JPS62195276A - Cell cultivation - Google Patents
Cell cultivationInfo
- Publication number
- JPS62195276A JPS62195276A JP3616386A JP3616386A JPS62195276A JP S62195276 A JPS62195276 A JP S62195276A JP 3616386 A JP3616386 A JP 3616386A JP 3616386 A JP3616386 A JP 3616386A JP S62195276 A JPS62195276 A JP S62195276A
- Authority
- JP
- Japan
- Prior art keywords
- culture
- cells
- medium
- indicates
- gas
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000004113 cell culture Methods 0.000 title claims description 7
- 239000007789 gas Substances 0.000 claims abstract description 28
- 239000007788 liquid Substances 0.000 claims abstract description 14
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000001301 oxygen Substances 0.000 claims abstract description 10
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 10
- 238000007664 blowing Methods 0.000 claims abstract description 7
- 238000012258 culturing Methods 0.000 claims description 7
- 238000003756 stirring Methods 0.000 abstract description 12
- 238000010008 shearing Methods 0.000 abstract description 8
- 229910001220 stainless steel Inorganic materials 0.000 abstract description 7
- 239000010935 stainless steel Substances 0.000 abstract description 7
- 239000012531 culture fluid Substances 0.000 abstract 4
- 239000006260 foam Substances 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 42
- 239000002609 medium Substances 0.000 description 37
- 239000000243 solution Substances 0.000 description 17
- 238000000034 method Methods 0.000 description 16
- 238000005273 aeration Methods 0.000 description 14
- 238000013019 agitation Methods 0.000 description 8
- 238000005187 foaming Methods 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 241000196324 Embryophyta Species 0.000 description 7
- 239000002775 capsule Substances 0.000 description 7
- 229920001817 Agar Polymers 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000008272 agar Substances 0.000 description 6
- 238000005286 illumination Methods 0.000 description 6
- 238000009423 ventilation Methods 0.000 description 6
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 238000011081 inoculation Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 239000003094 microcapsule Substances 0.000 description 4
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 4
- FAIXYKHYOGVFKA-UHFFFAOYSA-N Kinetin Natural products N=1C=NC=2N=CNC=2C=1N(C)C1=CC=CO1 FAIXYKHYOGVFKA-UHFFFAOYSA-N 0.000 description 3
- 210000004102 animal cell Anatomy 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- QANMHLXAZMSUEX-UHFFFAOYSA-N kinetin Chemical compound N=1C=NC=2N=CNC=2C=1NCC1=CC=CO1 QANMHLXAZMSUEX-UHFFFAOYSA-N 0.000 description 3
- 229960001669 kinetin Drugs 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 description 3
- 238000005507 spraying Methods 0.000 description 3
- JLIDBLDQVAYHNE-YKALOCIXSA-N (+)-Abscisic acid Chemical compound OC(=O)/C=C(/C)\C=C\[C@@]1(O)C(C)=CC(=O)CC1(C)C JLIDBLDQVAYHNE-YKALOCIXSA-N 0.000 description 2
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 2
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 206010020649 Hyperkeratosis Diseases 0.000 description 2
- UFWIBTONFRDIAS-UHFFFAOYSA-N Naphthalene Chemical compound C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 239000005708 Sodium hypochlorite Substances 0.000 description 2
- 229930003270 Vitamin B Natural products 0.000 description 2
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 2
- 239000004327 boric acid Substances 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229960002713 calcium chloride Drugs 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 239000000460 chlorine Substances 0.000 description 2
- 229910052801 chlorine Inorganic materials 0.000 description 2
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 description 2
- 239000000498 cooling water Substances 0.000 description 2
- WIVXEZIMDUGYRW-UHFFFAOYSA-L copper(i) sulfate Chemical compound [Cu+].[Cu+].[O-]S([O-])(=O)=O WIVXEZIMDUGYRW-UHFFFAOYSA-L 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 230000008020 evaporation Effects 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 2
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 2
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 2
- CDUFCUKTJFSWPL-UHFFFAOYSA-L manganese(II) sulfate tetrahydrate Chemical compound O.O.O.O.[Mn+2].[O-]S([O-])(=O)=O CDUFCUKTJFSWPL-UHFFFAOYSA-L 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 239000004323 potassium nitrate Substances 0.000 description 2
- 235000010333 potassium nitrate Nutrition 0.000 description 2
- 229910000160 potassium phosphate Inorganic materials 0.000 description 2
- 235000011009 potassium phosphates Nutrition 0.000 description 2
- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 description 2
- 229960004172 pyridoxine hydrochloride Drugs 0.000 description 2
- 235000019171 pyridoxine hydrochloride Nutrition 0.000 description 2
- 239000011764 pyridoxine hydrochloride Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000000661 sodium alginate Substances 0.000 description 2
- 235000010413 sodium alginate Nutrition 0.000 description 2
- 229940005550 sodium alginate Drugs 0.000 description 2
- 235000011121 sodium hydroxide Nutrition 0.000 description 2
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 2
- RWVGQQGBQSJDQV-UHFFFAOYSA-M sodium;3-[[4-[(e)-[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]-2-methylcyclohexa-2,5-dien-1-ylidene]methyl]-n-ethyl-3-methylanilino]methyl]benzenesulfonate Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=C1 RWVGQQGBQSJDQV-UHFFFAOYSA-M 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 235000019156 vitamin B Nutrition 0.000 description 2
- 239000011720 vitamin B Substances 0.000 description 2
- RKUNBYITZUJHSG-FXUDXRNXSA-N (S)-atropine Chemical compound C1([C@@H](CO)C(=O)O[C@H]2C[C@H]3CC[C@@H](C2)N3C)=CC=CC=C1 RKUNBYITZUJHSG-FXUDXRNXSA-N 0.000 description 1
- OVSKIKFHRZPJSS-UHFFFAOYSA-N 2,4-D Chemical compound OC(=O)COC1=CC=C(Cl)C=C1Cl OVSKIKFHRZPJSS-UHFFFAOYSA-N 0.000 description 1
- URDCARMUOSMFFI-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]ethyl-(2-hydroxyethyl)amino]acetic acid Chemical compound OCCN(CC(O)=O)CCN(CC(O)=O)CC(O)=O URDCARMUOSMFFI-UHFFFAOYSA-N 0.000 description 1
