JPH0249579A - Culture medium - Google Patents
Culture mediumInfo
- Publication number
- JPH0249579A JPH0249579A JP1044393A JP4439389A JPH0249579A JP H0249579 A JPH0249579 A JP H0249579A JP 1044393 A JP1044393 A JP 1044393A JP 4439389 A JP4439389 A JP 4439389A JP H0249579 A JPH0249579 A JP H0249579A
- Authority
- JP
- Japan
- Prior art keywords
- medium
- cells
- wheat gluten
- glutamine
- decomposition product
- 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
- 239000001963 growth medium Substances 0.000 title description 8
- 241000209140 Triticum Species 0.000 claims abstract description 34
- 235000021307 Triticum Nutrition 0.000 claims abstract description 34
- 108010068370 Glutens Proteins 0.000 claims abstract description 28
- 235000021312 gluten Nutrition 0.000 claims abstract description 28
- 238000000354 decomposition reaction Methods 0.000 claims abstract description 22
- 230000002255 enzymatic effect Effects 0.000 claims abstract description 18
- 229940041514 candida albicans extract Drugs 0.000 claims abstract description 12
- 239000012138 yeast extract Substances 0.000 claims abstract description 12
- 229940069820 wheat gluten extract Drugs 0.000 claims description 2
- 150000001413 amino acids Chemical class 0.000 abstract description 12
- 210000004102 animal cell Anatomy 0.000 abstract description 10
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 abstract description 10
- 230000001954 sterilising effect Effects 0.000 abstract description 3
- 238000004659 sterilization and disinfection Methods 0.000 abstract description 3
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 2
- 230000003247 decreasing effect Effects 0.000 abstract 1
- 239000000463 material Substances 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 43
- 210000004027 cell Anatomy 0.000 description 37
- 239000000047 product Substances 0.000 description 25
- 229940024606 amino acid Drugs 0.000 description 10
- 235000001014 amino acid Nutrition 0.000 description 10
- 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 description 10
- 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 10
- 239000000203 mixture Substances 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 8
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 8
- 229940088598 enzyme Drugs 0.000 description 8
- 108010082495 Dietary Plant Proteins Proteins 0.000 description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 6
- 229940082569 selenite Drugs 0.000 description 6
- MCAHWIHFGHIESP-UHFFFAOYSA-L selenite(2-) Chemical compound [O-][Se]([O-])=O MCAHWIHFGHIESP-UHFFFAOYSA-L 0.000 description 6
- 229940088594 vitamin Drugs 0.000 description 6
- 235000013343 vitamin Nutrition 0.000 description 6
- 239000011782 vitamin Substances 0.000 description 6
- 229930003231 vitamin Natural products 0.000 description 6
- 102000004877 Insulin Human genes 0.000 description 5
- 108090001061 Insulin Proteins 0.000 description 5
- 229930182555 Penicillin Natural products 0.000 description 5
- 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 5
- 238000012258 culturing Methods 0.000 description 5
- 229940125396 insulin Drugs 0.000 description 5
- 239000013028 medium composition Substances 0.000 description 5
- 229940049954 penicillin Drugs 0.000 description 5
- 229960005322 streptomycin Drugs 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 4
- 239000012981 Hank's balanced salt solution Substances 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 4
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 4
- 239000004365 Protease Substances 0.000 description 4
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 4
- 229960003589 arginine hydrochloride Drugs 0.000 description 4
- 229960002433 cysteine Drugs 0.000 description 4
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 4
- 235000018417 cysteine Nutrition 0.000 description 4
- 239000001103 potassium chloride Substances 0.000 description 4
- 235000011164 potassium chloride Nutrition 0.000 description 4
- 229940076788 pyruvate Drugs 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 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 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 3
- 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 3
- 239000007995 HEPES buffer Substances 0.000 description 3
- 108091005804 Peptidases Proteins 0.000 description 3
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 102000004338 Transferrin Human genes 0.000 description 3
- 108090000901 Transferrin Proteins 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 235000012000 cholesterol Nutrition 0.000 description 3
- 239000012141 concentrate Substances 0.000 description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 3
- 229910000397 disodium phosphate Inorganic materials 0.000 description 3
- 235000019800 disodium phosphate Nutrition 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 229930182830 galactose Natural products 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- DKAGJZJALZXOOV-UHFFFAOYSA-N hydrate;hydrochloride Chemical compound O.Cl DKAGJZJALZXOOV-UHFFFAOYSA-N 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
- 235000019341 magnesium sulphate Nutrition 0.000 description 3
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 3
- 235000019419 proteases Nutrition 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000012581 transferrin Substances 0.000 description 3
- 150000003722 vitamin derivatives Chemical class 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 108091005658 Basic proteases Proteins 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 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 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 2
- 208000005718 Stomach Neoplasms Diseases 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 235000003704 aspartic acid Nutrition 0.000 description 2
- LFYJSSARVMHQJB-QIXNEVBVSA-N bakuchiol Chemical compound CC(C)=CCC[C@@](C)(C=C)\C=C\C1=CC=C(O)C=C1 LFYJSSARVMHQJB-QIXNEVBVSA-N 0.000 description 2
- 239000007640 basal medium Substances 0.000 description 2
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 2
- ZCCIPPOKBCJFDN-UHFFFAOYSA-N calcium nitrate Chemical compound [Ca+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ZCCIPPOKBCJFDN-UHFFFAOYSA-N 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 2
