CN112899223A - Preparation method of uterine blood stem cells - Google Patents
Preparation method of uterine blood stem cells Download PDFInfo
- Publication number
- CN112899223A CN112899223A CN201911228749.0A CN201911228749A CN112899223A CN 112899223 A CN112899223 A CN 112899223A CN 201911228749 A CN201911228749 A CN 201911228749A CN 112899223 A CN112899223 A CN 112899223A
- Authority
- CN
- China
- Prior art keywords
- stem cells
- uterine
- serum
- growth factor
- acid
- 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
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 187
- 210000004369 blood Anatomy 0.000 title claims abstract description 62
- 239000008280 blood Substances 0.000 title claims abstract description 62
- 238000002360 preparation method Methods 0.000 title claims abstract description 47
- 230000035755 proliferation Effects 0.000 claims abstract description 66
- 230000001506 immunosuppresive effect Effects 0.000 claims abstract description 40
- 238000012216 screening Methods 0.000 claims abstract description 26
- 210000004027 cell Anatomy 0.000 claims description 64
- 239000012679 serum free medium Substances 0.000 claims description 51
- 235000014113 dietary fatty acids Nutrition 0.000 claims description 30
- 229930195729 fatty acid Natural products 0.000 claims description 30
- 239000000194 fatty acid Substances 0.000 claims description 30
- 150000004665 fatty acids Chemical class 0.000 claims description 30
- 239000002609 medium Substances 0.000 claims description 30
- 210000002966 serum Anatomy 0.000 claims description 30
- 150000003904 phospholipids Chemical class 0.000 claims description 28
- 102000009024 Epidermal Growth Factor Human genes 0.000 claims description 18
- 101800003838 Epidermal growth factor Proteins 0.000 claims description 18
- 102000018233 Fibroblast Growth Factor Human genes 0.000 claims description 18
- 108050007372 Fibroblast Growth Factor Proteins 0.000 claims description 18
- 238000012258 culturing Methods 0.000 claims description 18
- 229940116977 epidermal growth factor Drugs 0.000 claims description 18
- 229940126864 fibroblast growth factor Drugs 0.000 claims description 18
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 claims description 18
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 claims description 18
- 238000004519 manufacturing process Methods 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 16
- 102100032528 C-type lectin domain family 11 member A Human genes 0.000 claims description 15
- 101710167766 C-type lectin domain family 11 member A Proteins 0.000 claims description 15
- 108010009583 Transforming Growth Factors Proteins 0.000 claims description 12
- 102000009618 Transforming Growth Factors Human genes 0.000 claims description 12
- YZXBAPSDXZZRGB-DOFZRALJSA-N arachidonic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O YZXBAPSDXZZRGB-DOFZRALJSA-N 0.000 claims description 10
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 claims description 10
- 230000002062 proliferating effect Effects 0.000 claims description 10
- 108010067225 Cell Adhesion Molecules Proteins 0.000 claims description 9
- AWUCVROLDVIAJX-UHFFFAOYSA-N alpha-glycerophosphate Natural products OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 claims description 7
- PORPENFLTBBHSG-MGBGTMOVSA-N 1,2-dihexadecanoyl-sn-glycerol-3-phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(O)=O)OC(=O)CCCCCCCCCCCCCCC PORPENFLTBBHSG-MGBGTMOVSA-N 0.000 claims description 6
- 102000004887 Transforming Growth Factor beta Human genes 0.000 claims description 6
- 108090001012 Transforming Growth Factor beta Proteins 0.000 claims description 6
- 231100001221 nontumorigenic Toxicity 0.000 claims description 6
- SECPZKHBENQXJG-FPLPWBNLSA-N palmitoleic acid Chemical compound CCCCCC\C=C/CCCCCCCC(O)=O SECPZKHBENQXJG-FPLPWBNLSA-N 0.000 claims description 6
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 claims description 6
- OYHQOLUKZRVURQ-NTGFUMLPSA-N (9Z,12Z)-9,10,12,13-tetratritiooctadeca-9,12-dienoic acid Chemical compound C(CCCCCCC\C(=C(/C\C(=C(/CCCCC)\[3H])\[3H])\[3H])\[3H])(=O)O OYHQOLUKZRVURQ-NTGFUMLPSA-N 0.000 claims description 5
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 claims description 5
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 claims description 5
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 claims description 5
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 claims description 5
- 239000005642 Oleic acid Substances 0.000 claims description 5
- 235000021314 Palmitic acid Nutrition 0.000 claims description 5
- 235000021355 Stearic acid Nutrition 0.000 claims description 5
- DTOSIQBPPRVQHS-PDBXOOCHSA-N alpha-linolenic acid Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(O)=O DTOSIQBPPRVQHS-PDBXOOCHSA-N 0.000 claims description 5
- 235000020661 alpha-linolenic acid Nutrition 0.000 claims description 5
- 235000021342 arachidonic acid Nutrition 0.000 claims description 5
- 229940114079 arachidonic acid Drugs 0.000 claims description 5
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 claims description 5
- 229960004488 linolenic acid Drugs 0.000 claims description 5
- KQQKGWQCNNTQJW-UHFFFAOYSA-N linolenic acid Natural products CC=CCCC=CCC=CCCCCCCCC(O)=O KQQKGWQCNNTQJW-UHFFFAOYSA-N 0.000 claims description 5
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 claims description 5
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 claims description 5
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 claims description 5
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 claims description 5
- 235000021313 oleic acid Nutrition 0.000 claims description 5
- 150000008104 phosphatidylethanolamines Chemical class 0.000 claims description 5
- 150000003905 phosphatidylinositols Chemical class 0.000 claims description 5
- 239000008117 stearic acid Substances 0.000 claims description 5
- TUNFSRHWOTWDNC-HKGQFRNVSA-N tetradecanoic acid Chemical compound CCCCCCCCCCCCC[14C](O)=O TUNFSRHWOTWDNC-HKGQFRNVSA-N 0.000 claims description 5
- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 claims description 4
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 claims description 4
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 claims description 4
- 241000124008 Mammalia Species 0.000 claims description 4
- ATBOMIWRCZXYSZ-XZBBILGWSA-N [1-[2,3-dihydroxypropoxy(hydroxy)phosphoryl]oxy-3-hexadecanoyloxypropan-2-yl] (9e,12e)-octadeca-9,12-dienoate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCC(O)CO)OC(=O)CCCCCCC\C=C\C\C=C\CCCCC ATBOMIWRCZXYSZ-XZBBILGWSA-N 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 244000005700 microbiome Species 0.000 claims description 4
- WRGQSWVCFNIUNZ-GDCKJWNLSA-N 1-oleoyl-sn-glycerol 3-phosphate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)COP(O)(O)=O WRGQSWVCFNIUNZ-GDCKJWNLSA-N 0.000 claims description 3
- 235000021319 Palmitoleic acid Nutrition 0.000 claims description 3
- 210000004204 blood vessel Anatomy 0.000 claims description 3
- SECPZKHBENQXJG-UHFFFAOYSA-N cis-palmitoleic acid Natural products CCCCCCC=CCCCCCCCC(O)=O SECPZKHBENQXJG-UHFFFAOYSA-N 0.000 claims description 3