- PBKADZMAZVCJMR-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid;dihydrate Chemical compound O.O.OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O PBKADZMAZVCJMR-UHFFFAOYSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 241001106067 Atropa Species 0.000 description 1
- 241001465356 Atropa belladonna Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 240000001829 Catharanthus roseus Species 0.000 description 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 1
- RKUNBYITZUJHSG-UHFFFAOYSA-N Hyosciamin-hydrochlorid Natural products CN1C(C2)CCC1CC2OC(=O)C(CO)C1=CC=CC=C1 RKUNBYITZUJHSG-UHFFFAOYSA-N 0.000 description 1
- 241000234435 Lilium Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 241000863480 Vinca Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 230000001195 anabolic effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- LLSDKQJKOVVTOJ-UHFFFAOYSA-L calcium chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Ca+2] LLSDKQJKOVVTOJ-UHFFFAOYSA-L 0.000 description 1
- 229940052299 calcium chloride dihydrate Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- FCRACOPGPMPSHN-UHFFFAOYSA-N desoxyabscisic acid Natural products OC(=O)C=C(C)C=CC1C(C)=CC(=O)CC1(C)C FCRACOPGPMPSHN-UHFFFAOYSA-N 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 150000004683 dihydrates Chemical class 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000007667 floating Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 229960003210 hyoscyamine Drugs 0.000 description 1
- 229930005342 hyoscyamine Natural products 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 229940064880 inositol 100 mg Drugs 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 230000005808 skin problem Effects 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 230000005068 transpiration Effects 0.000 description 1
- 239000000052 vinegar Substances 0.000 description 1
- 235000021419 vinegar Nutrition 0.000 description 1
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/06—Nozzles; Sprayers; Spargers; Diffusers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/02—Means for regulation, monitoring, measurement or control, e.g. flow regulation of foam
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Sustainable Development (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Analytical Chemistry (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【発明の詳細な説明】 産業上の利用分野 本発明は、細胞の培養法における改良に係る。[Detailed description of the invention] Industrial applications The present invention relates to improvements in cell culture methods.
本発明方法は、動物、植物、微生物の培養において利用
することができるので医薬品、食品、発酵工業などの分
野において利用できる。The method of the present invention can be used in the cultivation of animals, plants, and microorganisms, and therefore can be used in fields such as pharmaceuticals, foods, and the fermentation industry.
従来の技術
従来、動植物の細胞や器官の培養ならびに微生物の培養
において通気攪拌を行う場合、インペラーによる攪拌と
培養槽下部に設置されたスパージャ−からの通気とによ
り通気攪拌を行うか、気泡塔を用いて通気攪拌する方法
が知られている。Conventional technology Conventionally, when aeration and agitation are performed in the culture of cells and organs of animals and plants, as well as in the culture of microorganisms, aeration and agitation are performed by agitation using an impeller and aeration from a sparger installed at the bottom of the culture tank, or by using a bubble column. A method of aeration and agitation is known.
発明が解決しようとする問題点
細胞培養に際し、攪拌をインペラーにより行う場合、攪
拌に伴う剪断力により細胞が破壊される。Problems to be Solved by the Invention When stirring is performed using an impeller during cell culture, the cells are destroyed by the shearing force accompanying the stirring.
また、気泡塔による通気攪拌の場合には培養液の発泡が
培養に悪影響を及ぼす。Furthermore, in the case of aeration and agitation using a bubble column, foaming of the culture solution has an adverse effect on the culture.
従って、“剪断力が少なく、しかも発泡を生じない通気
攪拌方法の開発が望まれている。Therefore, it is desired to develop an aeration stirring method that uses less shearing force and does not cause foaming.
問題膚を解決するための手段
本発明者は、培養液表面に酸素を含む気体を吹付ける方
法によれば、剪断力や発泡を生ぜず、培養液を攪拌し、
酸素を供給することができることを見出し本発明を完成
した。Means for Solving Skin Problems The present inventor has discovered that a method of spraying oxygen-containing gas onto the surface of a culture solution does not generate shearing force or foaming, stirs the culture solution,
They discovered that oxygen can be supplied and completed the present invention.
本発明によると、効率的な攪拌および充分な酸素の供給
ができ、しかも剪断力による細胞の破壊、発泡による培
養液表面の発泡も生じない培養系で細胞を培養すること
ができる。According to the present invention, cells can be cultured in a culture system that allows efficient stirring and sufficient oxygen supply, and does not cause cell destruction due to shear force or foaming on the surface of the culture medium due to foaming.
以下本発明の詳細な説明する。The present invention will be explained in detail below.
本発明は、細胞を液体中で培養するに際し、培養液表面
に酸素を含む気体を吹付けながら培養を行うことによる
細胞の培養法を提供する。The present invention provides a method for culturing cells in which cells are cultured in a liquid while blowing oxygen-containing gas onto the surface of the culture solution.
本発明で用いる培養槽としては、円筒状、球状など攪拌
に適するものであれば竪型、横型のいずれの形でもよい
。好ましくは培養槽中心底部を盛り上げ攪拌効率を上げ
たものが用いられる。The culture tank used in the present invention may have any shape, such as a cylindrical shape or a spherical shape, as long as it is suitable for stirring, either vertical or horizontal. Preferably, a culture tank is used in which the bottom of the center of the culture tank is raised to improve stirring efficiency.
本発明方法で細胞とは、動物の細胞、植物の細胞または
器官、微生物の細胞を意味する。In the method of the present invention, cells refer to animal cells, plant cells or organs, and microbial cells.
細胞としては浮遊性、接着依存性のいずれでもよく、動
物、植物、微生物のいずれの種類にも本発明方法は適用
できる。The cells may be either floating or adhesion-dependent, and the method of the present invention can be applied to any type of animal, plant, or microorganism.
培養のための培地としては、これら細胞が生育できるも
のならば、使用する細胞に合わせていかなるものも用い
ることができる。As a culture medium, any medium can be used depending on the cells used as long as these cells can grow.
培養液表面に酸素を含む気体を吹付ける方法としては、
培養液表面より０．１〜３００ｍｍ上部に液面に対して
０°〜９０°好ましくは５°〜８０″の傾きをもった気
体吹出口（ノズル）を設け、この気体吹出口より気体を
培養液表面に吹付（子ることにより行うことができく。The method of spraying oxygen-containing gas onto the surface of the culture solution is as follows:
A gas outlet (nozzle) with an inclination of 0° to 90°, preferably 5° to 80'' relative to the liquid level is provided 0.1 to 300 mm above the surface of the culture solution, and gas is supplied from this gas outlet to the culture medium. This can be done by spraying onto the surface of the liquid.
