WO2008108549A1 - Procédé de conservation structurelle d'hémocytes à long terme reposant sur une technique de lyophilisation cellulaire - Google Patents
Procédé de conservation structurelle d'hémocytes à long terme reposant sur une technique de lyophilisation cellulaire Download PDFInfo
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- WO2008108549A1 WO2008108549A1 PCT/KR2008/001119 KR2008001119W WO2008108549A1 WO 2008108549 A1 WO2008108549 A1 WO 2008108549A1 KR 2008001119 W KR2008001119 W KR 2008001119W WO 2008108549 A1 WO2008108549 A1 WO 2008108549A1
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- Prior art keywords
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- hemocytes
- cell
- trehalose
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- 238000000034 method Methods 0.000 title claims abstract description 45
- 210000003677 hemocyte Anatomy 0.000 title claims abstract description 16
- 230000007774 longterm Effects 0.000 title claims abstract description 15
- 238000004321 preservation Methods 0.000 title claims description 7
- 238000004108 freeze drying Methods 0.000 title abstract description 12
- 230000001413 cellular effect Effects 0.000 title description 3
- 229940000351 hemocyte Drugs 0.000 title description 2
- 210000004027 cell Anatomy 0.000 claims abstract description 73
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims abstract description 51
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims abstract description 51
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims abstract description 51
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 45
- GZIFEOYASATJEH-VHFRWLAGSA-N δ-tocopherol Chemical compound OC1=CC(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1 GZIFEOYASATJEH-VHFRWLAGSA-N 0.000 claims abstract description 34
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims abstract description 28
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims abstract description 28
- AGBQKNBQESQNJD-SSDOTTSWSA-N (R)-lipoic acid Chemical compound OC(=O)CCCC[C@@H]1CCSS1 AGBQKNBQESQNJD-SSDOTTSWSA-N 0.000 claims abstract description 17
- GZIFEOYASATJEH-UHFFFAOYSA-N D-delta tocopherol Natural products OC1=CC(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1 GZIFEOYASATJEH-UHFFFAOYSA-N 0.000 claims abstract description 17
- YJPIGAIKUZMOQA-UHFFFAOYSA-N Melatonin Natural products COC1=CC=C2N(C(C)=O)C=C(CCN)C2=C1 YJPIGAIKUZMOQA-UHFFFAOYSA-N 0.000 claims abstract description 17
- AGBQKNBQESQNJD-UHFFFAOYSA-N alpha-Lipoic acid Natural products OC(=O)CCCCC1CCSS1 AGBQKNBQESQNJD-UHFFFAOYSA-N 0.000 claims abstract description 17
- 235000010389 delta-tocopherol Nutrition 0.000 claims abstract description 17
- 235000019136 lipoic acid Nutrition 0.000 claims abstract description 17
- DRLFMBDRBRZALE-UHFFFAOYSA-N melatonin Chemical compound COC1=CC=C2NC=C(CCNC(C)=O)C2=C1 DRLFMBDRBRZALE-UHFFFAOYSA-N 0.000 claims abstract description 17
- 229960003987 melatonin Drugs 0.000 claims abstract description 17
- 229960002663 thioctic acid Drugs 0.000 claims abstract description 17
- 239000002446 δ-tocopherol Substances 0.000 claims abstract description 17
- 102000004877 Insulin Human genes 0.000 claims abstract description 15
- 108090001061 Insulin Proteins 0.000 claims abstract description 15
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 claims abstract description 15
- 235000000069 L-ascorbic acid Nutrition 0.000 claims abstract description 15
- 239000002211 L-ascorbic acid Substances 0.000 claims abstract description 14
- 229960005070 ascorbic acid Drugs 0.000 claims abstract description 14
- 229940125396 insulin Drugs 0.000 claims abstract description 14
- 239000000872 buffer Substances 0.000 claims abstract description 13
- 241000282414 Homo sapiens Species 0.000 claims abstract description 10
- 210000003743 erythrocyte Anatomy 0.000 claims description 46
- 206010018910 Haemolysis Diseases 0.000 claims description 18
- 230000008588 hemolysis Effects 0.000 claims description 18
- 150000003180 prostaglandins Chemical class 0.000 claims description 5
- 239000000463 material Substances 0.000 abstract description 31
- 238000005534 hematocrit Methods 0.000 abstract description 7
- 238000001035 drying Methods 0.000 abstract description 5
- GMVPRGQOIOIIMI-UHFFFAOYSA-N (8R,11R,12R,13E,15S)-11,15-Dihydroxy-9-oxo-13-prostenoic acid Natural products CCCCCC(O)C=CC1C(O)CC(=O)C1CCCCCCC(O)=O GMVPRGQOIOIIMI-UHFFFAOYSA-N 0.000 abstract 1
- 229960000711 alprostadil Drugs 0.000 abstract 1
- 239000007853 buffer solution Substances 0.000 abstract 1
- GMVPRGQOIOIIMI-DWKJAMRDSA-N prostaglandin E1 Chemical compound CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1CCCCCCC(O)=O GMVPRGQOIOIIMI-DWKJAMRDSA-N 0.000 abstract 1