- 230000002435 cytoreductive effect Effects 0.000 claims description 3
- 229960002969 oleic acid Drugs 0.000 claims description 3
- 230000000717 retained effect Effects 0.000 claims description 3
- 102000008395 cell adhesion mediator activity proteins Human genes 0.000 claims 1
- 239000000654 additive Substances 0.000 abstract description 27
- 239000001963 growth medium Substances 0.000 abstract description 14
- 239000003814 drug Substances 0.000 abstract description 3
- 230000001172 regenerating effect Effects 0.000 abstract description 3
- 230000000996 additive effect Effects 0.000 description 23
- 238000002054 transplantation Methods 0.000 description 17
- 238000002560 therapeutic procedure Methods 0.000 description 13
- 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
- 241001465754 Metazoa Species 0.000 description 9
- 210000001744 T-lymphocyte Anatomy 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 9
- 102000016289 Cell Adhesion Molecules Human genes 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 210000003719 b-lymphocyte Anatomy 0.000 description 7
- 239000007640 basal medium Substances 0.000 description 7
- 230000006870 function Effects 0.000 description 7
- 210000002865 immune cell Anatomy 0.000 description 7
- 239000000463 material Substances 0.000 description 6
- 102000004877 Insulin Human genes 0.000 description 5
- 108090001061 Insulin Proteins 0.000 description 5
- 229940125396 insulin Drugs 0.000 description 5
- 210000000822 natural killer cell Anatomy 0.000 description 5
- 241000282414 Homo sapiens Species 0.000 description 4
- 239000003963 antioxidant agent Substances 0.000 description 4
- 230000003078 antioxidant effect Effects 0.000 description 4
- 235000006708 antioxidants Nutrition 0.000 description 4
- 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 4
- 238000012136 culture method Methods 0.000 description 4
- 230000004069 differentiation Effects 0.000 description 4
- 150000002632 lipids Chemical class 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 239000004017 serum-free culture medium Substances 0.000 description 4
- 102000016359 Fibronectins Human genes 0.000 description 3
- 108010067306 Fibronectins Proteins 0.000 description 3
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 3
- 102000004338 Transferrin Human genes 0.000 description 3
- 108090000901 Transferrin Proteins 0.000 description 3
- 238000011109 contamination Methods 0.000 description 3
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 3
- 229960003957 dexamethasone Drugs 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 239000003862 glucocorticoid Substances 0.000 description 3
- 239000003102 growth factor Substances 0.000 description 3
- 239000012642 immune effector Substances 0.000 description 3
- 229940121354 immunomodulator Drugs 0.000 description 3
- 229940125721 immunosuppressive agent Drugs 0.000 description 3
- 239000003018 immunosuppressive agent Substances 0.000 description 3
- 239000004094 surface-active agent Substances 0.000 description 3
- 239000012581 transferrin Substances 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Natural products OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 102000004506 Blood Proteins Human genes 0.000 description 2
- 108010017384 Blood Proteins Proteins 0.000 description 2
- 108010039419 Connective Tissue Growth Factor Proteins 0.000 description 2
- 102000015225 Connective Tissue Growth Factor Human genes 0.000 description 2
- ZAKOWWREFLAJOT-CEFNRUSXSA-N D-alpha-tocopherylacetate Chemical compound CC(=O)OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C ZAKOWWREFLAJOT-CEFNRUSXSA-N 0.000 description 2
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 2
- 102000013275 Somatomedins Human genes 0.000 description 2
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 2
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 2
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 238000011316 allogeneic transplantation Methods 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 230000003110 anti-inflammatory effect Effects 0.000 description 2
- 235000010323 ascorbic acid Nutrition 0.000 description 2
- 239000011668 ascorbic acid Substances 0.000 description 2
- -1 ascorbic acid compound Chemical class 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 238000010322 bone marrow transplantation Methods 0.000 description 2
- 239000000919 ceramic Substances 0.000 description 2
- 235000012000 cholesterol Nutrition 0.000 description 2
- 210000004443 dendritic cell Anatomy 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 208000024908 graft versus host disease Diseases 0.000 description 2
- 210000005260 human cell Anatomy 0.000 description 2
- 230000002519 immonomodulatory effect Effects 0.000 description 2
- 230000006058 immune tolerance Effects 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 239000002075 main ingredient Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000007910 systemic administration Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 230000017423 tissue regeneration Effects 0.000 description 2
- 229940042585 tocopherol acetate Drugs 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 229940072107 ascorbate Drugs 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 150000008105 phosphatidylcholines Chemical class 0.000 description 1
- 229940067605 phosphatidylethanolamines Drugs 0.000 description 1
- 229940067626 phosphatidylinositols Drugs 0.000 description 1
- 150000008106 phosphatidylserines Chemical class 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 231100000588 tumorigenic Toxicity 0.000 description 1
- 230000000381 tumorigenic effect Effects 0.000 description 1
Images
Classifications
-
- 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
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
-
- 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
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
-
- 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
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
-
- 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
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- 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
- C12N2501/10—Growth factors
- C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
-
- 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
- C12N2501/10—Growth factors
- C12N2501/135—Platelet-derived growth factor [PDGF]
-
- 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
- C12N2501/10—Growth factors
- C12N2501/15—Transforming growth factor beta (TGF-β)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Developmental Biology & Embryology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Rheumatology (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of regenerative medicine, in particular to a preparation method of uterine blood stem cells, which mainly completes the preparation work of the uterine blood stem cells through a proliferation step, a screening step and a pre-proliferation step. Wherein, because the culture medium is added with specific additives, the prepared uterine blood stem cells have improved immunosuppressive action while maintaining immunosuppressive ability.