気体は吹出す前に無菌処理を施す場合もある。The gas may be sterilized before being blown out.
気体吹出口は、デッドスペースを生ずることなく効率的
な攪拌を行うことができれば培養液表面上どこに設置し
てもよい。円筒形または球型をした培養槽の場合は、気
体吹出口より出た気体が培養液表面に当った部位から円
運動を生じるので、培養槽全体が十分に攪拌される。The gas outlet may be installed anywhere on the surface of the culture solution as long as it can perform efficient stirring without creating dead space. In the case of a cylindrical or spherical culture tank, the gas discharged from the gas outlet generates circular motion from the portion where it hits the culture solution surface, so that the entire culture tank is sufficiently agitated.
吹出口は、液面の上下に対応できるように位置を移動で
きる方式が好ましい。また培養槽の規模が大きくなった
ときは、吹出口の数を増すこと、またはスリット式の吹
出口とすることによって対応することができる。It is preferable that the outlet be movable in position so as to correspond to the rise and fall of the liquid level. Furthermore, when the scale of the culture tank increases, this can be handled by increasing the number of blow-off ports or using a slit-type blow-off port.
吹出される気体は、培養対象となる細胞の種類により種
々のものが選ばれるが、一般に空気が使用される。気体
には、酸素、炭素ガス、窒素その他の気体成分を空気に
補充したものを使用することができる。Various types of gas are selected depending on the type of cells to be cultured, but air is generally used. As the gas, air supplemented with oxygen, carbon gas, nitrogen, or other gas components can be used.
吹出される気体の通気量は、培養対象細胞の酸素要求度
、培養槽の大きさなどによって選ばれる。The amount of gas blown out is selected depending on the oxygen demand of the cells to be cultured, the size of the culture tank, etc.
動物細胞のとき、たとえば直径８５ｍｍ、深さ８０ｍｍ
程度の培養槽においては、約１．３１７ｍ１ｎ以上の通
気を行えばよい。植物細胞や器官培養のとき、たとえば
直径３０ｃｍ、高さ４０ｃｍ程度の培養槽で培養液の深
さ３０ｃｍでは、約１ （ｌ ｌ ／ｍｉｎ以上の通気
を行えばよい。For animal cells, for example, the diameter is 85 mm and the depth is 80 mm.
In a culture tank of about 100 ml, aeration of about 1.317 ml or more is sufficient. When culturing plant cells or organs, for example, in a culture tank with a diameter of 30 cm and a height of 40 cm and a culture solution depth of 30 cm, aeration at a rate of about 1 l/min or more is sufficient.
培養槽には、通気による蒸散に対応するためのコンデン
サーを設置するのが好ましい。また培養槽として必要な
一般的設備、たとえば菌体接種口、ジャケット、センサ
一孔、センサー類、サンプリング穴などは適宜設置する
。It is preferable to install a condenser in the culture tank to cope with transpiration caused by ventilation. In addition, general equipment necessary for a culture tank, such as a bacterial inoculation port, a jacket, a sensor hole, sensors, sampling holes, etc., are installed as appropriate.
本発明方法は、一般的な培養装置の補助的手段としても
用いることができる。たとえば、インペラーを用いる通
気攪拌培養において、剪断力を生じない程度にインペラ
ーの回転速度を抑え、不足する攪拌を本発明の方法によ
って補う。このようにして剪断を回避して通気攪拌の効
率を上げることができる。The method of the present invention can also be used as an auxiliary means for general culture equipment. For example, in aerated agitation culture using an impeller, the rotational speed of the impeller is suppressed to an extent that no shearing force is generated, and insufficient agitation is compensated for by the method of the present invention. In this way, shearing can be avoided and the efficiency of aeration stirring can be increased.
また気泡塔において、発泡を少なくするために上昇する
気体量を抑え、不足する通気、攪拌を本発明方法を併用
することにより補うことができる。Furthermore, in a bubble column, the amount of gas rising can be suppressed to reduce foaming, and insufficient aeration and stirring can be compensated for by using the method of the present invention in combination.
本発明方法は、通常の動物、植物、微生物等の細胞の液
体培養に用いることができるほか、これら細胞をマイク
ロカプセルに包んで行う培養方法にも用いることができ
る。この培養においては、通常のインペラーを用いる攪
拌での剪断力を著しく軽減することができる。The method of the present invention can be used for ordinary liquid culture of cells of animals, plants, microorganisms, etc., and can also be used for a culture method in which these cells are wrapped in microcapsules. In this culture, the shearing force required for agitation using a normal impeller can be significantly reduced.
本発明方法に用いられる動物細胞培養装置の一例を第１
１！Ｉに示す。第１図の培養槽は、底部の盛り上がった
ガラス容器で、接種口およびジャケットを取り付けた５
０ Ｑｍｌ容量の培養槽セある。上部より無菌フィル
ターのついたステンレス製のチューブを差し込み、その
先端にエアーノズルをつける。エアーノズルは培養液表
面から０．１〜３００ｍｍ離した位置に液面と０°〜９
０°好ましくは５＠〜８０°の角度で設置する。ノズル
としては、断面積１〜ｌＱｍｍ’の円形または偏平状ス
テンレス針を用い、エアーポンプによって１〜２０１
／ｍｉ＋の流量で気体を培養液面に吹付ける。An example of the animal cell culture apparatus used in the method of the present invention is shown in the first example.
1! Shown in I. The culture tank shown in Figure 1 is a glass container with a raised bottom and a 5-inch inoculation port and jacket.
I have a culture tank with a capacity of 0 Qml. Insert a stainless steel tube with a sterile filter from the top and attach an air nozzle to the tip. The air nozzle is placed at a distance of 0.1 to 300 mm from the surface of the culture solution, at an angle of 0° to 9°.
It is installed at an angle of 0°, preferably 5@ to 80°. As a nozzle, use a circular or flat stainless steel needle with a cross-sectional area of 1 to 1Qmm', and use an air pump to
Gas is blown onto the culture solution surface at a flow rate of /mi+.