- 206010017758 gastric cancer Diseases 0.000 description 2
- 238000002523 gelfiltration Methods 0.000 description 2
- 229960000367 inositol Drugs 0.000 description 2
- 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 2
- 210000004185 liver Anatomy 0.000 description 2
- 229940040511 liver extract Drugs 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000012533 medium component Substances 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 2
- 235000019799 monosodium phosphate Nutrition 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- 201000011549 stomach cancer Diseases 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- QIJRTFXNRTXDIP-UHFFFAOYSA-N (1-carboxy-2-sulfanylethyl)azanium;chloride;hydrate Chemical compound O.Cl.SCC(N)C(O)=O QIJRTFXNRTXDIP-UHFFFAOYSA-N 0.000 description 1
- FBOUIAKEJMZPQG-AWNIVKPZSA-N (1E)-1-(2,4-dichlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pent-1-en-3-ol Chemical compound C1=NC=NN1/C(C(O)C(C)(C)C)=C/C1=CC=C(Cl)C=C1Cl FBOUIAKEJMZPQG-AWNIVKPZSA-N 0.000 description 1
- AUNIQBYZVOKWJK-UHFFFAOYSA-N 2-[1-(2-hydroxyethyl)piperazin-2-yl]ethanesulfonic acid Chemical compound OCCN1CCNCC1CCS(O)(=O)=O AUNIQBYZVOKWJK-UHFFFAOYSA-N 0.000 description 1
- 239000001763 2-hydroxyethyl(trimethyl)azanium Substances 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 241000282552 Chlorocebus aethiops Species 0.000 description 1
- 235000019743 Choline chloride Nutrition 0.000 description 1
- 101001012217 Conus geographus Con-Ins G3 Proteins 0.000 description 1
- 101001012221 Conus tulipa Con-Ins T3 Proteins 0.000 description 1
- AUNGANRZJHBGPY-UHFFFAOYSA-N D-Lyxoflavin Natural products OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- 150000008540 L-glutamines Chemical class 0.000 description 1
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 1
- AFCARXCZXQIEQB-UHFFFAOYSA-N N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CCNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 AFCARXCZXQIEQB-UHFFFAOYSA-N 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
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- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 1
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- DFPAKSUCGFBDDF-ZQBYOMGUSA-N [14c]-nicotinamide Chemical compound N[14C](=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-ZQBYOMGUSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- 229960004050 aminobenzoic acid Drugs 0.000 description 1
- 210000001691 amnion Anatomy 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000001557 animal structure Anatomy 0.000 description 1
- -1 anocohalamine Chemical compound 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 229960003121 arginine Drugs 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 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
- 230000001580 bacterial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- FAPWYRCQGJNNSJ-UBKPKTQASA-L calcium D-pantothenic acid Chemical compound [Ca+2].OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O.OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O FAPWYRCQGJNNSJ-UBKPKTQASA-L 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 229960002079 calcium pantothenate Drugs 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229960003178 choline chloride Drugs 0.000 description 1
- SGMZJAMFUVOLNK-UHFFFAOYSA-M choline chloride Chemical compound [Cl-].C[N+](C)(C)CCO SGMZJAMFUVOLNK-UHFFFAOYSA-M 0.000 description 1
- 229960001305 cysteine hydrochloride Drugs 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 229960000304 folic acid Drugs 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- MGIUUAHJVPPFEV-ABXDCCGRSA-N magainin ii Chemical compound C([C@H](NC(=O)[C@H](CCCCN)NC(=O)CNC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O)C1=CC=CC=C1 MGIUUAHJVPPFEV-ABXDCCGRSA-N 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000003801 milling Methods 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 235000008160 pyridoxine Nutrition 0.000 description 1
- 239000011677 pyridoxine Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 229960002477 riboflavin Drugs 0.000 description 1
- 235000019192 riboflavin Nutrition 0.000 description 1
- 239000002151 riboflavin Substances 0.000 description 1
- 235000011121 sodium hydroxide Nutrition 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- 235000019157 thiamine Nutrition 0.000 description 1
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 description 1
- 239000011721 thiamine Substances 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 229960004799 tryptophan Drugs 0.000 description 1
- 229940011671 vitamin b6 Drugs 0.000 description 1
Abstract
Description
【発明の詳細な説明】
産業上の利用分野
本発明は小麦グルテン酵素分解物を含有する培地に関す
る。本培地は動物細胞培地用培地として有用である。DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a medium containing enzymatically degraded wheat gluten. This medium is useful as a medium for animal cell culture.
従来の技術
従来、動物細胞用培地として、アミノ酸類、ビタミン類
、糖類、無機塩等を含有する培地（例えば、ＲＰＭ１１
６４０培地、イーグルのＭＥＭ培地など）が知られてい
る。しかしながら、これら培地を蒸気滅菌すると、培地
中のグルタミンが分解してしまい、グルタミンとして利
用されない欠点がある。これを改良するものとして、加
熱に対して安定であり、かつ動物細胞にＬ−グルタミン
として利用されるし一グルタミン誘導体（Ｌ−アミノ酸
−Ｌ−グルタミン）を含有する培地が知られている（特
開昭６１．−２７１９８５号公報）。Conventional technology Conventionally, as a culture medium for animal cells, a culture medium containing amino acids, vitamins, sugars, inorganic salts, etc. (for example, RPM11
640 medium, Eagle's MEM medium, etc.) are known. However, when these mediums are steam sterilized, the glutamine in the medium is decomposed and cannot be utilized as glutamine. To improve this, a medium is known that is stable against heat and contains a monoglutamine derivative (L-amino acid-L-glutamine), which is utilized by animal cells as L-glutamine (especially (Kokai No. 61.-271985).
発明が解決しようとする課題
培地を作成する際に、前記Ｌ−グルタミン誘導体以外に
培地成分として、培地に多種類のアミノ酸を加える必要
がある。Problems to be Solved by the Invention When preparing a culture medium, it is necessary to add various types of amino acids to the culture medium as medium components in addition to the L-glutamine derivative.
蒸気滅菌が可能でグルタミンの培地への別添加は必要が
なく、かつ添加するアミノ酸の数を減少させた安価な動
物細胞培養用培地が求められる。There is a need for an inexpensive animal cell culture medium that can be steam sterilized, does not require the separate addition of glutamine to the medium, and has a reduced number of added amino acids.