Description
Technical Field
The invention relates to the technical field of regenerative medicine, in particular to a preparation method of uterine blood stem cells.
Background
It is well known that in order to provide safer and stable quality of uterine stem cells, serum-free media which produce no or little contamination with heterologous proteins during culture are suitable, and that uterine stem cells are not only known to be low in immunogenicity but also to affect various immune effector cells. However, the use of immunosuppressive agents is associated with problems such as side effects of immunosuppressive agents. Uterine stem cells have immunosuppressive capabilities, so if uterine stem cells can be used, no immunosuppressive agent is needed. Therefore, it is highly desirable to produce immunosuppressive hematopoietic stem cells in serum-free or low serum cultures with a low risk of foreign protein and synthetic medium contamination.
Disclosure of Invention
The invention provides a preparation method of a uterine blood stem cell, which completes the preparation and culture processes of the uterine blood stem cell by adding a specific additive into a culture medium, maintains the immunosuppressive capability and improves the immunosuppressive action.
In order to achieve the purpose, the invention provides the following technical scheme: a preparation method of uterine blood stem cells is characterized by comprising the following steps:
a proliferation step comprising proliferating uterine stem cells in a first serum-free medium containing fibroblast growth factor, platelet-derived factor, transforming growth factor-beta; the first serum-free medium further comprises transforming growth factor-beta, stem cell growth factor, epidermal growth factor, at least one phospholipid, and at least one fatty acid;
a screening step of screening the uterine stem cells expanded in the expanding step for the uterine stem cells having retained or improved immunosuppressive ability;
a pre-propagation step comprising culturing the uterine stem cells in another serum-free medium containing fibroblast growth factor, platelet-derived factor, epidermal growth factor, at least one phospholipid and at least one fatty acid, said serum-free medium being free of stem cell growth factor and transforming growth factor-beta;
the culture time of the pre-proliferation step and the proliferation step is 48 days to 68 days, and the cell preparation comprises uterine stem cells for maintaining or improving the immunosuppressive ability; wherein, in the pre-proliferation step, the uterine stem cells are subcultured at least once.
Preferably, before the screening step, the method further comprises the following steps: a serum culturing step, which comprises culturing the uterine blood stem cells subjected to the proliferation step in a medium containing serum.
Preferably, it comprises screening the uterine blood stem cells expanded in the expanding step for non-tumorigenic uterine blood stem cells.
Preferably, in the step of expanding, the uterine blood stem cells are expanded by using a cultured blood vessel suitable for the expansion of the uterine blood stem cells.
Preferably, the serum-free medium further comprises a cell adhesion molecule during the propagation step.
Preferably, the uterine stem cells are subcultured at least once during the expansion step.
Preferably, in the proliferation step, the manner of subculturing is to use a cytoreductive agent containing no components derived from mammals or microorganisms to exfoliate the uterine stem cells.
Preferably, the method further comprises the following steps: selecting a culture vessel suitable for the proliferation of the uterine blood stem cells.
Preferably, the phospholipid is selected from phosphatidic acid, lysophosphatidic acid, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol.
Preferably, the fatty acid is selected from the group consisting of linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoleic acid, palmitic acid, and stearic acid.