本発明方法に用いられるスパージャ−を備えた植物細胞
培養装置の一例を第２図に示す。第２図の培養槽は、球
形のガラス容器で、接種口およびスパージャ−を備えた
２Ｏｆｆ容看の培養槽である。An example of a plant cell culture apparatus equipped with a sparger used in the method of the present invention is shown in FIG. The culture tank shown in FIG. 2 is a spherical glass container with a 2-off capacity and is equipped with an inoculation port and a sparger.
上部より無菌フィルターのついたステンレス製のチュー
ブを差し込み、その先端にエアージェットノズルをつけ
る。該ノズルは培養液表面から０．１〜３００ｍｍ離れ
た位置に液面と０°〜９０°好ましくは５゛〜８０゛の
角度で設置する。ノズルとしては断面積１〜ｌＱｍｍ２
の円形または扁平状ステンレス針を用いて、エアーポン
プによって１０〜４０１ ／ｍｉｎの流量で気体を培養
液面に吹付ける。また気体の蒸散を防ぐためにコンデン
サーを取り付け、その上部には微生物汚染を防ぐために
無菌フィルターを取り付ける。Insert a stainless steel tube with a sterile filter from the top and attach an air jet nozzle to the tip. The nozzle is installed at a distance of 0.1 to 300 mm from the surface of the culture solution at an angle of 0 to 90 degrees, preferably 5 to 80 degrees. The nozzle has a cross-sectional area of 1 to 1Qmm2.
Using a circular or flat stainless steel needle, gas is blown onto the surface of the culture solution using an air pump at a flow rate of 10 to 401/min. A condenser is also installed to prevent gas evaporation, and a sterile filter is installed above it to prevent microbial contamination.
スパージャ−を使う場合、スパージャ−からは、発泡を
抑えるために０．１〜５１１ ／＋ｔ＋ｉｎの範囲で空
気を通気する。When a sparger is used, air is vented through the sparger in a range of 0.1 to 511/+t+in to suppress foaming.
これら培養槽は単なる例示であり、規模の拡大、縮少、
付属装置の付加、削除等は自由にできる。These culture tanks are merely examples;
Additional devices can be added or deleted freely.
実施例１
第１図に示した培養槽を用いて、マウスミエローマ細胞
であるＭＰＣ−１１株（＾ＴＣＣＣＣＬ１６７）のカプ
セル内培養を下記のとおり行った。Example 1 Using the culture tank shown in FIG. 1, mouse myeloma cells MPC-11 strain (^TCCCCL167) were cultured in a capsule as described below.
ＲＰＭＩ−１６４０培地（日水製薬社製）、ＤＭＥ培地
（ダルベヅコウ変法ＭＥＭ培地、日水製薬社製）、ハム
ＦＩＯ培地（フローラボ社ＩＩ）の基礎培地を２：１：
１（ν／ｖ）で混合した培地に１０％（ｗ／ｖ）新生修
生血清、４ｍＭグルタミン、２５ｘｒ／＋ｎｌストレプ
トマイシン、２５１１／ｍｌペニシリン、１６ｍＭヘペ
ス緩衡液、０．０１％（ｗ／ｖ）重曹を加え、ｐ Ｈ７
，０とした培地（以下ＲＤＦ培地という）を細胞培養に
用いた。The basal media of RPMI-1640 medium (Nissui Pharmaceutical Co., Ltd.), DME medium (Darbezukou modified MEM medium, Nissui Pharmaceutical Co., Ltd.), and Ham's FIO medium (Flow Lab II) were mixed at a ratio of 2:1:
Medium mixed at 1 (v/v) with 10% (w/v) nascent serum, 4mM glutamine, 25xr/+nl streptomycin, 2511/ml penicillin, 16mM Hepes buffer, 0.01% (w/v) Add baking soda to pH 7
, 0 (hereinafter referred to as RDF medium) was used for cell culture.
ＭＰＣ−１１株の細胞を６．０Ｘ１０’個／ｍｌとなる
ように２％アルギン酸ソーダを含む生理食塩水に分散し
、注射筒に入れて、２％塩化カルシウムを含む生理食塩
水中に滴下した。この操作により細胞は約６００μｍの
径をもったアルギン酸ソーダの球状カプセル（マイクロ
カプセル）内に包括された。このときのカプセル内の生
存細胞密度は５．’７１ Ｘ １０’個／ｍｌテ生存率
１；！ ９４．１９６テアった。Cells of the MPC-11 strain were dispersed at 6.0 x 10' cells/ml in physiological saline containing 2% sodium alginate, placed in a syringe, and dropped into physiological saline containing 2% calcium chloride. Through this operation, the cells were encapsulated in spherical capsules (microcapsules) of sodium alginate having a diameter of about 600 μm. The density of viable cells in the capsule at this time is 5. '71 x 10' cells/ml survival rate 1;! 94.196 tore.
マイクロカプセルを濾過し、０．１％（１１／Ｖ）ポリ
Ｌ−ＩＪジンを含むＲＤＦ培地で洗浄した。The microcapsules were filtered and washed with RDF medium containing 0.1% (11/V) polyL-IJ gin.
第１図で示した培養槽にＲＤＦ培地４０ Ｑｍｌを入れ
、これにカプセル内容量として４ Ｑｍｌの前記洗浄マ
イクロカプセルを接種した。内径０．９ｍｍの円形エア
ーノズルを培養液表面から３〜４ｍｍの位置に設置し、
１．８１７ｍ１ｎの速度で００２５％を含む空気を吹付
けながら、３７℃で３４７時間培養した。40 Qml of RDF medium was placed in the culture tank shown in FIG. 1, and the washed microcapsules with a capsule content of 4 Qml were inoculated therein. A circular air nozzle with an inner diameter of 0.9 mm was installed at a position of 3 to 4 mm from the surface of the culture solution.
The cells were cultured at 37° C. for 347 hours while blowing air containing 0.0025% at a rate of 1.817 ml.
培養後のカプセル内細胞密度は２．１７ Ｘ １０”
／ｍｌで、生存率は７２，１％であった。The cell density inside the capsule after culturing is 2.17 x 10”
/ml, the survival rate was 72.1%.
対照として、球状の攪拌子（回転数５Ｏｒｐｍ）を用い
る通常の培養フラスコを用いる以外は、上記と同じ条件
で培養したが、この場合はカプセルが破損してしまいカ
プセル培養ができなかった。As a control, culture was carried out under the same conditions as above, except that a normal culture flask with a spherical stirrer (rotation speed: 5 rpm) was used, but in this case, the capsule was damaged and capsule culture could not be performed.