- XEYBRNLFEZDVAW-UHFFFAOYSA-N prostaglandin E2 Natural products CCCCCC(O)C=CC1C(O)CC(=O)C1CC=CCCCC(O)=O XEYBRNLFEZDVAW-UHFFFAOYSA-N 0.000 abstract 1
- 239000000523 sample Substances 0.000 abstract 1
- 230000006641 stabilisation Effects 0.000 abstract 1
- 238000011105 stabilization Methods 0.000 abstract 1
- 238000002474 experimental method Methods 0.000 description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- 210000003527 eukaryotic cell Anatomy 0.000 description 12
- 239000002953 phosphate buffered saline Substances 0.000 description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 239000003963 antioxidant agent Substances 0.000 description 9
- 235000006708 antioxidants Nutrition 0.000 description 9
- 230000003078 antioxidant effect Effects 0.000 description 7
- 210000000170 cell membrane Anatomy 0.000 description 7
- 239000006285 cell suspension Substances 0.000 description 7
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 230000008859 change Effects 0.000 description 6
- 229960005322 streptomycin Drugs 0.000 description 6
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Natural products 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 6
- 102000001554 Hemoglobins Human genes 0.000 description 5
- 108010054147 Hemoglobins Proteins 0.000 description 5
- 210000000601 blood cell Anatomy 0.000 description 5
- 230000005779 cell damage Effects 0.000 description 5
- 208000037887 cell injury Diseases 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 230000006378 damage Effects 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 4
- 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 4
- 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 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 230000002949 hemolytic effect Effects 0.000 description 4
- 230000003647 oxidation Effects 0.000 description 4
- 238000007254 oxidation reaction Methods 0.000 description 4
- 230000001681 protective effect Effects 0.000 description 4
- 229930024421 Adenine Natural products 0.000 description 3
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 3
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 3
- 229930195725 Mannitol Natural products 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- 229960000643 adenine Drugs 0.000 description 3
- RJGDLRCDCYRQOQ-UHFFFAOYSA-N anthrone Chemical compound C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 RJGDLRCDCYRQOQ-UHFFFAOYSA-N 0.000 description 3
- 150000002016 disaccharides Chemical class 0.000 description 3
- 239000000594 mannitol Substances 0.000 description 3
- 235000010355 mannitol Nutrition 0.000 description 3
- 239000010452 phosphate Substances 0.000 description 3
- 239000003223 protective agent Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 235000000346 sugar Nutrition 0.000 description 3
- 150000008163 sugars Chemical class 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000005034 decoration Methods 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 description 2
- 229940071106 ethylenediaminetetraacetate Drugs 0.000 description 2
- 210000001723 extracellular space Anatomy 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000008303 genetic mechanism Effects 0.000 description 2
- 231100000053 low toxicity Toxicity 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229940056360 penicillin g Drugs 0.000 description 2
- 230000035699 permeability Effects 0.000 description 2
- 150000003254 radicals Chemical class 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- 241000233866 Fungi Species 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 229920001612 Hydroxyethyl starch Polymers 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 150000000996 L-ascorbic acids Chemical class 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 229940050526 hydroxyethylstarch Drugs 0.000 description 1
- 239000004026 insulin derivative Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0641—Erythrocytes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/36—Lipids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/38—Vitamins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/33—Insulin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/80—Neurotransmitters; Neurohormones
- C12N2501/825—Serotonine (5-HT); Melatonine
Definitions
- the field of this invention belongs under cell preservation using lyophilization technique. If this method is utilized for preservation of eukaryotic cell, lyophilized cell can be stabilized structurally and can be preserved without cell damage over a long period of time.