The invention has the beneficial effects that: the uterine stem cells contained in the cell preparation produced by the present invention maintain the function of affecting immune effector cells, which function originally possessed the function of the uterine stem cells. The uterine stem cells have an immunomodulatory effect and an immune tolerance effect, and therefore the cell preparation produced by the present invention can be expected to be used for therapy to show those effects. Furthermore, it is also expected that the uterine stem cells of the cell preparation produced according to the present invention also play a role in the anti-inflammatory action of the uterine stem cells in transplantation therapy. The cell preparations produced by the present invention can be used for the treatment of local diseases in which the uterine stem cells are administered to a site requiring transplantation of the uterine stem cells. In addition, by administering the uterine stem cells to the whole body, that is, administering the uterine stem cells to veins and the like, and transporting the uterine stem cells to the whole body, it is possible to more effectively treat strong immune rejection by immunization. Acute GVHD caused by bone marrow transplantation, etc. Therefore, it is expected to significantly improve the survival rate of human beings. Furthermore, the cell preparation produced by the present invention is a cell preparation produced by serum-free culture. Therefore, useful growth factors or differentiation factors are not non-specifically adsorbed together with serum proteins onto a graft material such as ceramics. Therefore, the tissue regeneration ability caused by transplantation of the cell preparation produced by the present invention is very high. This results in a high therapeutic effect. Needless to say, treatment by combination of both local administration and systemic administration may be performed. The immunosuppressive ability of the uterine stem cells contained in the cell preparation produced by the present invention is maintained or improved, and the immune rejection caused when the transplantation is carried out is suppressed by the uterine stem cells. Thus, uterine stem cells can be used in therapy to transplant non-self tissues or cells to another person. The uterine stem cells can be preferably used not only for allogeneic transplantation by human tissues or human cells but also for xenogeneic transplantation by tissues other than animals or animals.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic diagram of the main steps of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it is to be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
According to fig. 1, a method for preparing uterine blood stem cells is characterized by comprising the following steps:
a proliferation step comprising proliferating uterine stem cells in a first serum-free medium containing fibroblast growth factor, platelet-derived factor, transforming growth factor-beta; the first serum-free medium further comprises transforming growth factor-beta, stem cell growth factor, epidermal growth factor, at least one phospholipid, and at least one fatty acid; a screening step of screening the uterine stem cells expanded in the expanding step for the uterine stem cells having retained or improved immunosuppressive ability; a pre-propagation step comprising culturing the uterine stem cells in another serum-free medium containing fibroblast growth factor, platelet-derived factor, epidermal growth factor, at least one phospholipid and at least one fatty acid, said serum-free medium being free of stem cell growth factor and transforming growth factor-beta; the culture time of the pre-proliferation step and the proliferation step is 48 days to 68 days, and the cell preparation comprises uterine stem cells for maintaining or improving the immunosuppressive ability; wherein, in the pre-proliferation step, the uterine stem cells are subcultured at least once. Before the screening step, the method also comprises the following steps: a serum culturing step, which comprises culturing the uterine blood stem cells subjected to the proliferation step in a medium containing serum. Further comprising selecting non-tumorigenic uterine blood stem cells from the uterine blood stem cells expanded in the expanding step. In the proliferation step, the uterine stem cells are proliferated by using a culture blood vessel suitable for the proliferation of the uterine stem cells. The serum-free medium further comprises a cell adhesion molecule during the propagation step.
The uterine stem cells are subcultured at least once during the expansion step. In the proliferation step, the manner of subculturing is to use a cytoreductive agent containing no components derived from mammals or microorganisms to exfoliate the uterine stem cells. Further comprising: selecting a culture vessel suitable for the proliferation of the uterine blood stem cells. The phospholipid is selected from phosphatidic acid, lysophosphatidic acid, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine and phosphatidylglycerol. The fatty acid is selected from the group consisting of linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoleic acid, palmitic acid, and stearic acid.
In the above-described configuration, the culture medium having the ability to greatly expand the uterine stem cells under serum-free conditions further has the effect of maintaining or improving the culture of the uterine stem cells having immunosuppressive ability. This makes it possible to produce cell preparations comprising uterine blood stem cells, which are used in particular in regenerative medicine.
A method for preparing a cell preparation containing uterine blood stem cells which maintain or improve their immunosuppressive ability under serum-free conditions. The method for producing a cell preparation according to the present invention comprises the steps of: (A) proliferating uterine stem cells in a serum-free medium comprising fibroblast growth factor, platelet-derived factor, transforming growth factor-beta, stem cell growth factor, epidermal growth factor, at least one phospholipid and at least one fatty acid; (B) selecting the uterine stem cells having maintained or improved immunosuppressive ability from the expanded uterine stem cells subjected to the step (A).
A diffusion step of proliferating the uterine stem cells by culturing the uterine stem cells in a serum-free medium containing fibroblast growth factor, platelet-derived factor, transforming growth factor-beta. Stem cell growth factor, epidermal growth factor, at least one phospholipid and at least one fatty acid. The basal medium constituting the serum-free medium used for the proliferation step is not particularly limited as long as the basal medium is a medium known for animal cells in the art. Serum-free media with fibroblast growth factor, platelet derived factor, transforming growth factor-beta, stem cell growth factor, epidermal growth factor, at least one phospholipid and at least one fatty acid added to the basal media can be used to proliferate the steps of one embodiment. The use of such a serum-free medium has a proliferation promoting effect which is equal to or greater than that of a serum-containing medium, while preventing the contamination with foreign proteins. This makes it possible to proliferate uterine stem cells desirably. In the proliferation step of the production method according to the present invention, examples of the phospholipid contained in the serum-free medium include phosphatidylacids, lysophosphatidylacids, phosphatidylinositols, phosphatidylserines, phosphatidylethanolamines, phosphatidylcholines and phosphatidylglycerols. These phospholipids may be used alone, or two or more kinds may be used in combination. In one embodiment, the serum-free medium may comprise phosphatidic acid and phosphatidylcholine in combination, and those phospholipids may be derived from animals or plants. Examples of the fatty acid contained in the serum-free medium include linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitic acid, and stearic acid. The additive for media according to this embodiment may contain any one of those fatty acids alone or two or more types of fatty acids in combination. The serum-free medium of the present embodiment may contain not only fatty acids but also cholesterol. The serum-free medium may further comprise at least two factors selected from the group consisting of connective tissue growth factor, vascular endothelial growth factor and an ascorbic acid compound. In one embodiment, the lipid antioxidant comprised in the serum-free medium may be tocopherol acetate. The serum-free medium may further comprise a surfactant. The serum-free medium may further comprise insulin, transferrin and selenate. The term "insulin" as used in this specification may also be an insulin-like growth factor and may be derived from a natural cell or may be genetically modified. The additive for a medium according to the present invention may further comprise dexamethasone or another glucocorticoid.
In the proliferation step, uterine blood stem cells isolated from animal tissues or cells by a conventionally known method are seeded on the above serum-free medium and cultured up to the uterine blood stem cells. Proliferating to the desired number of cells. As the culture conditions, preferred are: the uterine blood stem cells were inoculated in 1ml of a medium and cultured at a culture temperature of 37 ℃ for 48 to 96 hours. By culturing the uterine stem cells as described above, a large number of uterine stem cells maintaining or improving the immunosuppressive ability can be efficiently produced.