実施例２
第１図に示した培養槽（５００ｍＮを用いて、マウスミ
エローマ細胞であるＭＰＣ−１１株（ＡＴＣＣＣＣＬ１
６７）の浮遊培養を行った。Example 2 Mouse myeloma cells MPC-11 strain (ATCCCCCL1
67) was carried out in suspension culture.
ＭＰＣ−１１株の細胞５Ｘ１０’個／＋ｎｌ （生存率
９２％）の４ Ｑｍｌを、４０ ＱｍｌのＲＤＦ培地を
含む培養槽に接種し、実施例１と同様に通気しつつ、３
７℃で７２時間培養した。4 Qml of 5 x 10' cells/+nl (survival rate 92%) of the MPC-11 strain were inoculated into a culture tank containing 40 Qml of RDF medium, and incubated with aeration in the same manner as in Example 1.
The cells were cultured at 7°C for 72 hours.
培養後の細胞は４Ｘ１０’細胞／ｍｌで、生存率は８６
％であった。After culture, the cells were 4X10' cells/ml, and the survival rate was 86
%Met.
実施例３
へカタユリの球根を１０％次亜塩素酸ソーダ（有効塩素
１％）および７０％エチルアルコールで表面殺菌後、５
ｍｍ〜１５１ＴＩＩＴ１ｍｍ−切り、下記第１表の組織
を有するムラシゲ・スクーグ固体寒天培地Ｉ Ｑｍｌを
含む直径２．５ ｃｍ深さ１２．５ ｃｍの試験管に試
験管当り１個置床させ、２５℃、２５００ルクスの連続
照明下で６０日間培養した。この培養によって球根が分
化し、−切片につき１〜５個の球根が分化した塊が得ら
れた。このようにして得られた球根を滅菌したメスとビ
ンセットでリン片を分割し、約１５００枚のリン片を集
めた。集めたリン片を、次亜塩素酸ソーダー水溶液（有
効塩素量０．５％）で１５分間殺菌し、無菌水で良く洗
った。第１表の培地から寒天を除去し、アブサイシン酸
を培地１１当り０．２ ｍｇ含有する液体培地１５１を
第２図に示した構造を有する内容！１２０１の培養槽に
入れ上記洗浄リン片を移植した。Example 3 After surface sterilization of Hekata lily bulbs with 10% sodium hypochlorite (1% available chlorine) and 70% ethyl alcohol,
mm ~ 151TIIT1 mm - cut, place one piece per test tube in a test tube with a diameter of 2.5 cm and a depth of 12.5 cm containing Qml of Murashige-Skoog solid agar medium I having the structure shown in Table 1 below, at 25°C. The cells were cultured for 60 days under continuous illumination of 2500 lux. This culture resulted in differentiation of the bulbs, and a cluster of 1 to 5 bulbs per section was obtained. The bulbs thus obtained were divided into pieces using a sterilized scalpel and a bottle set, and about 1,500 pieces were collected. The collected pieces of phosphorus were sterilized for 15 minutes with a sodium hypochlorite aqueous solution (available chlorine amount: 0.5%) and thoroughly washed with sterile water. Agar is removed from the medium in Table 1, and liquid medium 151 containing 0.2 mg of abscisic acid per medium 11 has the structure shown in Figure 2! It was placed in a culture tank No. 1201, and the washed phosphorus pieces were transplanted.
２５℃、５．０００ルクス連続照明下、通気量２０１７
ｍ１ｎで培地表面に空気を吹きつけつつ３０日間通気、
攪拌した。３０日目量後は吹付通気に加えスパージャ−
による通気（５ｊｉ！／ｍ１ｎ）を行った。６０日間培
養すると、約２．６５０個の球根が得られた。25℃, 5,000 lux continuous lighting, ventilation amount 2017
Aerate for 30 days while blowing air on the medium surface with m1n,
Stirred. After the 30th day, use a sparger in addition to spray aeration.
Aeration (5ji!/m1n) was performed. After culturing for 60 days, approximately 2,650 bulbs were obtained.
第１表 ムラシゲ・スクーグ培地
硝酸アンモニウム １．６５Ｏｎ＋ｇ硝酸カリ
ウム １．９００ｍｇ塩化カルシウム・２
水塩 ４４０ｍｇ硫酸マグネシウム・７水塩 ３
７０＋＋＋ｇリン酸第−カリウム １７０＋
ｎｇＮ ａ２ ＨＥＤＴＡ ・２水塩 ３７．３
ｍｇ硫酸第一鉄・７水塩 ２７．８ ｍｇ
ホ ウ 酸
６．２ ｍｇ硫酸マンガン・４水塩 ２２．３
ｍｇ硫酸亜鉛・７水塩 ８．６ ｍｇ
ヨウ化カリウＡ 、０．８３ｍｇモ
リブデン酸ソーダ・２水塩 ０．２５ ｍｇ硫酸第
一銅 ０．０２５ ｍｇ塩化コバ
ルト ０．０２５ｍｇビタミンＢ、
０．４０ｍｇイノシトール
１００ｍｇ塩酸ピリドキシン
０．５０ ｍｇニコチン酸
０．５０ ｍｇグリシン ２
．００ ｍｇシュークロース ３０．０
ｍｇナフタレン酢酸 ０．１ ｍｇ
寒 天 ８．０
ｍｇ上記の成分を脱イオン水に溶かして１７！とし、０
．１規定のカセイソーダ水溶液でｐＨを６．２に調節し
、培養容器に分注した後１２１℃で２０分間殺菌する。Table 1 Murashige-Skoog medium Ammonium nitrate 1.65 On+g Potassium nitrate 1.900 mg Calcium chloride 2
Water salt 440mg Magnesium sulfate heptahydrate 3
70+++g potassium phosphate 170+
ngN a2 HEDTA ・dihydrate 37.3
mg Ferrous sulfate heptahydrate 27.8 mg
Boric acid
6.2 mg Manganese sulfate tetrahydrate 22.3
mg Zinc sulfate heptahydrate 8.6 mg
Potassium iodide A, 0.83 mg Sodium molybdate dihydrate 0.25 mg Cuprous sulfate 0.025 mg Cobalt chloride 0.025 mg Vitamin B,
0.40mg inositol 100mg pyridoxine hydrochloride
0.50 mg nicotinic acid
0.50 mg glycine 2
．． 00 mg sucrose 30.0
mg naphthalene acetic acid 0.1 mg
Agar 8.0
mg Dissolve the above ingredients in deionized water and make 17! and 0
．． The pH was adjusted to 6.2 with a 1N aqueous solution of caustic soda, and the mixture was dispensed into culture vessels and sterilized at 121°C for 20 minutes.