- Method related with this study is to load high treahlose level in the extracellular space and then to increases trehalose concentration of cell inside.
- This method has advantage that cell membrane is not manipulated artificially. But there is a problem that cell damage increases because of high treahalose level in itself. If erythrocjtes are incubated during 24 hours, the half degree of the cells become hemolytic. This hemolytic erythrocyte should be removed one by one to use clinically, but this technique is very difficult. Moreover non-hemolytic erythrocyte can not be regarded healthy for its weakness. So using this method, technique that stabilze the cell and cell membrane should be developed.
- Vacuum condition can do protection of microoiganisms by minimizing oxidation, but there are a problems in case that preserve eukaryotic cell; it is impossible to make high vacuum condition that gas does not exist totally, the minimum gas induce eukaryotic cell to cell oxidation because eukaryotic cell don't have genetic mechanism that stoping cellular function in the dehydrated state. This oxidation produce free radical, it destroys cell oiganelle and cell membrane. Method that solve this problem is to find the stable materials for preserving eukaryotic cell. Disclosure of Invention Technical Problem
- the first subject that should solve is as following. When cells are loaded with trehalose of high concentration, cell damage should be reduced to clinical usable level. To use clinically, next two conditions must be satisfied; First, The rate of cell damage must become around 5%. Second, trehalose concentration of cell inside must become more than 6OmM. It is not difficult to make cell model loaded to high trehalose level. Simply trehalose and antioxidant have only to be put on the incubated cells. But it is very difficult that determinate concentration and preparation of trehalose and antioxidant. Several kinds of antioxidant must be put on one by one and observe result one after another. Previous our study revealed that insulin increased antioxidant function on hemolysis of red blood cell. So, mixture of antioxidant and insulin were loaded on the same cell model.
- glycerol is selected among several materials.
- Third, water absorptiveness of glycerol is very high, but if there does not exits water in storage receptacle, there will be no circumstance to absorb water. And glycerol exits liquid state in room temperature. Fourth, glycerol is very low toxicity and low cell permeability. Fifth, glycerol has no explosiveness, and there is no any change of state in room temperature.
- the model of cells to preserve is human erythrocytes.
- the antioxidants are selected all materials including with ⁇ -tocopherol, ⁇ -lipoic acid, melatonin, N- acetyl-L-cysteine, L-ascorbic acid which is good effect from previous hemolysis experiment. Insulin is used as material which rises an effect of antioxidant.
- PBS Phosphate-buffered saline, 300mOsm, pH 7.2
- ADSOL (4ffim ⁇ sm) contained 11 ImM glucose, 2mM adenine, 154mM NaCl and 4ImM mannitol.
- Buffer PBS(T) contained PBS and indicated amount of trehalose.
- Buffer ADSOL(T) contained ADSOL and K-phosphate and indicated amount of trehalose.
- ADSOL(T) (80OmM) contained 80OmM endotoxin- free trehalose (Hayashibara biochemical lab.
- ⁇ - tocopherol was prepared as aqueous solution including l%(v/v) ethanol.
- ⁇ -lipoic acid and melatonin was prepared as aqueous solution including 0.5%(v/v) ethanol. This use of ethanol did not show no significant change in previous study of red blood cell hemolysis. Order of experiments, measuring methods, results of experiment were as followings
- erythrocytes were collected from the bottom portion of the packed red cells. Then the cells were washed in PBS and centrifuged at 515g, lOmin three times. At each spin, erythrocytes were collected same method. Finally the erythrocytes were stored in PBS at 4 0 C with 30% hematocrit and used within 1 day.