In the proliferation step, the culture vessel used for the culture is not particularly limited, provided that the uterine blood stem cells can be proliferated. In order to more efficiently proliferate bone marrow uterine stem cells, it is preferable to subject the bone marrow uterine stem cells to be proliferated to a proliferation step in the proliferation step. Examples of the method for selecting a culture vessel suitable for the proliferation of the target cells include a method for selecting an optimal culture vessel by the proliferation of the target cells. More specifically, a plurality of culture vessels are prepared, and target cells are propagated under the same conditions except for the kind of culture vessel. On the fourteenth day of culture, the number of cells in each vessel was measured by a well-known method. It can be determined that the culture vessel with the largest number of cells is most suitable for proliferation of the target cells. Further, in the case where the proliferation rate of proliferating target cells is high, in the case where a culture vessel suitable for proliferation of proliferating target cells has been found, the culture vessel can be used in the proliferation step of the production method according to the present invention. In contrast, in the case where a culture vessel suitable for proliferation of the target cells has not been found, the production method of the present invention may further include a "culture vessel selection step" before the proliferation step. The cell adhesion molecule content in serum-free medium is preferably 1 to 50. mu.g/ml at the final concentration, and more preferably 5. mu.g/ml at the final concentration. In one embodiment, where the cell adhesion molecule is fibronectin, fibronectin is added such that the final concentration of fibronectin in serum-free medium is 5 μ g/ml. This can improve the adhesion efficiency of the proliferating target cells with respect to the culture vessel.
In the step of expanding, the uterine stem cells may be subcultured at least once. Uterine stem cells proliferate anchorage-dependent. For example, in the case where the uterine blood stem cells are locally unevenly expanded, the culture conditions of the uterine blood stem cells can be improved by subculturing the uterine blood stem cells in the middle of the expansion step. The subculture of the uterine blood stem cells may be carried out by any method, and may be carried out by a known method for subculturing uterine blood stem cells. In order to obtain a good cell state of the uterine stem cells after the subculture, when the uterine stem cells are stem cells, it is preferable to exfoliate the uterine stem cells using a cell-depleting agent which does not contain components derived from mammals and microorganisms.
A screening step of screening the uterine stem cells having been subjected to proliferation for the uterine stem cells having maintained or improved immunosuppressive ability. And (5) carrying out the steps. The uterine stem cells expanded in serum-free medium during the expansion step maintain or even improve their immunosuppressive capacity at least. By screening such uterine stem cells with an immunosuppressive ability as a reference, it is possible to select a uterine stem cell having an immunosuppressive ability maintained or enhanced. The uterine stem cells thus selected are used as a cell preparation.
Serum-free media are free of contaminating proteins and allow the growth of uterine stem cells without depriving them of function as stem cells. In addition, the proliferation potency of the uterine stem cells cultured in a serum-free medium is higher than that of the uterine stem cells cultured in a medium containing fetal bovine serum, and also has a high activity indicating an immunosuppressive effect. In the case where uterine stem cells cultured in a serum-free medium are used for transplantation therapy, cells having stem cell function and increased immunosuppressive ability and proliferating single cells have high activity. Thus, a synergistic effect of function and immunosuppressive ability is desired.
In the screening step, the thus-proliferated uterine stem cells and immune cells are co-cultured in a serum-free medium that has been used for the proliferation step, and the number of immune cells in the medium is evaluated after the culture. Co-cultivation is carried out. This allows the screening of uterine stem cells that maintain or improve their immunosuppressive ability. In the case where B cells or activated NK cells are used as immune cells, the activated NK cells are evaluated. This allows the screening of uterine stem cells whose immunosuppressive capacity is maintained or increased. In addition, in the case where dendritic cells are used as immune cells, differentiation, maturation, activation, and the like of the dendritic cells are evaluated. This allows the screening of uterine stem cells whose immunosuppressive capacity is maintained or increased. The immune cells used for such screening are preferably T cells, B cells or activated NK cells, more preferably T cells or B cells, most preferably T cells. This allows the screening of uterine stem cells whose immunosuppressive capacity is maintained or increased. The immune cells used for such screening are preferably T cells, B cells or activated NK cells, more preferably T cells or B cells, most preferably T cells. This allows the screening of uterine stem cells whose immunosuppressive capacity is maintained or increased. The immune cells used for such screening are preferably T cells, B cells or activated NK cells, more preferably T cells or B cells, most preferably T cells.
A second screening step, wherein the proliferation in serum-free medium in the proliferation step selectively grows the uterine blood stem cells, thereby increasing the number of non-tumorigenic uterine blood stem cells. Such uterine stem cells are screened by using tumorigenicity as a reference. This allows selection of uterine stem cells that are not tumorigenic. Meanwhile, the thus selected uterine stem cells are used as a cell preparation, which enables transplantation therapy of uterine stem cells having no tumorigenicity. Thus, the present invention also provides a method for producing non-tumorigenic uterine blood stem cells containing a cell preparation under serum-free conditions.
And a serum culture step of culturing the uterine blood stem cells which have been subjected to the proliferation step in a serum-containing medium. In the serum culture step, the uterine blood stem cells cultured under serum-free conditions in the proliferation step are cultured in a serum-containing medium. In the case of performing the serum culture step, the uterine blood stem cells are subjected to the screening step after being subjected to the serum culture step.
A conventionally known serum-containing medium can be used as the serum-containing medium used in the serum culturing step, for example, a 10% FBS-containing medium prepared by adding 10% FBS to a basal medium. In the serum culture step, the uterine blood stem cells obtained in the proliferation step are inoculated and cultured in a serum-containing medium. By culturing the uterine stem cells as described above, the step of screening the uterine stem cells cultured in a serum-containing medium is performed, and the uterine stem cells that maintain or improve the immunosuppressive ability are selected.