課題を解決するだめの手段
本発明は小麦グルテン酵素分解物を含有する培地および
該培地に酵母エキスを添加した培地を提供する。Means for Solving the Problems The present invention provides a medium containing an enzymatically decomposed product of wheat gluten and a medium in which yeast extract is added to the medium.
本発明の培地は動物細胞の培養に用いられる培地の構成
成分として、少なくとも小麦クルテン酵素分解物が含ま
れていればいずれの培地でもよい。The medium of the present invention may be any medium used for culturing animal cells as long as it contains at least an enzymatically decomposed product of wheat kurten.
例えば、糖類、ビタミン類および無機物を含む培地に小
麦クルテン酵素分解物を添加した培地又は糖類および無
機物に小麦グルテン酵素分解物および酵母エキスを添加
した培地である。さらに、通常の動物細胞培養に用いら
れる基礎培地に■小麦グルテン酵素分解物又は■液分解
物および酵母エキスを添加した培地も本発明の培地とし
てあげられる。さらに、本発明の培地にアミノ酸類、抗
生物質、その他動物細胞を生育させる物質などを添加し
てもよい。For example, a medium containing saccharides, vitamins, and inorganic substances to which enzymatic decomposition product of wheat gluten is added, or a medium containing saccharides and minerals to which enzymatic decomposition product of wheat gluten and yeast extract are added. Furthermore, a medium prepared by adding (1) enzymatic decomposition product of wheat gluten or (2) liquid decomposition product and yeast extract to the basal medium used for normal animal cell culture can also be mentioned as the medium of the present invention. Furthermore, amino acids, antibiotics, and other substances for growing animal cells may be added to the medium of the present invention.
本発明の培地の糖類としては、グルコース、ガラクトー
ス、フルクトースなどがあげられる。Examples of sugars in the medium of the present invention include glucose, galactose, and fructose.
ビタミン類としては、ビオチン、葉酸、ｐ−アミノ安息
香酸、ニコチンアミド、パントテン酸カルシウム、ピリ
ドキシン、リボフラビン、チアミン、ンアノコハラミン
、塩化コリン、イノシトール、アスコルビン酸、チミジ
ンなどがあげられる。Examples of the vitamins include biotin, folic acid, p-aminobenzoic acid, nicotinamide, calcium pantothenate, pyridoxine, riboflavin, thiamine, anocohalamine, choline chloride, inositol, ascorbic acid, and thymidine.
無機物としては、塩化す） ＩＪつｔ・、塩化カリウム
、リン酸−水素ナトリウム、リン酸二水素ナトリウム、
硫酸マグ２、シウト、硝酸カルシウム、塩化カルンウｌ
１、塩化マク不シウム、亜セレン酸などがあげられる。Examples of inorganic substances include chloride), potassium chloride, sodium hydrogen phosphate, sodium dihydrogen phosphate,
Sulfuric acid mag 2, sulfuric acid, calcium nitrate, chloride
1. Macunium chloride, selenite, etc.
アミノ酸類としては、システィン、アルギニン、トリプ
上ファンなどがあげられる。Examples of amino acids include cysteine, arginine, and tryptophan.
抗生物質としては、ペニシリン、ストレプトマイシン、
カナマイシンなどがあげられる。Antibiotics include penicillin, streptomycin,
Examples include kanamycin.
動物細胞を生育させる物質としては牛胎児血清、修生血
清、馬血清などの動物血清、豚肝臓抽出濃縮物などの動
物臓器抽出物、インスリン、トランスフェリン、コレス
テロールなどのホルモン類などがあげられる。基礎培地
としては、一般の動物細胞の培養に用いられるものであ
ればいずれでもよい。例えば、ＲＰＭＩ’１６４０培地
、イークルのＭＥＭ培地、ハｌ、のＦ１２、ダルベツコ
変法イクル培地、１９９培地などがあげられる。Examples of substances for growing animal cells include animal serum such as fetal bovine serum, amended serum, and horse serum, animal organ extracts such as pig liver extract concentrate, and hormones such as insulin, transferrin, and cholesterol. As the basal medium, any medium used for culturing general animal cells may be used. Examples include RPMI'1640 medium, Ekl's MEM medium, Hal's F12, Dulbecco's modified Ikl's medium, and 199 medium.
本発明の培地に添加される小麦クルテン酵素分解物とし
ては、小麦クルテンを蛋白質分解酵素で分解したもので
あればいずれも用いられる。例えば、市販の小麦クルテ
ン酵素分解物、後記方法によって得られる小麦クルテン
酵素分解物等が用いられる。好ましくは、第、１図に示
すゲル濾過高性能液体クロマトグラフィーによる分子量
分布を有し、かつ後記第１表に示すアミノ酸組成を有す
る小麦グルテン酵素分解物、さらに好ましくは、第２図
に示すゲル濾過高性能液体クロマトグラフィーによる分
子量分布を有するものが用いられる。As the enzymatic decomposition product of wheat kurten to be added to the culture medium of the present invention, any product obtained by decomposing wheat kurten with a protease can be used. For example, commercially available enzymatically decomposed wheat kurten products, enzymatically decomposed wheat kurten products obtained by the method described below, and the like can be used. Preferably, a wheat gluten enzymatic decomposition product having a molecular weight distribution determined by gel filtration high performance liquid chromatography as shown in FIG. 1 and an amino acid composition shown in Table 1 below, more preferably a gel shown in FIG. Those having a molecular weight distribution determined by filtration and high performance liquid chromatography are used.