- the loaded cells (0.5ml 30% hematocrit) were centrifugated at 350g for 3min and were mixed with 4ml of 80% methanol. The mixture was incubated at 85 0 C for 45min followed by centrifugation at 20Og for lOmin. The supernatant was collected and evaporated using a vacuum centrifuge(Labconco, USA), and dry residue was dissolved in 3mL of nano-pure water. This sample (3mL) were mixed with 6mL of anthrone reagent [2% anthrone in concentrated sulfuric acid], heated to 100 0 C for 3min, and allowed to cool.
- anthrone reagent 2% anthrone in concentrated sulfuric acid
- Absorbance at 620nm was measured on a spectrophotometer (Hitachi U2000, Japan)at room temperature and compared with a standard curve. Since the anthrone method detects all sugars, unloaded control erythrocytes were always treated in parallel. These values, normalized for cell count, were subtracted from the trehalose specifically and to avoid artifact due to endogenous sugars. Data are shown for at least three independent experiments.
- Figure 1 presents the result of incubated erythrocytes in 80OmM trehalose buffer without cell protection protocol for 15 hours in 37 0 C.
- erythrocytes were incubated during 15 hours ( ⁇ +4. ImM)
- this result showed that trehalose concentration of cell interior becomed above 6OmM.
- Figure 2 presents degree of erythrocytes hemolysis in 80OmM trehalose buffer without cell protection protocol in 37 0 C.
- erythrocytes were incubated for 15 hours(25 ⁇ 3.1%), erythrocytes showed 25% hemolysis.
- This result showed that erythrocytes were available to use clinically if only hemolytic erythrocytes were removed.
- this result showed that 5% hemolysis happened when 3 hours passed. Therefore, cell protection protocol must decrease cell damage up to result of 3 hours hemolysis statistically.
- Figure 3 presents the degree of hemolysis under each conditions. A(5.2+0.69%) and
- Figure 4 presents the result of incubated erythrocytes in 80OmM trehalose buffer with the final cell protection protocol ( ⁇ - tocopherol, ⁇ - lipoic acid, melatonin, N- acetyl-L-cysteine, L-ascorbic acid, insulin) in 37 0 C. It showed that concentration of cell inside beconed above 6OmM when erythrocytes were incubated for 15 hours (68+3.ImM). This value showed that there was no statistical significance to compared to value of result which was incubated for 15 hours (69+4.ImM) in Figure 1.
- the model of cells to preserve is human hemocytes.
- the names and concentration of cell protection material are 5 x 10 '4 M L-ascorbic acid, 5 x 10 '5 M ⁇ -lipoic acid, 5 x 10 ⁇ 5 M Melatonin, 3 x 10 4 M N-acetyl-L-cysteine, 10 5 M ⁇ -tocopherol, and 5 x 10 5 M insulin.
- the 10 5 M prostaglandin El is also used because prostaglandin El did role of stabilizing platelet in our previous experiment.
- PBS Phosphate-buffered saline, 300mOsm, pH 7.2
- ADSOL 462mOsm
- ADSOL contained 11 ImM glucose, 2mM adenine, 154mM NaCl and 4ImM mannitol.
- Buffer PBS(T) contained PBS and indicated amount of trehalose.
- Buffer ADSOL(T) contained ADSOL and K-phosphate and indicated amount of trehalose.
- ADSOL(T) (80OmM) contained 80OmM endotoxin-free trehalose (Hayashibara biochemical lab.
- Lyophilizing buffer contained lOOmOsmol ADSOL(24mM glucose, 0.43mM adenine, 33mM NaCl, 89mM mannitol), 10OmM trehalose, 66mM K-phosphate, 15% high molecular weight hydroxyethyl starch (HES, B. Braun Medical, Irvine, CA) and 2.5% human serum albumin (HSA, Sigma- Aldrich).
- ⁇ -tocopherol was prepared as aqueous solution including l%(v/v) ethanol.
- ⁇ -lipoic acid, melatonin and prostaglandin were prepared as aqueous solution including 0.5%(v/v) ethanol.
- erythrocytes were collected from the bottom portion of the packed red cells, leukocytes were collected from the buffy coat, and platelets were collected from upper layer of sample. Each hemocytes were collected to same tube. Then the cells were washed in PBS and centrifuged at 515g, lOmin three times. At each spin, blood corpuscles were collected same method. Washed erythrocytes were stored in PBS at 4 0 C with 30% hematocrit and used within 1 day.
- lyophilizer (Labconco,USA) The conditions of lyophilizer (Labconco,USA) were set as following. Hemocytes were incubated for 15 hours and were added with lyophilization buffer until the hematocrit reached to 5%. Then, the hemocytes were cooled at a rate of I 0 C per minute until they got to -4O 0 C, in which temperature the hemocytes were incubated for 30 minutes. Primary drying were performed at -3O 0 C for 7 hours at a vacuum of 30mTorr. During secondary drying which were performed for 6 hours, shelf temperature were increased from -3O 0 C to 2O 0 C at a rate of 0.8 0 C per minute using a vacuum of SOmTorr. After then, samples were put into a specially manufactured container. The air in the container were removed and the container were filled with anhydrous glycerol(>99.5%) for a long-term storage.
- Fig 5 presents well-preserved erythrocytes generally as time passes with little damage of erythrocytes. For the 6 months, cell morphology was showed most well- preserved aspect. Also, because there is no living things that live in the anhydrous glycerol(>99.5%), if basic aseptic method is performed, the contamination to mi- crooiganism, fungus, virus will be not concerned about. However, there are some problem that totally recover function. This problem is next subject that must solve. [41] Fig 6 presents sample that be utilized for remembering deceased people in funeral.
- the decoration container have lyophilized erythrocytes of the deceased. Even if all of deceased disappears with death, his blood corpuscle cells will be kept over a long period of time. After this is utilized for funeral ceremony, keeping can be with family or deceased.
- Fig 7 presents sample that be utilized for commemoration between lovers.
- the decoration container is personal ornaments which is portable commemoration material of lover.
- Figure 1 shows a graph illustraing the change of trehalose level at intervals of 3 hours when to erythrocytes were added with 80OmM trehalose in 37 0 C.
- Figure 2 shows a graph illustraing the degree of hemolysis at intervals of 3 hours when to erythrocytes were added with 80OmM trehalose in 37 0 C.
- Figure 3 shows a graph illustrating the degree of hemolysis occurred in 15 hours observation depending on each situation of cell protection protocols when to erythrocytes were added with 80OmM trehalose in 37 0 C.
- Figure 4 shows a graph illustrating the change of trehalose level at intervals of 3 hours observation depending on final cell protection protocols when to erythrocytes were added with 80OmM trehalose in 37 0 C.
- Figure 5 shows the result of long-term storage of lyophilized cells observed depending on time under the phase contrast microscope.
- Figure 6 shows a sample of lyophilized cells stored using this technique which can be utilized for rememberig deceased people.
- Figure 7 shows a sample of lyophilized cells stored using this technique which can be utilized for commemoration between lovers.
- T3 3 hours, T ⁇ 6 hours, T9: 9 hours, T12 12 hours, T15: 15 hours
- C The condition that erythrocytes were added with 80OmM trehalose, 5 x 10 4 M L- ascorbic acid for 15 hours in 37 0 C.
- D The condition that erythrocytes were added with 80OmM trehalose, 5 x 10 5 M ⁇ - lipoic acid for 15 hours in 37 0 C.
- E The condition that erythrocytes were added with 80OmM trehalose, 5 x 10 5 M
- F The condition that erythrocytes were added with 80OmM trehalose, 3 x 10 4 M N- acetyl-L-cysteine for 15 hours in 37 0 C.
- G The condition that erythrocytes were added with 80OmM trehalose, 10 5 M ⁇ - tocopherol for 15 hours.
- H The condition that erythrocytes were added with 80OmM trehalose, 5 x 10 4 M L- ascorbic acid, 5 x 10 5 M ⁇ -lipoic acid, 5 x 10 5 M Melatonin, 3 x 10 4 M N- acetyl-L-cysteine, 10 '5 M ⁇ -tocopherol for 15 hours in 37 0 C.
- the lyophilized cell can be preserved for remembering deceased people in funeral.
- the lyophilized cell can be preserved for remembering deceased people in funeral.
- the lyophilized cell can be preserved for commemoration between lovers.
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Abstract
L'invention concerne un procédé permettant de conserver des cellules de manière structurelle pendant une période courte ou longue. Ce procédé repose sur une technique de lyophilisation. Le procédé de l'état de la technique permet de préserver des cellules humaines lyophilisées pendant longtemps mais il présente des limites. Notre institut de recherches a découvert une manière de conserver les cellules lyophilisées de manière structurelle et sûre pendant une période courte ou longue. Le modèle de cellule à conserver est l'hémocyte humain. Il existe deux catégories de procédé de stockage : un procédé de stockage à court terme et un procédé de stockage à long terme. Pour conserver les hémocytes humains à long terme, on fait incuber les hémocytes dans une solution tampon de tréhalose 800nM à 37°C pendant 15 heures. Les noms et la concentration de la matière de protection cellulaire sont: acide L-ascorbique 5 × 10-4M; acide α-lipoïque 5 × 10-5M, mélatonine 5 × 10-5M; N-acétyl-L-cystéine3 × 10-4M; δ-tocophérol 10-5M; insuline 5 × 10-5M et prostaglandine E1 10-5M. Le stockage à long terme commence par la lyophilisation d'hémocytes incubés auquels on ajoute du glycérol plus tard. Les hémocytes incubent pendant 15 heures et sont ajoutés au tampon de lyophilisation jusqu'à ce que l'hématocrite atteigne 5 %. Puis, les hémocytes sont refroidis à une vitesse de 1°C par minute jusqu'à ce qu'ils atteignent -40°C, température à laquelle ils incubent pendant 30 minutes. Un séchage primaire s'effectue à -30°C pendant 7 h sous un vide de 30mTorr. Pendant le séchage secondaire qui dure six heures, la température de conservation augmente de -30 °C à 20 °C à une vitesse de 0, 8°C par minute, avec un vide de 50mTorr. Enfin, des échantillons sont placés dans un contenant spécialement conçu à cet effet. L'air du contenant est retiré et le contenant est rempli de glycérol anhydre (≥99,5%) pour un stockage à long terme. Même si de nombreuses études sont nécessaires pour retrouver les caractéristiques fonctionnelles de manière parfaite, le procédé de l'invention permet d'obtenir une stabilisation structurelle permettant un stockage semi-permanent. Le procédé de l'invention, destiné à stocker des cellules lyophilisées de manière semi-permanente, peut être utilisé de plusieurs façons dans nos vies quotidiennes. Par exemple, il peut être utilisé pour se souvenir de personnes décédées, à des funérailles, ou pour une commémoration entre des amoureux.
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KR1020070021350A KR100868602B1 (ko) | 2007-03-05 | 2007-03-05 | 인간 적혈구가 고농도 트리할로즈에 노출되었을 때발생하는 세포 용혈 현상을 최소화하는 방법 |
KR10-2007-0021350 | 2007-03-05 | ||
KR1020070023828A KR20080083376A (ko) | 2007-03-12 | 2007-03-12 | 세포의 동결건조 기술을 이용해 인간의 혈구세포를구조적으로 장기간 보관하는 방법 |
KR10-2007-0023828 | 2007-03-12 |
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Cited By (9)
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WO2013087936A1 (fr) * | 2011-12-16 | 2013-06-20 | Galderma Research & Development | Association d'un agoniste des récepteurs des prostaglandines et d'un agoniste du récepteur mc1r pour le traitement et/ou la prévention de désordres de la pigmentation |
WO2015191633A1 (fr) * | 2014-06-10 | 2015-12-17 | Biomatrica, Inc. | Stabilisation de polypeptides non dénaturés, d'acides nucléiques, et d'exosomes dans un échantillon de sang à des températures ambiantes |
US9376709B2 (en) | 2010-07-26 | 2016-06-28 | Biomatrica, Inc. | Compositions for stabilizing DNA and RNA in blood and other biological samples during shipping and storage at ambient temperatures |
US9725703B2 (en) | 2012-12-20 | 2017-08-08 | Biomatrica, Inc. | Formulations and methods for stabilizing PCR reagents |
US9845489B2 (en) | 2010-07-26 | 2017-12-19 | Biomatrica, Inc. | Compositions for stabilizing DNA, RNA and proteins in saliva and other biological samples during shipping and storage at ambient temperatures |
US10064404B2 (en) | 2014-06-10 | 2018-09-04 | Biomatrica, Inc. | Stabilization of thrombocytes at ambient temperatures |
US10568317B2 (en) | 2015-12-08 | 2020-02-25 | Biomatrica, Inc. | Reduction of erythrocyte sedimentation rate |
US11604026B2 (en) | 2019-03-14 | 2023-03-14 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
US11634257B2 (en) | 2017-10-09 | 2023-04-25 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
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US5242792A (en) * | 1991-02-25 | 1993-09-07 | The United States Of America As Represented By The Secretary Of The Navy | Method for the preservation of red blood cells by lyophilization using glycerol or inositol with disaccharides |
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Cited By (20)
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US9999217B2 (en) | 2010-07-26 | 2018-06-19 | Biomatrica, Inc. | Compositions for stabilizing DNA, RNA, and proteins in blood and other biological samples during shipping and storage at ambient temperatures |
US9376709B2 (en) | 2010-07-26 | 2016-06-28 | Biomatrica, Inc. | Compositions for stabilizing DNA and RNA in blood and other biological samples during shipping and storage at ambient temperatures |
US9845489B2 (en) | 2010-07-26 | 2017-12-19 | Biomatrica, Inc. | Compositions for stabilizing DNA, RNA and proteins in saliva and other biological samples during shipping and storage at ambient temperatures |
WO2013087936A1 (fr) * | 2011-12-16 | 2013-06-20 | Galderma Research & Development | Association d'un agoniste des récepteurs des prostaglandines et d'un agoniste du récepteur mc1r pour le traitement et/ou la prévention de désordres de la pigmentation |
US9364455B2 (en) | 2011-12-16 | 2016-06-14 | Galderma Research & Development | Combination of a prostaglandin receptor agonist and an MC1R receptor agonist for the treatment and/or prevention of pigmentation disorders |
US9725703B2 (en) | 2012-12-20 | 2017-08-08 | Biomatrica, Inc. | Formulations and methods for stabilizing PCR reagents |
US11672247B2 (en) | 2014-06-10 | 2023-06-13 | Biomatrica, Inc. | Stabilization of thrombocytes at ambient temperatures |
US10064404B2 (en) | 2014-06-10 | 2018-09-04 | Biomatrica, Inc. | Stabilization of thrombocytes at ambient temperatures |
US10772319B2 (en) | 2014-06-10 | 2020-09-15 | Biomatrica, Inc. | Stabilization of thrombocytes at ambient temperatures |
WO2015191633A1 (fr) * | 2014-06-10 | 2015-12-17 | Biomatrica, Inc. | Stabilisation de polypeptides non dénaturés, d'acides nucléiques, et d'exosomes dans un échantillon de sang à des températures ambiantes |
US10568317B2 (en) | 2015-12-08 | 2020-02-25 | Biomatrica, Inc. | Reduction of erythrocyte sedimentation rate |
US11116205B2 (en) | 2015-12-08 | 2021-09-14 | Biomatrica, Inc. | Reduction of erythrocyte sedimentation rate |
US11634257B2 (en) | 2017-10-09 | 2023-04-25 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
US11609042B2 (en) | 2019-03-14 | 2023-03-21 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
US11609043B2 (en) | 2019-03-14 | 2023-03-21 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
US11604026B2 (en) | 2019-03-14 | 2023-03-14 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
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US11747082B2 (en) | 2019-03-14 | 2023-09-05 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
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