As described above, the uterine stem cells are pre-cultured in a serum-free medium and then cultured in a serum-containing medium. Therefore, the immunosuppression obtained by the uterine stem cells is equal to or greater than that of the uterine stem cells which are pre-cultured and cultured in a serum-containing medium. In addition, uterine stem cells show earlier immunosuppressive effects. In addition, since serum is used only when uterine stem cells are cultured, the serum content is low. This enables low serum culture.
A pre-diffusion step comprising the steps of: (D) comprises fibroblast growth factor, platelet derived factor, epidermal growth factor, at least one phospholipid and at least one fatty acid. In one aspect, the serum-free medium preferably comprises a lipid antioxidant, similar to serum-free medium. In addition, the serum-free medium may further comprise a surfactant. In addition, the serum-free medium may further comprise insulin, transferrin and selenate. The serum-free medium may further comprise dexamethasone or another glucocorticoid. These components have also been described in the serum-free medium of the "propagation step", and therefore the description thereof is omitted here.
In the pre-proliferation step, uterine blood stem cells isolated from animal tissues by a conventionally known method are inoculated in a serum-free medium and cultured until the number of the uterine blood stem cells is proliferated. To the desired number of uterine stem cells. The uterine stem cells subjected to the pre-expansion step are not particularly limited. However, the uterine stem cells are preferably primary ones, i.e., preferably cells that have not been subcultured after collection from animal tissue.
In order to efficiently proliferate the uterine stem cells in the pre-proliferation step, it is preferable that the pre-proliferation step is performed with a culture vessel suitable for proliferation of a specific type of uterine stem cells to be proliferated therein. A pre-proliferation step. The method of selecting a culture vessel suitable for the pre-proliferation of the proliferation target cells has been described in the "proliferation step", and thus the description thereof is omitted here.
In the case where the target cells do not adhere sufficiently to the culture vessel before proliferation, the serum-free medium may further contain a cell adhesion molecule in the pre-proliferation step, similarly to the proliferation step. The cell adhesion molecules have been described in the "proliferation step" and thus the description thereof is omitted here.
The uterine stem cells may be passaged at least once during the pre-expansion step, which is similar to the expansion step. By culturing the uterine stem cells in the middle of the pre-expansion step, the culture conditions can be improved.
A culture container selection step for selecting a culture container suitable for the proliferation of the uterine blood stem cells before the proliferation step. The method of selecting a culture vessel suitable for the proliferation of uterine blood stem cells has been described in the "proliferation step", and thus the description thereof is omitted here.
Serum-free additive for media used for the preparation of cell preparations, the additive for media according to the invention comprises fibroblast growth factor, platelet-derived factor, transforming growth factor-beta, stem cell growth factor, epidermal growth factor, at least one phospholipid and at least one fatty acid. By adding the additive for a culture medium according to the present invention to a conventionally known basal medium, a conventionally known basal medium can be used as a serum-free medium for production. A uterine blood stem cell containing a cell preparation which maintains or enhances its immunosuppressive ability. Examples of the phospholipid contained in the additive for media according to the present invention include phosphatidic acid, lysolipidic acid, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol. These phospholipids may be used alone, or two or more kinds may be used in combination. In one embodiment, an additive for a medium according to the present invention comprises a combination of phosphatidic acid and phosphatidylcholine. In addition, those phospholipids may be derived from animals or plants. According to this embodiment, examples of the fatty acid contained in the additive for media include linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitic acid, and stearic acid. The additive for media according to this embodiment may contain any one of those fatty acids alone or two or more types of fatty acids in combination. The medium additive of the present embodiment may contain cholesterol in addition to the fatty acid. In one embodiment, the serum-free medium may further comprise at least two factors selected from the group consisting of connective tissue growth factor, vascular endothelial growth factor and ascorbate compound. In one aspect, the additive for a medium according to the invention preferably comprises a lipid antioxidant. In one embodiment, the lipid antioxidant comprised in the additive for media according to this embodiment may be tocopherol acetate. The additive for media according to the present invention may further comprise a surfactant. The supplement for culture medium according to the present invention may further comprise insulin, transferrin and selenate. The term "insulin" as used in this specification may also be an insulin-like growth factor and may be derived from a natural cell or may be genetically modified. The additive for a medium according to the present invention may further comprise dexamethasone or another glucocorticoid.
A serum-free culture kit for animal cells for producing a cell preparation containing uterine stem cells having maintained or improved immunosuppressive ability. The additive for a culture medium according to the present invention comprises fibroblast growth factor, platelet-derived factor, transforming growth factor-beta, stem cell growth factor, epidermal growth factor, at least one phospholipid and at least one fatty acid. . The additive for culture media according to the present invention may further include a cell adhesion molecule. The kit for an additive for a culture medium according to the present invention may contain fibroblast growth factor, platelet-derived factor, transforming growth factor-beta, stem cell growth factor, epidermal growth factor, at least one phospholipid and at least one fatty acid in one container or contain these components, respectively. The kit for an additive for a culture medium according to the present invention may further comprise a cell adhesion molecule. By using the kit of the additive for a culture medium of the present invention in a conventionally known basal medium, the additive can be added to the conventionally known basal medium to prepare a serum-free culture medium for producing a cell preparation containing uterine stem cells, the immunosuppressive ability of which is maintained or improved. In the specification, the term "composition" refers to a composition in which one main ingredient is contained in one material, and the term "kit" refers to a kit in which at least one of a plurality of main ingredients is contained in a material other than one material. Including other materials of various primary constituents. Therefore, it is easily understood that the growth factors, at least one phospholipid and at least one fatty acid included in the kit for additives for culture media according to the present invention are the same as those described in the additives for culture media. The kit according to the present invention is a kit for producing a cell preparation containing uterine blood stem cells maintaining or improving their immunosuppressive ability, and includes at least the additive for culture medium according to the present invention. In addition, the kit according to the present invention may further comprise additives for a medium containing fibroblast growth factor, platelet-derived factor, epidermal growth factor, at least one phospholipid and at least one fatty acid. Serum-free medium for the preparation of cells for the production of a cell preparation containing uterine blood stem cells maintained or improved in their immunosuppressive capacity. The culture medium according to the invention comprises fibroblast growth factor, platelet derived factor, transforming growth factor-beta, stem cell growth factor, epidermal growth factor, at least one phospholipid and at least one fatty acid. The medium of the present invention can be used as a serum-free medium for producing a cell preparation containing uterine stem cells which maintains or enhances the immunosuppressive ability. The medium according to the present invention comprises fibroblast growth factor, platelet-derived factor, transforming growth factor-beta, stem cell growth factor, epidermal growth factor, at least one phospholipid and at least one fatty acid, and these components may be added to the base simultaneously. The medium may be added separately. That is, it can be said that the medium according to the present invention only needs to contain components contained in the medium additive or components contained in the kit.
