high, it was demonstrated that the islet cell function of culture of the present invention
Good, amount of insulin secretion increased significantly (P < 0.05), compensate for the islet cells of general culture (1640 culture medium) because apoptosis is led
Cause the deficiency of insulin secretion decline.
Optimal Medium of the invention reduces apoptosis rate, increases cell recoveries, reduces Apoptosis quantity,
Islet cells group after culture is set to be crushed reduction, coating is more complete, and the pancreas islet short period cultivates more mature, equal islet cell
Amount of insulin secretion is obviously improved, and establishes stability and high efficiency, and cell acquisition amount is big, cell high activity, high survival degree, and cell mass
Sizeable youth's porcine islet cultivating system.
Advantage of the present invention is summarized as follows:
1, Apoptosis declines, and the rate of recovery increases；
2, islet cells group is broken is reduced, and cell mass coating is complete, is more suitable for transplanting；
3, beta Cell of islet vigor increases；
4, same amount of islet cells amount of insulin secretion increases；
5, culture cell has sensitive glucose stimulation responses, and function is more mature than the islet cells generally cultivated.
Detailed description of the invention
Fig. 1 is to detect F10 culture medium, EGM-2 culture medium and culture medium of the present invention under ordinary optical microscope to cultivate 3 respectively
It islet cells group MIcrosope image, amplification factor 100 ×, length of the scale is 200 μm in figure；
Fig. 2 is that the culture medium case study on implementation 1,2,3 of the present invention of detection various concentration under ordinary optical microscope cultivates 3 respectively
It islet cells group MIcrosope image, amplification factor 100 ×；
Fig. 3 is the islet cells that DTZ detects ordinary culture medium (1640) and culture medium of the present invention cultivates 5 days and 7 days respectively
Group's MIcrosope image, amplification factor 100 ×, length of the scale is 200 μm in figure；
Fig. 4 be AO/EB detect ordinary culture medium (1640) and culture medium of the present invention cultivate respectively 5 days and 7 days pancreas islet it is thin
Born of the same parents group's MIcrosope image, amplification factor 40 ×, length of the scale is 400 μm in figure；
Fig. 5 is that islet cells motility rate comparison in 5 days is cultivated in comparison case 1；
Fig. 6 is that islet cells motility rate comparison in 5 days is cultivated in comparison case 2.
Specific embodiment
It is intended to further illustrate, is not intended to limit the present invention with reference to embodiments.
Material and reagent: 50ml centrifuge tube (Jie Te), 15ml centrifuge tube (Jie Te), 10ml syringe, incubator (Sai Mo
Fly), 1640 culture mediums, M199 culture medium, glutamine, glutathione, Swine serum, HBSS, antibiotic (life), DTZ, AO/
EB dyeing liquor, AnnexinV/PI kit (life), 0.22 μm of filter (Merck millipore) etc..
Mentioned reagent consumptive material is purchased from sigma company without specified otherwise.
Case study on implementation 1, culture medium and cultural method test of the present invention
The present invention takes the preoperative intraperitoneal injection 10IU/kg heparin sodium of pancreas in young pig, and the present invention is for separated purifying
The inoculum density of young porcine islet in the medium is 5000-10000IEQ/25-30mL, and culture medium is (in M199 culture medium
Add glutathione 25mM, glutamine 25mM, ITS0.5%, niacinamide 20mM, 9 μM of watermiscible vitamin E, heparin
60U/L, 4- (2- aminoethyl) benzene sulfonyl fluorine hydrochloride 0.25mM, 0.1 μM of Exenatide.And account for culture volume percentage
10% Swine serum) in 15cm not adhere-wall culture ware, it is placed in 37 DEG C, 5%CO2, culture in 95% air jet flow case, preparation
Cell in first day replacement culture dish and culture medium, hereafter every other day replacement is primary, in culture collection in the 5th day and the 7th day
Cell count simultaneously does coherent detection.