実施例４
ベラドンナ（Ａｔｒｏｐａ ｂｅｌａｄｏｎｎａ）の茎
を約５ｃｍの１００分間殺菌し、無菌水で良く水洗した
後に５〜ｌＱｍｍの切片に切った。第１表に示した培地
からナフタレン酢酸を除き、４ ＰＵ （Ｎ −２−ｃ
ｈｌｏｒ。Example 4 A stem of belladonna (Atropa belladonna) was sterilized for 100 minutes in a length of about 5 cm, thoroughly washed with sterile water, and then cut into sections of 5 to 1Q mm. Naphthalene acetic acid was removed from the medium shown in Table 1, and 4 PU (N -2-c
hlor.
−４−ｐｙｒｉｄｙｌ−Ｎ−ｐｈｅｎｙｌｕｒｅａ）を
培地１１当り１ｍｇの濃度で添加したムラシゲ・スクー
グ寒天培地ｌ Ｑｍｌを含有する直径２４ｍｍ長さ１２
５ｍｍの試験管に上記切片を移植した。２２℃、２．５
００ルクス連続照明下で６０日間培養すると、多数のシ
ュートを有する組織が得られた。この組織を、第２表の
組成を有するムラシゲ・スクーグの改良培地１’ＯＱｍ
ｌを含有する３ ０９ｍｇ容のコニカルビーカーに移植
した。２２℃、５．ＯＱＯルクス連続照明下、毎分１２
６回転で３０日間振とう培養してさらに生育させた。-4-pyridyl-N-phenylurea) at a concentration of 1 mg per medium 11.Containing 1 Qml of Murashige-Skoog agar medium 24 mm in diameter and 12 in length
The above sections were transplanted into 5 mm test tubes. 22℃, 2.5
When cultured for 60 days under 00 lux continuous illumination, tissues with numerous shoots were obtained. This tissue was cultured in 1'OQm of Murashige-Skoog's improved medium having the composition shown in Table 2.
The cells were transferred to a 309 mg conical beaker containing 1. 22℃, 5. Under OQO lux continuous illumination, 12 per minute
Further growth was carried out by shaking culture at 6 rotations for 30 days.
３０日目量生育した組織を無菌的に取り出し、ピンセッ
トとメスを用いて組織から発生した根のみ無菌的に採取
した。これらの根を再度、第２表の組成を有する新しく
作成した液体培地１００ｍｇを含有するコニカルビーカ
ーに移植して、３０日間培養し、根が多数分枝しながら
生育した根の塊りを得た。この根の塊りをピンセットと
メスを用いて無菌的に分割し、同一の培地（第２表）お
よび培養方法で５同化代増殖を繰り返して根のみを増殖
させた。増殖した根の塊りをコニカルビーカー１０本か
ら直径２０ｃｍ、深さ４ｃｍのべ） １７皿に無菌的に
取り出し、メスで細断し、根の断片を多数得た。これら
根の断片を第２表の組成ををする液体培地１５１を含む
、第２図からスパーシアーを除いた構造を有する内容積
２Ｏｆｆの培養漕に移植した。２５℃、５．ＯＯＱルク
ス連続照明下、通気ｆｆｔ ２０１ ／ｍｉｎで、培地
表面に空気を吹きつけながら通気、攪拌した。４５日間
培養すると根が分枝しながら生育し、新鮮重量として約
２．７ ｋｇの根を収穫することができた。根の粗抽出
液をシリカゲル薄槽クロマトグラフィ（メルク社製：製
品番号５６３１）にスポットし、クロロホルム：エタノ
ール：２８％アンモニア水＝８５：１４：１の混合溶媒
で展開し、乾燥後ドラーゲンドルフ試薬で発色したとこ
ろ、ヒヨスチアミンに一致するスポットの発色が認めら
れた。The tissue that had grown on the 30th day was aseptically removed, and only the roots that had developed from the tissue were aseptically collected using tweezers and a scalpel. These roots were again transplanted into a conical beaker containing 100 mg of a newly prepared liquid medium having the composition shown in Table 2, and cultured for 30 days to obtain a root mass in which the roots grew with multiple branches. . This root mass was aseptically divided using tweezers and a scalpel, and propagated for 5 anabolic generations using the same medium (Table 2) and culture method to propagate only the roots. The proliferated root mass was aseptically taken out from 10 conical beakers into a dish with a diameter of 20 cm and a depth of 4 cm) and chopped with a scalpel to obtain a large number of root fragments. These root fragments were transplanted into a culture tank containing a liquid medium 151 having the composition shown in Table 2 and having an internal volume of 2Off and having a structure similar to that shown in FIG. 2 except for the sparse. 25℃, 5. Under continuous OOQ lux illumination, the medium was aerated and stirred at fft 201 /min while blowing air onto the surface of the medium. After culturing for 45 days, the roots grew while branching, and it was possible to harvest approximately 2.7 kg of fresh roots. The crude root extract was spotted on silica gel thin tank chromatography (manufactured by Merck & Co., product number 5631), developed with a mixed solvent of chloroform: ethanol: 28% ammonia water = 85:14:1, and after drying, Dragendorff reagent was applied. When the color was developed, a spot color matching that of hyoscyamine was observed.