A culture method for preparing cells, the culture method according to the present invention comprising a step wherein uterine stem cells are cultured in a serum-free medium containing fibroblast growth factor, platelet-derived factor, transforming growth factor-beta, stem cell growth factor, epidermal growth factor, at least one phospholipid and at least one fatty acid. In the culture method according to the present invention, serum-free medium may be used to culture the uterine stem cells. The culture method according to the present invention may further comprise the step of culturing the uterine blood stem cells in a serum-free medium containing fibroblast growth factor, platelet-derived factor, epidermal growth factor at 20 ℃ before the culturing step. At least one phospholipid and at least one fatty acid.
As a further use, it is also possible to proliferate uterine blood stem cells at the same rate as or higher than that of the uterine blood stem cells. Culturing the cells in a serum-containing medium, whereby the immunosuppressive ability of the expanded uterine stem cells is maintained or enhanced. In addition, non-tumorigenic uterine stem cells expanded in the present invention are selectively increased. In the present invention, the differentiation ability of the uterine stem cells is expanded. Therefore, when a cell preparation containing such uterine stem cells is administered to a patient, not only good transplantation therapy but also immune rejection can be effectively suppressed due to the uterine stem cells, which is a big problem of transplantation therapy. This makes it possible to reduce the burden on the patient and achieve stable treatment. Many differences in serum are not to be considered, compared to conventional cell preparations produced by using serum. This makes it possible to achieve a stable cure rate in the transplantation therapy. Compared with the conventional cell preparation produced by using serum. This makes it possible to achieve a stable cure rate in the transplantation therapy. Compared with the conventional cell preparation produced by using serum. This makes it possible to achieve a stable cure rate in the transplantation therapy.
It can be said that the uterine stem cells contained in the cell preparation produced by the present invention maintain the function of affecting immune effector cells, which originally had the function of the uterine stem cells. The uterine stem cells have an immunomodulatory effect and an immune tolerance effect, and therefore the cell preparation produced by the present invention can be expected to be used for therapy to show those effects. Furthermore, it is also expected that the uterine stem cells of the cell preparation produced according to the present invention also play a role in the anti-inflammatory action of the uterine stem cells in transplantation therapy. The cell preparations produced by the present invention can be used for the treatment of local diseases in which the uterine stem cells are administered to a site requiring transplantation of the uterine stem cells. In addition, by administering the uterine stem cells to the whole body, that is, administering the uterine stem cells to veins and the like, and transporting the uterine stem cells to the whole body, it is possible to more effectively treat strong immune rejection by immunization. Acute GVHD caused by bone marrow transplantation, etc. Therefore, it is expected to significantly improve the survival rate of human beings. Furthermore, the cell preparation produced by the present invention is a cell preparation produced by serum-free culture. Therefore, useful growth factors or differentiation factors are not non-specifically adsorbed together with serum proteins onto a graft material such as ceramics. Therefore, the tissue regeneration ability caused by transplantation of the cell preparation produced by the present invention is very high. This results in a high therapeutic effect. Needless to say, treatment by combination of both local administration and systemic administration may be performed. The immunosuppressive ability of the uterine stem cells contained in the cell preparation produced by the present invention is maintained or improved, and the immune rejection caused when the transplantation is carried out is suppressed by the uterine stem cells. Thus, uterine stem cells can be used in therapy to transplant non-self tissues or cells to another person. The uterine stem cells can be preferably used not only for allogeneic transplantation by human tissues or human cells but also for xenogeneic transplantation by tissues other than animals or animals.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (10)
1. A preparation method of uterine blood stem cells is characterized by comprising the following steps:
a pre-propagation step comprising culturing the uterine stem cells in another serum-free medium containing fibroblast growth factor, platelet-derived factor, epidermal growth factor, at least one phospholipid and at least one fatty acid, said serum-free medium being free of stem cell growth factor and transforming growth factor-beta;
a proliferation step comprising proliferating uterine stem cells in a first serum-free medium containing fibroblast growth factor, platelet-derived factor, transforming growth factor-beta; the first serum-free medium further comprises transforming growth factor-beta, stem cell growth factor, epidermal growth factor, at least one phospholipid, and at least one fatty acid;
a screening step of screening the uterine stem cells expanded in the expanding step for the uterine stem cells having retained or improved immunosuppressive ability;
the culture time of the pre-proliferation step and the proliferation step is 48 days to 68 days, and the cell preparation comprises uterine stem cells for maintaining or improving the immunosuppressive ability; wherein, in the pre-proliferation step, the uterine stem cells are subcultured at least once.
2. The method for producing uterine blood stem cells according to claim 1, characterized in that: before the screening step, the method also comprises the following steps: a serum culturing step, which comprises culturing the uterine blood stem cells subjected to the proliferation step in a medium containing serum.
3. The method for producing uterine blood stem cells according to claim 1, characterized in that: comprising selecting non-tumorigenic uterine blood stem cells from the uterine blood stem cells expanded in the expanding step.
4. The method for producing uterine blood stem cells according to claim 1, characterized in that: in the proliferation step, the uterine stem cells are proliferated by using a culture blood vessel suitable for the proliferation of the uterine stem cells.