Case study on implementation 2
The present invention takes the preoperative intraperitoneal injection 10IU/kg heparin sodium of pancreas in young pig, and the present invention is for separated purifying
The inoculum density of young porcine islet in the medium is 5000-10000IEQ/25-30mL, and culture medium is (in M199 culture medium
Add glutathione 50mM, glutamine 50mM, ITS 1%, niacinamide 45mM, 20 μM of watermiscible vitamin E, heparin
50U/L, 4- (2- aminoethyl) benzene sulfonyl fluorine hydrochloride 0.01mM, 0.1 μM of Exenatide.And account for culture volume percentage
10% Swine serum) in 15cm not adhere-wall culture ware, it is placed in 37 DEG C, 5%CO2, culture in 95% air jet flow case, preparation
Cell in first day replacement culture dish and culture medium, hereafter every other day replacement is primary, in culture collection in the 5th day and the 7th day
Cell count simultaneously does coherent detection.
Case study on implementation 3
The present invention takes the preoperative intraperitoneal injection 10IU/kg heparin sodium of pancreas in young pig, and the present invention is for separated purifying
The inoculum density of young porcine islet in the medium is 5000-10000IEQ/25-30mL, and culture medium is (in M199 culture medium
Add glutathione 2mM, glutamine 2mM, ITS 0.06%, niacinamide 2mM, 0.1 μM of watermiscible vitamin E, heparin
100U/L, 4- (2- aminoethyl) benzene sulfonyl fluorine hydrochloride 0.2mM, 0.1 μM of Exenatide and accounts for culture volume percentage
10% Swine serum) in 15cm not adhere-wall culture ware, it is placed in 37 DEG C, 5%CO2, culture in 95% air jet flow case, preparation
Cell in first day replacement culture dish and culture medium, hereafter every other day replacement is primary, in culture collection in the 5th day and the 7th day
Cell count simultaneously does coherent detection.
As a result:
Various concentration culture medium of the present invention shows preferable culture effect in 3 case study on implementation (see Fig. 2).Pancreas islet
After cell mass culture 3 days, cell mass film forming is preferably, unicellular few, and about 200 μm of cell mass diameter.Cell in case 1 and case 3
Group is bigger than the cell mass in case 2, illustrates that the protease inhibitors of higher concentration has certain protection to the film forming of cell mass
Effect.
Embodiment 4, the present invention are compared in general culture medium (RPMI 1640) culture effect, the results are shown in Table 1.
Test detection means:
Cell mass counts: collecting culture cell, 1000rpm is centrifuged 1 minute, abandons supernatant, and physiological saline is resuspended, is settled to
50mL takes 50 μ L suspensions, and dithizone (DTZ) dyes, and counts under microscope, and quantity is as total multiplied by 1000 times.
Dead cell ratio living: taking the acutase enzymic digestion of 1500-2000IEQ islet cells group is individual cells,
By flow cytometer to living cells and dead cell counts after AnnexinV/PI dyeing.
Insulin measurement: commercially available chemical luminescence reagent kit.
The detection of β cell viability: taking 1500-2000IEQ islet cells acutase enzymic digestion is individual cells, NEWPORT
GREEN specifically contaminates β cell, and overflow-type cell instrument detects activity β cell.
Case study on implementation 3 of the present invention cultivates 5 days active islet cells motility rates and reaches 93.48 ± 1.25%, than what is generally cultivated
Islet cells (< 75%) is high；Active islet cells motility rate reaches 92.30 ± 0.56% within culture of the present invention 7 days, significantly larger than generally
Culture medium (< 35%)；5 days β cell percentages 80.13 ± 1.69% of culture of the present invention, also than general culture medium (< 76%)
It is high；7 days β cell percentages of culture of the present invention reach 85.96 ± 1.56%, than general culture medium (< 35%) height；In addition,
Culture of the present invention 5 days SI glucose stimulus indexes (3.74 ± 1.35) are also much higher than general culture medium (2.13 ± 1.11).
And the islet cells group particle diameter of culture medium culture 5 days of the present invention is greater than 100 μm mostly, cell mass coating is complete
It is whole, it is crushed the islet cells that islet cells group (diameter < 50 μm) are less than general culture medium culture 5 days, is particularly suited for transplanting.Such as
Cell is used acridine orange (AO) and ethidium bromide (EB) to dye by Fig. 3 simultaneously, and living cells is under fluorescence microscope 488nm exciting light
In green fluorescence, dead cell takes on a red color fluorescence under 546nm exciting light, and picture is islet cells respectively in ordinary culture medium and this
Cultivated 5 days and 7 days in invention improved culture medium, amplification 40 × picture, in Fig. 4 as can be seen that the present invention cultivate basal cell
Group's periphery coating is complete, and cell mass is uniform in size, and for most diameter in 100-200nm, dead cell is few, and superposition picture is almost seen
Less than dead cell；Ordinary culture medium cell mass size distribution is uneven, and coating is imperfect, and cell mass edge is unsmooth, is easily broken
It is dead for individual cells.
1 islet cells motility rate of table
2 β cell content of table
3 SI value of table
Compare case 1
Young porcine islet inoculum density in the medium for separated purifying is 5000-10000IEQ/25-
30mL, culture medium (add glutathione 2mM, glutamine 2mM, ITS 0.06%, niacinamide 2mM, water in M199 culture medium
0.1 μM of soluble vitamin E, heparin 100U/L, 4- (2- aminoethyl) benzene sulfonyl fluorine hydrochloride 0.2mM, 0.1 μM of Exenatide, resist
Bad hematic acid 1mM and the Swine serum for accounting for culture volume percentage 10%) in 15cm not adhere-wall culture ware, be placed in 37 DEG C,
5%CO2, culture in 95% air jet flow case, the cell of preparation is in first day replacement culture dish and culture medium, hereafter every other day
Replacement is primary, in culture collection cell count in the 5th day and does coherent detection.
As a result:
As a result as shown in figure 5, using 3 culture medium culture youth porcine islet of case study on implementation of the present invention to the 5th day motility rate
It is 93.48 ± 1.25%, compared to addition ascorbic acid group motility rate 85.30 ± 1.24%, there is significant difference.Show long-term
In incubation, each matching component effect of culture medium of the present invention is more excellent.
Compare case 2
Young porcine islet inoculum density in the medium for separated purifying is 5000-10000IEQ/25-
30mL, culture medium (add glutathione 2mM, glutamine 2mM, ITS 0.06%, niacinamide 2mM, water in M199 culture medium
0.1 μM of soluble vitamin E, heparin 100U/L, 4- (2- aminoethyl) benzene sulfonyl fluorine hydrochloride 0.2mM, 0.1 μM of Exenatide, N-
Acetylcysteine 3mM and the Swine serum for accounting for culture volume percentage 10%) in 15cm not adhere-wall culture ware, it is placed in
37 DEG C, 5%CO2, culture in 95% air jet flow case, the cell of preparation is in first day replacement culture dish and culture medium, hereafter often
Replacement is primary every two days, in culture collection cell count in the 5th day and does coherent detection.
As a result:
As a result as shown in fig. 6, using 3 culture medium culture youth porcine islet of case study on implementation of the present invention to the 5th day motility rate
It is 93.48 ± 1.25%, compared to addition N-acetylcystein motility rate 76.30 ± 2.62%, there is significant difference.Show
During long-term cultivation, each matching component effect of culture medium of the present invention is more excellent.
Claims (7)
1. a kind of youth's porcine islet culture medium, which is characterized in that be to contain glutathione 1- in basal medium M199
50mM, glutamine 1-50mM, insulin transferrins 0.01-1%, niacinamide 1-50mM, watermiscible vitamin E 0.1-
20 μM, heparin 1-100U/L, 4- (2- aminoethyl) benzene sulfonyl fluorine hydrochloride 0.01-0.5mM, 0.01-0.1 μM of Exenatide.
2. youth's porcine islet culture medium according to claim 1, which is characterized in that contained in minimal medium M199
There is a glutathione 1-25mM, glutamine 1-25mM, ITS 0.01-0.5%, niacinamide 1-25mM, watermiscible vitamin E
0.1-10 μM, heparin 50-100U/L, 4- (2- aminoethyl) benzene sulfonyl fluorine hydrochloride 0.01-0.25mM, Exenatide 0.05-0.1
μM。
3. youth's porcine islet culture medium according to claim 2, which is characterized in that contained in minimal medium M199
There are a glutathione 2mM, glutamine 2mM, ITS 0.06%, niacinamide 2mM, 0.1 μM of watermiscible vitamin E, heparin
100U/L, 4- (2- aminoethyl) benzene sulfonyl fluorine hydrochloride 0.2mM, 0.1 μM of Exenatide.
4. youth's porcine islet culture medium according to claim 1, which is characterized in that also contain in minimal medium M199
There are Swine serum, the preferably Swine serum of 10% volume.
5. the described in any item culture mediums of claim 1-4 are cultivating the application in young porcine islet.
6. application according to claim 5, the inoculum density of young porcine islet in the medium is 5000-
10000IEQ/25-30mL, in 37 DEG C, 5%CO2, culture in 95% air jet flow case.
7. application according to claim 5, in first day replacement culture medium and culture dish when culture, hereafter every other day more
It changes once, cultivates to 5-7 days.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910194216.9A CN109880793B (en) | 2019-03-14 | 2019-03-14 | Young pig islet cell culture medium and using method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910194216.9A CN109880793B (en) | 2019-03-14 | 2019-03-14 | Young pig islet cell culture medium and using method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109880793A true CN109880793A (en) | 2019-06-14 |
| CN109880793B CN109880793B (en) | 2020-01-10 |
Family
ID=66932327
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910194216.9A Active CN109880793B (en) | 2019-03-14 | 2019-03-14 | Young pig islet cell culture medium and using method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109880793B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111808797A (en) * | 2020-07-28 | 2020-10-23 | 湖南赛诺生物科技股份有限公司 | Application of Necrostatin-1 and preparation for promoting maturation of islet cells of newborn pigs |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003044181A1 (en) * | 2001-11-19 | 2003-05-30 | University Of Miami | Improvement of viability and function of pancreatic islets |
| CN103695367A (en) * | 2013-12-24 | 2014-04-02 | 湖南赛诺生物科技有限责任公司 | Improved culture medium for neonatal pig islet cells and use method of same |
| CN103911340A (en) * | 2013-01-04 | 2014-07-09 | 广州康睿生物医药科技有限公司 | Culture solution for promoting islet cell proliferation and preparation method thereof |
-
2019
- 2019-03-14 CN CN201910194216.9A patent/CN109880793B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003044181A1 (en) * | 2001-11-19 | 2003-05-30 | University Of Miami | Improvement of viability and function of pancreatic islets |
| CN103911340A (en) * | 2013-01-04 | 2014-07-09 | 广州康睿生物医药科技有限公司 | Culture solution for promoting islet cell proliferation and preparation method thereof |
| CN103695367A (en) * | 2013-12-24 | 2014-04-02 | 湖南赛诺生物科技有限责任公司 | Improved culture medium for neonatal pig islet cells and use method of same |
Non-Patent Citations (1)
| Title |
|---|
| 张育森等: "胰岛移植过程中胰岛培养的研究现状", 《中国组织工程研究与临床康复》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109880793B (en) | 2020-01-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4439521A (en) | Method for producing self-reproducing mammalian pancreatic islet-like structures | |
| TW200300449A (en) | Organism-culture apparatus and organism-culture method | |
| CN105462884B (en) | The isolation and purification method of mycoplasma anatis culture medium and mycoplasma anatis | |
| CN103409368A (en) | A method for purifying cardiomyocytes or programmed cardiomyocytes derived from stem cells or fetuses | |