ゲル濾過高性能液体クロマトグラフィーの測定条件
使用機器 、 島原製作所製 ＬＣ−６Ａカ ラ
ム ： ＴＳＫ Ｇｅｌ Ｇ３０００Ｓ
ｌｔｌ ＸＬ溶 媒 ０．５Ｍ食塩／４０ｍＭ
リン酸バッファ（ｐ）１８）
１ｍｌ／ｍｉｎ
８０ｎｍ
（第１図）
流 速
紫外部吸収
分子量マーカ
溶出位置（分）
８．４９７
１０．６６７
分子量
４３．０００
１３．７００
分子量マーカー（第２図）
溶出位置（分） 分子量
１０、６６７ １３．７００
培地に添加される小麦グルテン酵素分解物の量としては
、培地１１当り０．１−１０ｇ、好ましくは０．２５−
３ｇ（乾物換算）の範囲である。Measurement conditions for gel filtration high performance liquid chromatography Equipment used: Shimabara Manufacturing LC-6A Kara
Mu: TSK Gel G3000S
ltl XL solvent 0.5M salt/40mM
Phosphate buffer (p)18) 1ml/min 80nm (Fig. 1) Flow rate Ultraviolet absorption molecular weight marker elution position (min) 8.497 10.667 Molecular weight 43.000 13.700 Molecular weight marker (Fig. 2) Elution Position (min) Molecular weight 10, 667 13.700 The amount of enzymatically degraded wheat gluten added to the medium is 0.1-10 g per 11 medium, preferably 0.25-
It is in the range of 3g (dry matter equivalent).
培地に添加される酵母エキスとしては、一般に市販品が
用いられ、添加量としては、培地１１当り０、１ ＝２
ｇ、好ましくは０．２５−１ｇ（乾物換算）の範囲で
ある。As the yeast extract added to the medium, commercially available products are generally used, and the amount added is 0 per 11 medium, 1 = 2
g, preferably in the range of 0.25-1 g (in terms of dry matter).
小麦グルテン分解物は市販品以外に下記の方法で得られ
たものも使用できる。In addition to commercially available wheat gluten decomposition products, those obtained by the following method can also be used.
小麦グルテンを酵素で処理して小麦グルテン酵素分解物
を得ることができる。Wheat gluten enzymatic decomposition products can be obtained by treating wheat gluten with enzymes.
使用する酵素としては、動物由来の酵素（トリプシン、
ペプシンなど）、植物由来の酵素（ブロメラン、パパイ
ンなど）、カビ由来の酵素（アルカリ性プロテアーセな
ど）、細菌由来の酵素（酸性プロテアーセなど）などが
あげられる。その使用量は、反応ｐＨ１反応温度、反応
時間などにもよるが、通常５０−５０万単位／小麦クル
テン１００ｇの範囲が好ましい。反応は各酵素の至適条
件下で行えばよいが、通常１０−７０℃で１−２４時間
行われる。The enzymes used include animal-derived enzymes (trypsin,
pepsin, etc.), plant-derived enzymes (bromelan, papain, etc.), mold-derived enzymes (alkaline protease, etc.), and bacterial-derived enzymes (acidic protease, etc.). The amount used depends on the reaction pH, reaction temperature, reaction time, etc., but is usually preferably in the range of 500,000 to 500,000 units/100 g of wheat gluten. The reaction may be carried out under optimal conditions for each enzyme, but is usually carried out at 10-70°C for 1-24 hours.
つぎに、本発明に用いる小麦クルテン酵素分解物のアミ
ノ酸組成の一例を第１表に示す。Next, Table 1 shows an example of the amino acid composition of the enzymatically degraded wheat kurten used in the present invention.
第 １ 表
（注）１．ＮＨ３遊離より推定してアスパラギン酸およ
びグルタミン酸の８０−９０％はアスパラギン酸
られる。Table 1 (Note) 1. Estimated from NH3 release, 80-90% of aspartic acid and glutamic acid is aspartic acid.
２、分析方法、小麦クルテン酵素分解物を塩酸により加
水分解した後、アミノ酸アナライザー（日立Ｌ８５００
）で分析した。2. Analysis method: After hydrolyzing the enzymatic decomposition product of wheat gluten with hydrochloric acid, an amino acid analyzer (Hitachi L8500
) was analyzed.
小麦グルテン酵素分析物の蒸気滅菌（１２０℃、１５分
間）時のｒ＋ Ｈ安定性について、ナマルバ細胞の増殖
性を基に検討した実験例を以下に示す。An experimental example in which the r+H stability of a wheat gluten enzyme analyte during steam sterilization (120° C., 15 minutes) was investigated based on the proliferation of Namalva cells is shown below.
実験例１
ナマルバ細胞を２． ＯＸ １０５ｃｅｌｌｓ／ｍｌに
なる様に下記組成の培地に懸濁させた懸濁戒名２ｍｌを
２４穴マイクロプレート （ヌンク社製）の名犬に添加
した後、５％ＣＯ２−９５％空気存在下、３７℃で２日
間培養した。培養後の細胞数を第２表に示す。Experimental example 1 Namalva cells 2. After adding 2 ml of the suspension in a medium with the following composition to a concentration of OX 105 cells/ml to a 24-well microplate (manufactured by Nunc), it was incubated at 37°C in the presence of 5% CO2-95% air. The cells were cultured for 2 days. Table 2 shows the number of cells after culture.