第２表 ムラシゲ・スクーグ改変培地
硝酸アンモニウム ８２５ｍｇ硝酸カリウム
９５０ｍｇ塩化カルシウム・２水塩
２２０ｍｇ硫酸マグネシウム・７水塩 １８５ｍ
ｇリン酸第−カリウム ゛ ８５ｍｇＮａ２
’ ＥＤＴＡ ・２水塩 １８．６５ ｍｇ硫酸第
一鉄・７水塩 １３．９■ホ ウ 酸
３．１■硫酸マン
ガン・４水塩 １１．１５ ｍｇ硫酸亜枦・７
水塩 ４．３ ｍｇジヨウカリウム
０．４１５ｍｇモリブデン酸ソーダ・
２水塩 ０．１２５ ｍｇ硫酸第一銅
０．０１２５ｍｇ塩化コバルト
０．０１２５■ビタミンＢ＋
０．２ｍｇイノシトール ５
０ ■塩酸ピリドキシン ０．２５■ヱ
コチン酸 ０．２５■グリシ
ン １．００ ｍｇシェークロ
ース ３０．０ ｍｇナフタレン酢ＷＩ
Ｑ、１ｍｇ上記の成分を脱イオン
水に溶かして１１とし、０、１規定のカセイソーダ水溶
液でｐＨを６．２に調節し、培養容器に分注した後１２
１℃で２０分間殺菌する。Table 2 Murashige-Skoog modified medium Ammonium nitrate 825 mg Potassium nitrate 950 mg Calcium chloride dihydrate
220mg magnesium sulfate heptahydrate 185m
g Potassium phosphate ゛ 85mgNa2
' EDTA dihydrate 18.65 mg Ferrous sulfate heptahydrate 13.9 ■ Boric acid
3.1 ■ Manganese sulfate tetrahydrate 11.15 mg sodium sulfate 7
Water salt 4.3 mg diiopotassium
0.415mg Sodium Molybdate
Dihydrate 0.125 mg Cuprous sulfate
0.0125mg cobalt chloride
0.0125■ Vitamin B+
0.2mg inositol 5
0 ■ Pyridoxine hydrochloride 0.25 ■ Ecotinic acid 0.25 ■ Glycine 1.00 mg Shakerose 30.0 mg Naphthalene vinegar WI
Q. Dissolve 1 mg of the above ingredients in deionized water to make 11, adjust the pH to 6.2 with 0 or 1 N caustic soda aqueous solution, dispense into culture vessels, and then prepare 12.
Sterilize for 20 minutes at 1°C.
実施例５
ニチニチソウ（Ｖｉｎｃａ ｒｏｓｅａ）の茎を長さ約
５ｃｍの長さに切り、実施例２と同様の操作で殺菌した
。Example 5 Stems of Vinca rosea were cut into pieces approximately 5 cm in length and sterilized in the same manner as in Example 2.
第１表に示した培地のナフタレン酢酸に代えて、２．４
−ジクロロフェノキシ酢酸とカイネチンを培地１１当り
、それぞれ０．５ ｍｇおよび０．１ｍｇ添加した培地
ＩＱｍｌを含む直径２４ｍｍ長さ１２５ｍｍの試験管に
上記ニチニチソウの茎の切片を移植し、２８℃、２．５
００ルクス連続照明下で培養した。In place of naphthalene acetic acid in the medium shown in Table 1, 2.4
- The above-mentioned periwinkle stem sections were transplanted into test tubes with a diameter of 24 mm and a length of 125 mm containing IQml of medium to which 0.5 mg and 0.1 mg of dichlorophenoxyacetic acid and kinetin were added per 11 medium, respectively, and incubated at 28°C for 2. 5
The cells were cultured under 00 lux continuous illumination.
２週間後、茎の断面よ、リカルスが形成された。Two weeks later, in the cross section of the stem, a recallus was formed.
これを無菌的に分取し、同じ組成の培地上に移植し、２
８℃、２．５００ルクス連続照明下で継代培養すると安
定したカルスが得られた。このカルスを、第１表に示し
た培地のナフタレン酢酸に代えて２．４−ジクロロフェ
ノキシ酢酸とカイネチンを培地１１当りそれぞれ０．５
■および０．１ｍｇ添加し、寒天を除いた液体培地１０
０ｍｇを含む３０９ｍｌ容三角フラスコに移植し、２８
℃、暗黒下、回転数１８Ｏｒｐｍで振とう培養した。こ
のようにして液体培地中で３０日日間化培養すると約半
年後には８日間で旺盛な生育を示すようになった。この
ようにして継代培養した細胞を種細胞とし、その１．５
１を第１表に示した培地のナフタレン酢酸に代えて、２
．４−ジクロロフェノキシ酢酸とカイネチンを培地１１
当りそれぞれ０．５ ｍｇおよび０．１ ｍｇを添加し
、寒天を除いた液体培地１５Ｉ２を含む、第２図からス
パージャ−を除いた構造を有する内容積２０１の培養槽
に移植した。２８℃、暗黒下、通気５１３０１１ ／ｍ
ｉｎ■で空気を培地表面に吹きつけながら通気、攪拌し
、１０日間培養すると細胞が生育して新鮮重量として、
３．７００ｇ、乾物量として２４０ｇの細胞を収穫する
ことができた。This was collected aseptically, transplanted onto a medium with the same composition, and
Stable callus was obtained by subculturing at 8° C. under continuous illumination of 2.500 lux. This callus was treated with 2.4-dichlorophenoxyacetic acid and kinetin in place of naphthaleneacetic acid in the medium shown in Table 1 at 0.5% each per 11 days of medium.
■ Liquid medium 10 with addition of 0.1mg and agar removed
Transferred to a 309 ml Erlenmeyer flask containing 0 mg,
Shaking culture was carried out at 18 °C in the dark at a rotational speed of 18 rpm. When cultured for 30 days in a liquid medium in this manner, it began to show vigorous growth in 8 days after about half a year. The cells subcultured in this way are used as seed cells, and the 1.5
1 in place of naphthalene acetic acid in the medium shown in Table 1, 2
．． 4-dichlorophenoxyacetic acid and kinetin in medium 11
0.5 mg and 0.1 mg were added, respectively, and the cells were transferred to a culture tank containing a liquid medium 15I2 without agar and having an internal volume 201 having the structure shown in FIG. 2 without the sparger. 28℃, darkness, ventilation 513011/m
Aerate and stir while blowing air onto the surface of the medium in ■. After culturing for 10 days, the cells will grow and the fresh weight will be expressed as
It was possible to harvest 3.700 g of cells, with a dry weight of 240 g.
発明の効果
本発明によれば、インペラーの剪断力による細胞の破壊
および気体の発泡による培養の抑制を回避して、効率よ
く細胞の培養を行うことができる。Effects of the Invention According to the present invention, cells can be efficiently cultured while avoiding destruction of cells due to shearing force of an impeller and suppression of culture due to gas foaming.
第１図は本発明方法に用いる培養装置を示す。
図中番号および記号は下記の意義を有する。括弧内は培
養液４ＱＯ〜５００ｍｌを用いるときのサイズの例を示
す。
■は、培養槽底部の盛り上がりを示す。
■は、細胞の接種口を示す。
■は、ジャケットを示す。
Ａは、恒温水の人口
Ｂは、恒温水の出口
■は、培養槽を示す。（培養槽は内径３５ｍｍ、高さ１
８３ｍｍ０）
■および■は、無菌フィルターを示す。
■は、通気ラインを示す。
■は、エアジェツトノズルを示す。（培養液面に応じて
上下動ができる。）
■は、気体の蒸散を防ぐためのコンデンサーを示す。
［相］は、ステンレス針を示す。（先端は３０°の角度
をもつ。内径０．９ｍｍ）
Ｃは、気体（５％炭酸ガス／空気）供給口を示す。
（通気は、１．８１ ／ｍｉｎで行う。）Ｄは、培養液
表面を示す。
Ｅは、気体出口を示す。
Ｆは、冷却水人口を示す。
Ｇは、冷却水出口を示す。
図中の数値は、ｍｍで表示された装置のサイズを示す。
第２図は、本発明方法に用いる培養装置において、スパ
ージャ−を併用する例を示す。図中番号は下記の意義を
有する。括弧内は具体的装置の一例の大きさを示す。
■は、球形培養槽を示す。（直径３５０ｍｍ）■は、ス
パージャ−を示す。
■は、通気ラインを示す。
■は、エアーポンプを示す。（通気は２０１ ／ｍｉｎ
とする）
■は、無菌フィルターを示す。
■は、細胞の接種口を示す。
■は、気体の流量計を示す。
＠は、エアージェットノズルを示す。（ノズルは液面に
対して５°の角度とする。）
■は、コンデンサーを示す。
■は、気体出口を示す。FIG. 1 shows a culture apparatus used in the method of the present invention. Numbers and symbols in the figures have the following meanings. The size in parentheses indicates an example of the size when using 4QO to 500ml of culture solution. ■ indicates a swell at the bottom of the culture tank. ■ indicates the cell inoculation port. ■ indicates a jacket. A indicates the constant temperature water population B indicates the constant temperature water outlet ■ indicates the culture tank. (The culture tank has an inner diameter of 35 mm and a height of 1
83mm0) ■ and ■ indicate sterile filters. ■ indicates a ventilation line. ■ indicates an air jet nozzle. (It can move up and down depending on the culture solution level.) ■ indicates a condenser to prevent gas evaporation. [Phase] indicates a stainless steel needle. (The tip has an angle of 30°. Inner diameter 0.9 mm) C indicates a gas (5% carbon dioxide gas/air) supply port. (Aeration is performed at 1.81/min.) D indicates the surface of the culture solution. E indicates the gas outlet. F indicates the cooling water population. G indicates a cooling water outlet. The numbers in the figure indicate the size of the device in mm. FIG. 2 shows an example in which a sparger is used in combination with the culture apparatus used in the method of the present invention. The numbers in the figure have the following meanings. The size of a specific device is shown in parentheses. ■ indicates a spherical culture tank. (Diameter: 350 mm) ■ indicates a sparger. ■ indicates a ventilation line. ■ indicates an air pump. (Ventilation is 201/min
) ■ indicates a sterile filter. ■ indicates the cell inoculation port. ■ indicates a gas flow meter. @ indicates an air jet nozzle. (The nozzle should be at an angle of 5° to the liquid level.) ■ indicates a condenser. ■ indicates the gas outlet.
Claims (1)
体を吹付けながら培養を行うことを特徴とする細胞培養
法。A cell culture method characterized by culturing cells in liquid while blowing oxygen-containing gas onto the surface of the culture solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3616386A JPS62195276A (en) | 1986-02-20 | 1986-02-20 | Cell cultivation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3616386A JPS62195276A (en) | 1986-02-20 | 1986-02-20 | Cell cultivation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS62195276A true JPS62195276A (en) | 1987-08-28 |
Family
ID=12462096
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3616386A Pending JPS62195276A (en) | 1986-02-20 | 1986-02-20 | Cell cultivation |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS62195276A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63164880A (en) * | 1986-08-08 | 1988-07-08 | Hitachi Ltd | Culture of animal cell and apparatus therefor |
| US4968331A (en) * | 1989-05-15 | 1990-11-06 | Nippon Steel Corporation | Organic polymer separation membrane having fluorene skeleton and oxygen enrichment device utilizing same |
| US4975190A (en) * | 1988-05-16 | 1990-12-04 | Nippon Steel Corporation | Organic polymer separation membrane having fluorene skeleton and oxygen enrichment device utilizing same |
| JP2014530618A (en) * | 2011-10-21 | 2014-11-20 | セル・メディカ・リミテッド | Equipment for aseptic growth of cells |
| CN108211696A (en) * | 2018-01-23 | 2018-06-29 | 青岛理工大学 | A kind of preparation facilities and method in indoor air purification beneficial bacteria room |
-
1986
- 1986-02-20 JP JP3616386A patent/JPS62195276A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63164880A (en) * | 1986-08-08 | 1988-07-08 | Hitachi Ltd | Culture of animal cell and apparatus therefor |
| US4975190A (en) * | 1988-05-16 | 1990-12-04 | Nippon Steel Corporation | Organic polymer separation membrane having fluorene skeleton and oxygen enrichment device utilizing same |
| US5116504A (en) * | 1988-05-16 | 1992-05-26 | Nippon Steel Corporation | Organic polymer separation membrane having fluorene skeleton and oxygen enrichment device utilizing same |
| US4968331A (en) * | 1989-05-15 | 1990-11-06 | Nippon Steel Corporation | Organic polymer separation membrane having fluorene skeleton and oxygen enrichment device utilizing same |
| JP2014530618A (en) * | 2011-10-21 | 2014-11-20 | セル・メディカ・リミテッド | Equipment for aseptic growth of cells |
| CN108211696A (en) * | 2018-01-23 | 2018-06-29 | 青岛理工大学 | A kind of preparation facilities and method in indoor air purification beneficial bacteria room |
| CN108211696B (en) * | 2018-01-23 | 2020-12-22 | 青岛理工大学 | Preparation device and method of indoor air purification beneficial bacteria room |
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