| CN101491228A (en) | Sea no-nucleus pearl incubation method | |
| CN103695367B (en) | A kind of live pig pancreatic island cell substratum of improvement and using method thereof | |
| CN101491229A (en) | Vitro culturing method of pearl shell mantle tissue and culture medium | |
| CN109619089A (en) | A kind of islet cells cryopreservation solution and its application method | |
| CN110301357A (en) | A kind of the regeneration seedling establishment method and special culture media of sponge gourd Unfertilized Ovaries embryoid | |
| CN109880793A (en) | A kind of youth's porcine islet culture medium and its application method | |
| CN103789268B (en) | A kind of method of separation and Culture hippocampal neurons and special culture solution thereof | |
| Alexopoulos | The laboratory cultivation of Stemonitis | |
| CN104593334A (en) | Method for producing ring II type DBN-SX07 strain virus for pigs by seroculturing PK-15 cells | |
| CN104630159A (en) | Method for producing hog cholera C-strain virus by culturing ST Cells in low serum | |
| CN112251397B (en) | Culture method of newborn pig islet cells | |
| CN108102986A (en) | Promote desert algae growth medium and its method for cultivating the growth of desert algae | |
| CN104651320A (en) | Method for producing porcine transmissible gastroenteritis Hua strain virus by low-serum culture of ST cells | |
| CN107326006B (en) | Special culture medium for differentiating human embryonic stem cells into endothelial cells and method thereof | |
| CN105907706A (en) | Isolation and culture method for primary rat colonic smooth muscle cells | |
| TW202122570A (en) | Novel spirulina platensis strain | |
| CN104928233A (en) | Culture solution and method of islet-like cells | |
| Rhodes | Symposium on microbial insecticides. II. Milky disease of the Japanese beetle | |
| CN103352024B (en) | In vitro cultivation method for insect mesenteron cells | |
| CN107299081B (en) | A kind of dedicated serum free medium of human embryo stem cell differentiation | |
| JP2011067203A (en) | Creation of silkworm-culturing cell line enabling serumless culture to be conducted, and use of the cell line |
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 | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A culture medium for pancreatic islet cells of young pigs and its application method Effective date of registration: 20201229 Granted publication date: 20200110 Pledgee: Jinzhou Branch of Hunan Ningxiang Rural Commercial Bank Co.,Ltd. Pledgor: HUNAN XENO LIFE SCIENCE Co.,Ltd. Registration number: Y2020980010070 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20230105 Granted publication date: 20200110 Pledgee: Jinzhou Branch of Hunan Ningxiang Rural Commercial Bank Co.,Ltd. Pledgor: HUNAN XENO LIFE SCIENCE Co.,Ltd. Registration number: Y2020980010070 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A culture medium for pancreatic islet cells of young pigs and its application method Effective date of registration: 20230112 Granted publication date: 20200110 Pledgee: Jinzhou Branch of Hunan Ningxiang Rural Commercial Bank Co.,Ltd. Pledgor: HUNAN XENO LIFE SCIENCE Co.,Ltd. Registration number: Y2023980031055 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20231124 Granted publication date: 20200110 Pledgee: Jinzhou Branch of Hunan Ningxiang Rural Commercial Bank Co.,Ltd. Pledgor: HUNAN XENO LIFE SCIENCE Co.,Ltd. Registration number: Y2023980031055 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A youth pig pancreatic island cell culture medium and its usage method Effective date of registration: 20231129 Granted publication date: 20200110 Pledgee: Jinzhou Branch of Hunan Ningxiang Rural Commercial Bank Co.,Ltd. Pledgor: HUNAN XENO LIFE SCIENCE Co.,Ltd. Registration number: Y2023980068297 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right |