培地組成
ハンクス液〔クルコースＩｇ／ｊＬ塩化ナトリウム８
ｇ ／ Ｉ、塩化カリウム０．４ｇ／ｊ！、リン酸水素
ナトリウム（無水＞４７．９ｍｇ／β、リン酸水素カリ
ウム（無水＞６０ｍｇ／β、硫酸マグネシウム（無水）
４８．８ｍｇ／ ＩＩ、塩化マグネシウム４６．８
ｍｇ／ｊｌ！、塩化カルシウム（無水）１４０ｍｇ／ｌ
及びフェノールレッド６ｍｇ／！、小麦グルテン酵素分
解物（植物性蛋白５Ｋ−５、千葉製粉■製） ０．５ｇ
／ｌ （ｐｆｌｌ、 ２． ４． ６． ８及び１０
で蒸気滅菌）、酵母エキス０．５ ｇ ／ Ｉ、システ
ィン塩酸塩−水塩６０ｍｇ／ｆｌ、トリプトファン１０
ｍｇ／β、アルギニン塩酸塩２００ｍｇ／β、ペニシリ
ン５０，０ＯＯＵ／ｊｌ！、ストレプトマイシン５０ｍ
ｇ／Ｉ！、ＨＥＰＥＳ （Ｎ−２−ヒドロキシエチルピ
ペラジン−２−エタンスルホン酸、フッドの緩衝液用試
薬＞ １０ｍＭ、インシュリン３ｍｇ／β、トランスフ
ェリン５ｍｇ／ｊ２． ピルビン酸５ｍＭ、亜セレン酸
０．１２５μＭおよびガラクト−２ニ炭酸水素ナトリウ
ムでｐＨを７．４に調節第 ２ 表
表から明らかな如く、小麦グルテン酵素分解物はｐＨ２
−８において蒸気滅菌されても、該分解物中のグルタミ
ンがほとんど分解されることなく、培地成分として、安
定して使用されることが判る。Medium composition Hank's solution [Curcose Ig/jL Sodium chloride 8
g/I, potassium chloride 0.4g/j! , sodium hydrogen phosphate (anhydrous >47.9 mg/β, potassium hydrogen phosphate (anhydrous >60 mg/β), magnesium sulfate (anhydrous)
48.8mg/II, magnesium chloride 46.8
mg/jl! , calcium chloride (anhydrous) 140mg/l
and phenol red 6mg/! , Wheat gluten enzymatic decomposition product (vegetable protein 5K-5, manufactured by Chiba Seifun ■) 0.5g
/l (pfll, 2. 4. 6. 8 and 10
steam sterilized), yeast extract 0.5 g/I, cysteine hydrochloride-water salt 60 mg/fl, tryptophan 10
mg/β, arginine hydrochloride 200mg/β, penicillin 50,0OOU/jl! , streptomycin 50m
g/I! , HEPES (N-2-hydroxyethylpiperazine-2-ethanesulfonic acid, Hood's buffer reagent>10mM, insulin 3mg/β, transferrin 5mg/j2. pyruvate 5mM, selenite 0.125μM and galacto-2 The pH was adjusted to 7.4 with sodium bicarbonate.As is clear from Table 2, the enzymatic decomposition product of wheat gluten had a pH of 2
It can be seen that even when steam sterilized in -8, glutamine in the decomposition product is hardly decomposed and can be stably used as a medium component.
次に小麦クルテン酵素分解物と酵母エキスとの混合によ
るナマルバ細胞の増殖性についての実験例を示す。Next, an experimental example will be shown regarding the proliferation of Namalva cells by mixing the enzymatic decomposition product of wheat kurten and yeast extract.
実験例２
実験例１において、ナマルバ細胞の濃度２．０×１０５
ｃｅ１１ｓ／ｒｒＩ！２を４． ２ Ｘ １ ０ ５ｃ
ｅｌｌｓ／ ｍｌに、培地組成の小麦グルテン酵素分解
物（植物性蛋白ＳＫ５、千葉製粉■製）０．５ｇ／ｆｆ
ｌを参考例１で得られた小麦クルテン酵素分解物（濃度
：０，０．５１．２．５．５及び１０ｇ＃）に代える以
外は実験例１と同様に培養した。培養後の細胞数を第３
表に示す。Experimental Example 2 In Experimental Example 1, the concentration of Namalva cells was 2.0×105
ce11s/rrI! 2 to 4. 2 X 1 0 5c
ells/ml, 0.5 g/ff of wheat gluten enzymatic decomposition product (vegetable protein SK5, manufactured by Chiba Flour Mills) in the medium composition.
Culture was carried out in the same manner as in Experimental Example 1, except that 1 was replaced with the enzymatically degraded wheat kurten obtained in Reference Example 1 (concentrations: 0, 0.51, 2, 5.5, and 10 g). The number of cells after culturing is
Shown in the table.
第 ３ 表 培養後の細胞数を第４表に示す。Table 3 Table 4 shows the number of cells after culture.
第 ４ 表
４．９
実験例３
実験例１において用いた培地中の小麦クルテン酵素分解
物及び酵母エキスの使用量を第３表に示す使用量に変え
る以外は実験例１と同様に培養した。Table 4 Table 4.9 Experimental Example 3 Culture was carried out in the same manner as in Experimental Example 1, except that the amounts of enzymatically decomposed wheat kurten and yeast extract in the medium used in Experimental Example 1 were changed to the amounts shown in Table 3.
細胞数： ｘｌＱ’ ｃｅｌｌｓ／ｍｌ実験例４
実験例１において、ナマルバ細胞の濃度２．０×１０５
ｃｅｌ Ｉｓ／ ｒｒ（Ｑを３． ＯＸ １０５ｃｅｌ
ｌｓ／ｍｆ！に、培養日数２日間を３日間に、及び小麦
グルテン酵素分解物（植物性蛋白５Ｋ−５、千葉製粉■
製）０．５ｇ／ｊ２を参考例１で得られた小麦グルテン
酵素分解物０．５ｇ／Ｉｌに代えて、さらに培地にリバ
ーコンセントレー）２０！−３（豚肝臓抽出濃縮物シグ
マ社製）０．２５ｇ／ｊ！、コレステロール０．１ｍｇ
／！もしくは１．０ ｍｇ／ Ｉｔ又はイノシトール１
０ｍｇ／ｆｌを添加する以外は実験例１と同様に培養し
た。Number of cells: xlQ' cells/ml Experimental Example 4 In Experimental Example 1, the concentration of Namalva cells was 2.0×105
cel Is/rr (Q 3. OX 105cel
ls/mf! In addition, the culture period was changed from 2 days to 3 days, and wheat gluten enzymatic decomposition product (vegetable protein 5K-5, Chiba Flour Milling)
0.5g/j2 of the wheat gluten enzymatic decomposition product obtained in Reference Example 1, and then added liver concentrae to the medium) 20! -3 (pig liver extract concentrate manufactured by Sigma) 0.25g/j! , cholesterol 0.1mg
/! or 1.0 mg/It or inositol 1
Culture was carried out in the same manner as in Experimental Example 1 except that 0 mg/fl was added.
培養後の細胞数を第５表に示す。Table 5 shows the number of cells after culture.
第 ５ 表
本発明の培地はナマルバ細胞、ヒト胃癌由来ＫＡＴＯ−
１１１細胞、ヒト子宮頚癌由来ＨｅＬａ細胞、ヒト羊膜
由来ＦＬ細抱、ヒト白血病由来ＨＬ６０細胞、アフリカ
ミドリザル腎由来Ｖｅｒ。Table 5 The culture medium of the present invention is derived from Namalva cells, human gastric cancer-derived KATO-
111 cells, human cervical cancer-derived HeLa cells, human amniotic membrane-derived FL cells, human leukemia-derived HL60 cells, African green monkey kidney-derived Ver.
細胞、マウス繊維芽細胞Ｌ９２９細胞などの培養に用い
ることができる。It can be used for culturing cells, mouse fibroblast L929 cells, etc.
以下に実施例を示す。Examples are shown below.
実施例１
ナマルバ細胞を５ Ｘ １０５ｃｅｌｌｓ／ｍｌになる
様に下記組成の培地に懸濁させた懸濁波谷２ｍｌを２４
穴マイクロプレート（ヌンク社製）の多穴に添加した後
、５％ＣＯ□−９５％空気存在下、３７℃で２日間培養
した。培養後の細胞数は？、 ＯＸ １０５ｃｅｌｌｓ
／ｍｌであった。Example 1 2 ml of Namalva cells suspended in a medium with the following composition at 5 x 105 cells/ml was added to 24
After adding to the multiwell microplate (manufactured by Nunc), the cells were cultured at 37° C. for 2 days in the presence of 5% CO□-95% air. What is the number of cells after culturing? , OX 105cells
/ml.
培地組成
ハンクス液、植物性蛋白５Ｋ−５０，５ｇ／ｊ！、シス
ティン塩酸塩−水塩６０ｍｇ／ｊ！、）リブトファン１
０ｍｇ／ｊ！、アルギニン塩酸塩２００ｍｇ／Ｉｌ、ペ
ニシリン５０，０ＯＯＵ／ｊ！、ストレプトマイシン５
０＋ｎｇ／β、ＨＥＰＥＳ １０ｍＭ、インシュリン３
ｍｇ／β、トランスフェリン５ｍｇ／ｊｉ！、ピルビン
酸５ｍＭ、亜セレン酸０．１２５μＭ、ガラクトス１ｇ
／Ｉｌ及びＲＰＭ１１６４０培地のビタミン混合物、ｐ
Ｈ７，４
実施例２
ナマルバ細胞を５． ＯＸ １０５ｃｅｌｌｓ／ｍｌに
なる様に下記組成の培地に懸濁させた懸濁液２５ｍ１を
Ｆ７５ｃｉｌ培養フラスコ （コーニング社製）に添加
した後、３７℃で３日間培養したところ、細胞数が１、
０７ Ｘ １０６ｃｅｌｌｓ／ｍｌに増殖した。ついで
、細胞数を再び５． ＯＸ １０５ｃｅｌｌｓ／ ｍｌ
に下げた後、同様に培養したところ、細胞数が１．０５
Ｘ １０６ｃｅｌｌｓ／ｍｌまで増殖した。Medium composition Hank's solution, vegetable protein 5K-50, 5g/j! , cysteine hydrochloride-water salt 60 mg/j! ,) Ributofan 1
0mg/j! , arginine hydrochloride 200 mg/Il, penicillin 50,0 OOU/j! , streptomycin 5
0+ng/β, HEPES 10mM, insulin 3
mg/β, transferrin 5mg/ji! , pyruvate 5mM, selenite 0.125μM, galactos 1g
/Il and vitamin mixture of RPM11640 medium, p
H7,4 Example 2 Namalva cells 5. OX 105 cells/ml was suspended in a medium with the following composition, and 25 ml of the suspension was added to an F75cil culture flask (manufactured by Corning) and cultured at 37°C for 3 days.
Proliferated to 0.07 x 106 cells/ml. Then, increase the number of cells again to 5. OX 105cells/ml
When cultured in the same manner after lowering the cell number to 1.05
Proliferated to X 106 cells/ml.
培地組成
ハンクス液、植物性蛋白５Ｋ−５０，５ｇ／β、酵母エ
キス０．５ｇ／Ａ、ＨＥＰＥＳ １０ｍＭ。Medium composition Hank's solution, vegetable protein 5K-50, 5g/β, yeast extract 0.5g/A, HEPES 10mM.
インシュリン３ｍｇ／１１ ）ランスフェリ２５ｍｇ／
１１ピルビン酸５ｍＭ、亜セレン酸０．１２５ ｍＭ及
ＩＪガラクトースＩｇ／β、ｐＨ７，４
実施例３
ヒト胃がん由来ＫＡＴＯＩＩＴ細胞を２．５ Ｘ １０
５ｃｅｌｌｓ／ｍ＋になる様に実施例２で用いた培地に
牛胎児血清４０ｇ／Ａを添加した培地で懸濁した懸濁液
１０ｍ１をＦ２５ｃＩＩｔ培地フラスコ（コーニング社
製）に添加した後、３７℃で５３日日間化培養を行った
。各培養日数の日にそれぞれ細胞数を測定した後、再度
細胞数を１０ Ｘ １０ ’ｃｅｌｌｓ／ｍｌに下げ培
養を続けた。その結果を第６表に示す。Insulin 3mg/11) Lanceferri 25mg/
11 Pyruvate 5mM, selenite 0.125mM and IJ galactose Ig/β, pH 7.4 Example 3 Human gastric cancer-derived KATOIIT cells were incubated at 2.5 x 10
After adding 10 ml of a suspension prepared by adding 40 g/A of fetal bovine serum to the medium used in Example 2 to give a concentration of 5 cells/m+ to an F25cIIt medium flask (manufactured by Corning), the suspension was incubated at 37°C. Daily culture was performed for 53 days. After measuring the cell number on each culture day, the cell number was lowered again to 10 x 10' cells/ml and culture was continued. The results are shown in Table 6.
第 ６ 表
実施例４
ナマルバ細胞を４．２ Ｘ １０５ｃｅｌｌｓ／−にな
る様に下記組成の培地５ｍｌに懸濁させた後、Ｆ２５Ｃ
ｎｌ培養フラスコ（コーニング社製）に添加し、３７℃
で第７表に示す如く継代培養を行った。その結果を第７
表に示す。Table 6 Example 4 After suspending Namalva cells to 4.2 x 105 cells/- in 5 ml of a medium with the following composition, F25C
Add to nl culture flask (Corning) and incubate at 37°C.
Subculture was performed as shown in Table 7. The results are shown in the 7th section.
Shown in the table.
培地組成
塩化ナトリウム６．８ｇ／Ａ、塩化カリウム０．４ｇ／
ｊ！、リン酸二水素ナトリウム１０８．７ｍｇ／ｆｌ、
硫酸マグネシウム９７．７ ｍｇ／ ｊ！、塩化カルシ
ウム０．２ｇ／Ｉｌ、グルコースＩｇ／Ｃフェノールレ
ッド６ｍｇ／β、トリプトファン１０ｍｇ／Ａ、アルギ
ニン塩酸塩０．２ｇ／ｊ２、システィン塩酸塩−水塩６
０ｍｇ／ｌ、参考例１で得られた小麦グルテン加水分解
２．５ｇ／ｊ２、酵母エキス０．５ｇ／Ｃリバーコンセ
ントレート２０２−３ ０．２５ｇ／Ｃコレステロール
０、１ ｍｇ／ Ｒ１炭酸水素ナトリウム１．５ｇ／＊
、ペニシリン５００００Ｕ／β、ストレプトマイシン５
０，０００μｇ／ＣＨＥＰＥＳ ２．３８３ｇ／Ｒ，
インシュリン３ｍｇ／１１ ）ランスフェリン５ｍｇ／
Ｊ、ピルビン酸ナトリウム５５０．２ｍｇ／１２、亜セ
レン酸１６．１３μｇ／ｊ！、ガラクトースＩｇ／Ｃｐ
Ｈ７，４
第 ７ 表
実施例５
ハンクス液〔グルコースＩｇ／ｊ７．塩化ナトリウム８
ｇ／Ｒ，塩化カリウム０．４ｇ／β、リン酸−水素ナト
リウム（無水）４７．９ｍｇ／ｊ、 リン酸二水素カリ
ウム（無水）６０ｍｇ／Ｉｌ、硫酸マグネシウム（無水
）４８．８ｍｇ７ｊ！、塩化マグネシラ１１４６、８
ｍｇ／ ｉ’、塩化カルシウム（無水）１４０ｍｇ／Ｉ
！及びフェノールレッド６ｍｇ／ｊＪに植物性蛋白ＳＫ
〜５ ０．５ｇ／矛、システィン塩酸塩−水塩６０ｍｇ
／ｊ！、トリプトファン１０ｍｇ／β、アルギニン塩酸
塩０．２ｇ／Ａ、ペニシリン５０，０ＯＯＵ／！、スト
レプトマイシン５０ ｍｇ／ Ｒ、）ＩＥＰＢ３１０
ｍＭ。Medium composition Sodium chloride 6.8g/A, potassium chloride 0.4g/A
j! , sodium dihydrogen phosphate 108.7 mg/fl,
Magnesium sulfate 97.7 mg/j! , calcium chloride 0.2g/Il, glucose Ig/C phenol red 6mg/β, tryptophan 10mg/A, arginine hydrochloride 0.2g/j2, cysteine hydrochloride hydrate 6
0 mg/l, wheat gluten hydrolysis obtained in Reference Example 1 2.5 g/j2, yeast extract 0.5 g/C liver concentrate 202-3 0.25 g/C cholesterol 0, 1 mg/R1 sodium bicarbonate 1 .5g/*
, penicillin 50,000 U/β, streptomycin 5
0,000μg/CHEPES 2.383g/R,
Insulin 3mg/11) Lanceferrin 5mg/
J, sodium pyruvate 550.2mg/12, selenite 16.13μg/j! , galactose Ig/Cp
H7,4 Table 7 Example 5 Hank's solution [Glucose Ig/j7. Sodium chloride 8
g/R, potassium chloride 0.4g/β, sodium hydrogen phosphate (anhydrous) 47.9mg/j, potassium dihydrogen phosphate (anhydrous) 60mg/Il, magnesium sulfate (anhydrous) 48.8mg7j! , Magnesilla Chloride 1146, 8
mg/i', calcium chloride (anhydrous) 140mg/I
! and phenol red 6mg/jJ plus vegetable protein SK
~5 0.5g/spear, cysteine hydrochloride-water salt 60mg
/j! , tryptophan 10 mg/β, arginine hydrochloride 0.2 g/A, penicillin 50,0 OOU/! , streptomycin 50 mg/R,) IEPB310
mM.
インシュリン３ｍｇ／ｊ、）ランスフェリン５ｍｇ／β
、ピルビン酸５ｍＭ、亜セレン酸 ０１２５μＭ１ガラ
トクトース１ ｇ／ｊ！及びＲＰＭ１１６４０培地のビ
タミン混合物を添加し培地を得た。Insulin 3mg/j,) Lanceferrin 5mg/β
, pyruvate 5mM, selenite 0125μM1 galatoctose 1 g/j! and a vitamin mixture of RPM11640 medium were added to obtain a medium.
実施例６
実施例５において、ＲＰＭ１１６４０培地のビタミン混
合物の代わりに酵母エキス０．５ｇ／ｊ２を用いる以外
は実施例５と同様にして培地を得た。Example 6 A medium was obtained in the same manner as in Example 5, except that 0.5 g/j2 of yeast extract was used instead of the vitamin mixture in the RPM11640 medium.
参考例１
小麦グルテンを５Ｗ／Ｖ％になる様に水溶液に懸濁した
後、塩酸でｐＨを４に調整した。該懸濁液にスミチーム
ＲＰ（酸性プロテアーセ：新日本化学社製）をグルテン
当りＩＷ／Ｖ％加え、５０℃で２時間振盪した後、カセ
イソーダでｐＨを７に調整した。これにスミチームＭＰ
（アルカリ性プロテアーゼ：新日本化学社製）をグルテ
ン当りＩＷ／Ｖ％加え、５０℃で２時間振盪後、遠心分
離（３０００ｒ、ｐ１ｍ８．１０分）し、上清を小麦ク
ルテン酵素分解物（グルテン濃度５Ｗ／Ｖ％）として得
た。Reference Example 1 Wheat gluten was suspended in an aqueous solution to a concentration of 5 W/V%, and then the pH was adjusted to 4 with hydrochloric acid. Sumiteam RP (acidic protease, manufactured by Shin Nippon Kagaku Co., Ltd.) was added to the suspension in an amount of IW/V% based on gluten, and after shaking at 50° C. for 2 hours, the pH was adjusted to 7 with caustic soda. Sumi team MP for this
(alkaline protease: manufactured by Shin Nippon Kagaku Co., Ltd.) was added at IW/V% per gluten, shaken at 50°C for 2 hours, centrifuged (3000r, p1m 8.10 minutes), and the supernatant was collected from wheat gluten enzymatic decomposition product (gluten concentration 5W/V%).
発明の効果
本発明の培地はグルタミンがペプチドの形で存在する為
、蒸気滅菌してもり゛ルタミンが分解されることがなく
グルタミンを別途添加する必要はなし）。又、小麦グル
テン酵素分解物中には多種類のアミノ酸が含まれている
ため、別途添加するアミノ酸の種類も少なくなり、経済
的にも安価な培地となる。Effects of the Invention Since the culture medium of the present invention contains glutamine in the form of a peptide, glutamine is not decomposed even after steam sterilization, so there is no need to separately add glutamine). In addition, since the enzymatic decomposition product of wheat gluten contains many types of amino acids, the number of types of amino acids that need to be added separately is reduced, resulting in an economically inexpensive medium.
第１及び２図は本発明の培地に添加される小麦グルテン
酵素分解物のゲルｐ過高性能液体クロマトグラフィーに
よる分子量分布パターンの一例を示す。
但し、数値は溶出時間（分）、カッコ内の数値は分子量
を示す。Figures 1 and 2 show an example of the molecular weight distribution pattern of the enzymatically decomposed wheat gluten product added to the medium of the present invention by gel p-superperformance liquid chromatography. However, the numerical value indicates the elution time (minutes), and the numerical value in parentheses indicates the molecular weight.
Claims (2)
する培地。(2) A medium containing enzymatic decomposition product of wheat gluten and yeast extract.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1044393A JPH0249579A (en) | 1988-02-29 | 1989-02-23 | Culture medium |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4705488 | 1988-02-29 | ||
| JP63-47054 | 1988-02-29 | ||
| JP1044393A JPH0249579A (en) | 1988-02-29 | 1989-02-23 | Culture medium |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0249579A true JPH0249579A (en) | 1990-02-19 |
Family
ID=26384289
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1044393A Pending JPH0249579A (en) | 1988-02-29 | 1989-02-23 | Culture medium |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0249579A (en) |
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|---|---|---|---|---|
| EP0672352A1 (en) * | 1994-03-16 | 1995-09-20 | Campina Melkunie B.V. | Processes for the preparation of glutamine-rich peptides and food preparations made therewith |
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1989
- 1989-02-23 JP JP1044393A patent/JPH0249579A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0672352A1 (en) * | 1994-03-16 | 1995-09-20 | Campina Melkunie B.V. | Processes for the preparation of glutamine-rich peptides and food preparations made therewith |
| NL9400418A (en) * | 1994-03-16 | 1995-11-01 | Campina Melkunie Bv | Processes for the preparation of glutamine-rich peptides and nutritional preparations made therewith. |
| US5741705A (en) * | 1995-02-23 | 1998-04-21 | Quest International Flavors & Food Ingredients Company, Division Of Indopco, Inc. | Method for in vitro cell growth of eucaryotic cells using low molecular weight peptides |
| US5885835A (en) * | 1995-02-23 | 1999-03-23 | Quest International Flavors & Food Ingredients Co., Division Of Indopco, Inc. | Kit for in vitro cell growth of eucaryotes using low molecular weight peptides containing L-glutamine |
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| EP1983044A2 (en) | 1996-10-10 | 2008-10-22 | Invitrogen Corporation | Animal cell culture media comprising plant-derived nutrients |
| US6103529A (en) * | 1996-10-10 | 2000-08-15 | Life Technologies, Inc. | Animal cell culture media comprising peptides derived from rice |
| WO2000003000A3 (en) * | 1998-07-10 | 2000-04-20 | Chugai Pharmaceutical Co Ltd | Serum-free medium for culturing animal cells |
| US6406909B1 (en) * | 1998-07-10 | 2002-06-18 | Chugai Seiyaku Kabushiki Kaisha | Serum-free medium for culturing animal cells |
| EP2351827A1 (en) | 2001-03-27 | 2011-08-03 | Life Technologies Corporation | Culture medium for cell growth and transfection |
| WO2005083058A1 (en) * | 2004-03-01 | 2005-09-09 | Ares Trading S.A. | Use of a serum-free cell culture medium for the production of il-18bp in mammalian cells |
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| WO2017195789A1 (en) * | 2016-05-10 | 2017-11-16 | 興人ライフサイエンス株式会社 | Composition for culture medium |
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