5. The method for producing uterine blood stem cells according to claim 1, characterized in that: the serum-free medium further comprises a cell adhesion molecule during the propagation step.
6. The method for producing uterine blood stem cells according to claim 1, characterized in that: the uterine stem cells are subcultured at least once during the expansion step.
7. The method for producing uterine blood stem cells according to claim 6, wherein: in the proliferation step, the manner of subculturing is to use a cytoreductive agent containing no components derived from mammals or microorganisms to exfoliate the uterine stem cells.
8. The method for producing uterine blood stem cells according to claim 4, wherein: further comprising: selecting a culture vessel suitable for the proliferation of the uterine blood stem cells.
9. The method for producing uterine blood stem cells according to claim 1, characterized in that: the phospholipid is selected from phosphatidic acid, lysophosphatidic acid, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine and phosphatidylglycerol.
10. The method for producing uterine blood stem cells according to claim 1, characterized in that: the fatty acid is selected from the group consisting of linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoleic acid, palmitic acid, and stearic acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911228749.0A CN112899223A (en) | 2019-12-04 | 2019-12-04 | Preparation method of uterine blood stem cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911228749.0A CN112899223A (en) | 2019-12-04 | 2019-12-04 | Preparation method of uterine blood stem cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112899223A true CN112899223A (en) | 2021-06-04 |
Family
ID=76111044
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911228749.0A Pending CN112899223A (en) | 2019-12-04 | 2019-12-04 | Preparation method of uterine blood stem cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112899223A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113817674A (en) * | 2021-09-18 | 2021-12-21 | 领航干细胞再生医学工程有限公司 | Culture medium suitable for efficient amplification of uterine blood stem cells and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101802174A (en) * | 2007-09-11 | 2010-08-11 | 北海道公立大学法人札幌医科大学 | Cell growth method and pharmaceutical preparation for tissue repair and regeneration |
| CN102791276A (en) * | 2010-03-10 | 2012-11-21 | 智再如股份有限公司 | Cell preparation containing mesenchymal stem cells, and method for producing same |
| CN108373995A (en) * | 2018-02-14 | 2018-08-07 | 浙江生创精准医疗科技有限公司 | A kind of stem cell conditioned medium, preparation method and use |
-
2019
- 2019-12-04 CN CN201911228749.0A patent/CN112899223A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101802174A (en) * | 2007-09-11 | 2010-08-11 | 北海道公立大学法人札幌医科大学 | Cell growth method and pharmaceutical preparation for tissue repair and regeneration |
| CN102791276A (en) * | 2010-03-10 | 2012-11-21 | 智再如股份有限公司 | Cell preparation containing mesenchymal stem cells, and method for producing same |
| CN108373995A (en) * | 2018-02-14 | 2018-08-07 | 浙江生创精准医疗科技有限公司 | A kind of stem cell conditioned medium, preparation method and use |
Non-Patent Citations (2)
| Title |
|---|
| 李宏;高钰琪;: "低氧对神经干细胞、造血干细胞增殖调控的影响", 中国微循环, no. 01, pages 215 * |
| 韩忠朝: "《干细胞临床进展》", 天津科技翻译出版有限公司, pages: 15 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113817674A (en) * | 2021-09-18 | 2021-12-21 | 领航干细胞再生医学工程有限公司 | Culture medium suitable for efficient amplification of uterine blood stem cells and application |
| CN113817674B (en) * | 2021-09-18 | 2023-08-22 | 领航干细胞再生医学工程有限公司 | Culture medium suitable for high-efficiency amplification of uterine blood stem cells and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US5945337A (en) | Method for culturing CD34+ cells in a serum-free medium | |
| US9394521B2 (en) | Cell preparation containing mesenchymal stem cells, and method for producing same | |
| RU2272839C2 (en) | Serum-free medium for cultured cells used for reconstruction of osseous and cartilage segments (variants) | |
| US4940666A (en) | Process and defined medium for growth of human epidermal keratinocyte cells | |
| US20070178074A1 (en) | Chondrocyte Culture Formulations | |
| KR20130112028A (en) | Serum-free chemically defined cell culture medium | |
| WO2004048557A1 (en) | Treatment of tissue with undifferentiated mesenchymal cells | |
| EP2840133B1 (en) | Method for manufacturing stem cell having appropriate size for intravascular administration | |
| KR20030032934A (en) | Human pancreatic epithelial progenitor cells and methods of isolation and use thereof | |
| EP2482761A1 (en) | Tissue transplant compositions and methods for use | |
| EP1857542A1 (en) | Method for in vitro amplification of adult stem cells | |
| JP4550582B2 (en) | Hair-derived cell culture | |
| Xing et al. | Engineered myocardial tissues constructed in vivo using cardiomyocyte-like cells derived from bone marrow mesenchymal stem cells in rats | |
| EP0953633A1 (en) | Cell culturing method and medium for producing proliferated, normal, differentiated human liver cells | |
| JP2022512963A (en) | Regenerative Abscopal effect | |
| US20020159984A1 (en) | Cultivation of cells for long term engraftment | |
| CN112899223A (en) | Preparation method of uterine blood stem cells | |
| US20050019310A1 (en) | Method for culturing and expansion of mammalian undifferentiated epidermal kerainocytes exhibiting stem cell characteristics | |
| CN113373108A (en) | Epidermal stem cell amplification culture medium and preparation method and application thereof | |
| CN108517313B (en) | Serum/plasma substitute for culturing and amplifying dental pulp stem cells | |
| WO2024063020A1 (en) | Intervertebral disc therapeutic agent | |
| WO2022098861A1 (en) | Compositions and methods for preparing reconstructed multilayered epithelial cell sheets | |
| Huie | Characterization of liver macrophage (Kupffer cell) ultrastructure and erythropoietin (Ep) production in vitro |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |