CN110669730A - 一种人外周血淋巴细胞培养基 - Google Patents
一种人外周血淋巴细胞培养基 Download PDFInfo
- Publication number
- CN110669730A CN110669730A CN201911011883.5A CN201911011883A CN110669730A CN 110669730 A CN110669730 A CN 110669730A CN 201911011883 A CN201911011883 A CN 201911011883A CN 110669730 A CN110669730 A CN 110669730A
- Authority
- CN
- China
- Prior art keywords
- culture medium
- peripheral blood
- mol
- ions
- bovine serum
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000001963 growth medium Substances 0.000 title claims abstract description 66
- 210000005104 human peripheral blood lymphocyte Anatomy 0.000 title claims abstract description 21
- 210000002966 serum Anatomy 0.000 claims abstract description 27
- -1 iron ions Chemical class 0.000 claims abstract description 22
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims abstract description 16
- 108010047620 Phytohemagglutinins Proteins 0.000 claims abstract description 14
- 239000012980 RPMI-1640 medium Substances 0.000 claims abstract description 14
- 229910052742 iron Inorganic materials 0.000 claims abstract description 14
- 230000001885 phytohemagglutinin Effects 0.000 claims abstract description 14
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 claims abstract description 12
- 229910001424 calcium ion Inorganic materials 0.000 claims abstract description 12
- 229920000669 heparin Polymers 0.000 claims abstract description 12
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 claims abstract description 12
- 229960001008 heparin sodium Drugs 0.000 claims abstract description 12
- RDEIXVOBVLKYNT-VQBXQJRRSA-N (2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2r,3r,6s)-3-amino-6-(1-aminoethyl)oxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-5-methyl-4-(methylamino)oxane-3,5-diol;(2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2r,3r,6s)-3-amino-6-(aminomethyl)oxan-2-yl]o Chemical compound OS(O)(=O)=O.O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H](CC[C@@H](CN)O2)N)[C@@H](N)C[C@H]1N.O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H](CC[C@H](O2)C(C)N)N)[C@@H](N)C[C@H]1N.O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N RDEIXVOBVLKYNT-VQBXQJRRSA-N 0.000 claims abstract description 11
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000007995 HEPES buffer Substances 0.000 claims abstract description 11
- 239000002253 acid Substances 0.000 claims abstract description 11
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 claims abstract description 11
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000010936 titanium Substances 0.000 claims abstract description 11
- 229910052719 titanium Inorganic materials 0.000 claims abstract description 11
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims abstract description 11
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 claims abstract description 8
- 239000000843 powder Substances 0.000 claims abstract description 4
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims abstract description 3
- 239000012888 bovine serum Substances 0.000 claims description 12
- 238000002156 mixing Methods 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 10
- 239000000243 solution Substances 0.000 claims description 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 7
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 6
- 229940098773 bovine serum albumin Drugs 0.000 claims description 6
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 6
- 239000012498 ultrapure water Substances 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 3
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 108010071390 Serum Albumin Proteins 0.000 claims 1
- LCKIEQZJEYYRIY-UHFFFAOYSA-N Titanium ion Chemical compound [Ti+4] LCKIEQZJEYYRIY-UHFFFAOYSA-N 0.000 claims 1
- 238000005303 weighing Methods 0.000 claims 1
- 210000004698 lymphocyte Anatomy 0.000 abstract description 11
- 230000008901 benefit Effects 0.000 abstract description 5
- 210000004369 blood Anatomy 0.000 abstract description 5
- 239000008280 blood Substances 0.000 abstract description 5
- 230000004663 cell proliferation Effects 0.000 abstract description 5
- 239000006143 cell culture medium Substances 0.000 abstract description 4
- 229910021645 metal ion Inorganic materials 0.000 abstract description 4
- 230000035755 proliferation Effects 0.000 abstract description 4
- 235000004252 protein component Nutrition 0.000 abstract description 4
- 230000009466 transformation Effects 0.000 abstract description 3
- 235000018102 proteins Nutrition 0.000 description 24
- 102000004169 proteins and genes Human genes 0.000 description 24
- 108090000623 proteins and genes Proteins 0.000 description 23
- 210000004027 cell Anatomy 0.000 description 20
- 230000010261 cell growth Effects 0.000 description 12
- 230000000694 effects Effects 0.000 description 12
- 239000000284 extract Substances 0.000 description 8
- 239000003102 growth factor Substances 0.000 description 8
- 241001465754 Metazoa Species 0.000 description 7
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 6
- 238000004113 cell culture Methods 0.000 description 6
- 230000012010 growth Effects 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 239000000126 substance Substances 0.000 description 5
- IAKHMKGGTNLKSZ-INIZCTEOSA-N (S)-colchicine Chemical compound C1([C@@H](NC(C)=O)CC2)=CC(=O)C(OC)=CC=C1C1=C2C=C(OC)C(OC)=C1OC IAKHMKGGTNLKSZ-INIZCTEOSA-N 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 239000001569 carbon dioxide Substances 0.000 description 3
- 229910002092 carbon dioxide Inorganic materials 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 239000011573 trace mineral Substances 0.000 description 3
- 235000013619 trace mineral Nutrition 0.000 description 3
- 239000011782 vitamin Substances 0.000 description 3
- 235000013343 vitamin Nutrition 0.000 description 3
- 229940088594 vitamin Drugs 0.000 description 3
- 229930003231 vitamin Natural products 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- HBOMLICNUCNMMY-KJFJCRTCSA-N 1-[(4s,5s)-4-azido-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)C(C)=CN1C1O[C@H](CO)[C@@H](N=[N+]=[N-])C1 HBOMLICNUCNMMY-KJFJCRTCSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical class [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- 102000000588 Interleukin-2 Human genes 0.000 description 2
- 108010002350 Interleukin-2 Proteins 0.000 description 2
- IPQKDIRUZHOIOM-UHFFFAOYSA-N Oroxin A Natural products OC1C(O)C(O)C(CO)OC1OC(C(=C1O)O)=CC2=C1C(=O)C=C(C=1C=CC=CC=1)O2 IPQKDIRUZHOIOM-UHFFFAOYSA-N 0.000 description 2
- 235000006484 Paeonia officinalis Nutrition 0.000 description 2
- 241001106477 Paeoniaceae Species 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 description 2
- MUMGGOZAMZWBJJ-DYKIIFRCSA-N Testostosterone Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 MUMGGOZAMZWBJJ-DYKIIFRCSA-N 0.000 description 2
- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical class [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 description 2
- 230000010100 anticoagulation Effects 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- IKIIZLYTISPENI-ZFORQUDYSA-N baicalin Chemical compound O1[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1OC(C(=C1O)O)=CC2=C1C(=O)C=C(C=1C=CC=CC=1)O2 IKIIZLYTISPENI-ZFORQUDYSA-N 0.000 description 2
- 229960003321 baicalin Drugs 0.000 description 2
- AQHDANHUMGXSJZ-UHFFFAOYSA-N baicalin Natural products OC1C(O)C(C(O)CO)OC1OC(C(=C1O)O)=CC2=C1C(=O)C=C(C=1C=CC=CC=1)O2 AQHDANHUMGXSJZ-UHFFFAOYSA-N 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 230000019522 cellular metabolic process Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 229960001338 colchicine Drugs 0.000 description 2
- 239000000084 colloidal system Substances 0.000 description 2
- 239000002158 endotoxin Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 229930182478 glucoside Natural products 0.000 description 2
- 150000008131 glucosides Chemical class 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 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 2
- 229920006008 lipopolysaccharide Polymers 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- 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 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- CZNVSLGYWMSMKE-OPDGVEILSA-K Ferric gluconate Chemical compound [Fe+3].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O CZNVSLGYWMSMKE-OPDGVEILSA-K 0.000 description 1
- 108010003471 Fetal Proteins Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 230000035519 G0 Phase Effects 0.000 description 1
- 102400000321 Glucagon Human genes 0.000 description 1
- 108060003199 Glucagon Proteins 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 102000018997 Growth Hormone Human genes 0.000 description 1
- 108010051696 Growth Hormone Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 244000241838 Lycium barbarum Species 0.000 description 1
- 235000015459 Lycium barbarum Nutrition 0.000 description 1
- NVNLLIYOARQCIX-MSHCCFNRSA-N Nisin Chemical compound N1C(=O)[C@@H](CC(C)C)NC(=O)C(=C)NC(=O)[C@@H]([C@H](C)CC)NC(=O)[C@@H](NC(=O)C(=C/C)/NC(=O)[C@H](N)[C@H](C)CC)CSC[C@@H]1C(=O)N[C@@H]1C(=O)N2CCC[C@@H]2C(=O)NCC(=O)N[C@@H](C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(NCC(=O)N[C@H](C)C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCSC)C(=O)NCC(=O)N[C@H](CS[C@@H]2C)C(=O)N[C@H](CC(N)=O)C(=O)N[C@H](CCSC)C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(N[C@H](C)C(=O)N[C@@H]3C(=O)N[C@@H](C(N[C@H](CC=4NC=NC=4)C(=O)N[C@H](CS[C@@H]3C)C(=O)N[C@H](CO)C(=O)N[C@H]([C@H](C)CC)C(=O)N[C@H](CC=3NC=NC=3)C(=O)N[C@H](C(C)C)C(=O)NC(=C)C(=O)N[C@H](CCCCN)C(O)=O)=O)CS[C@@H]2C)=O)=O)CS[C@@H]1C NVNLLIYOARQCIX-MSHCCFNRSA-N 0.000 description 1
- 108010053775 Nisin Proteins 0.000 description 1
- 108010001441 Phosphopeptides Proteins 0.000 description 1
- 108010039918 Polylysine Proteins 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 229920002385 Sodium hyaluronate Polymers 0.000 description 1
- 102000011923 Thyrotropin Human genes 0.000 description 1
- 108010061174 Thyrotropin Proteins 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- QVYARBLCAHCSFJ-UHFFFAOYSA-N butane-1,1-diamine Chemical compound CCCC(N)N QVYARBLCAHCSFJ-UHFFFAOYSA-N 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- MASNOZXLGMXCHN-ZLPAWPGGSA-N glucagon Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 MASNOZXLGMXCHN-ZLPAWPGGSA-N 0.000 description 1
- 229960004666 glucagon Drugs 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000008518 lycium barbarum polysaccharide Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000008558 metabolic pathway by substance Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000003226 mitogen Substances 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 230000026211 mitotic metaphase Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000004309 nisin Substances 0.000 description 1
- 235000010297 nisin Nutrition 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 235000018343 nutrient deficiency Nutrition 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229920000656 polylysine Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 239000000186 progesterone Substances 0.000 description 1
- 229960003387 progesterone Drugs 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 229940010747 sodium hyaluronate Drugs 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000003270 steroid hormone Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 229960003604 testosterone Drugs 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 230000004102 tricarboxylic acid cycle Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
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
- C12N5/0634—Cells from the blood or the immune system
-
- 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
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
-
- 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
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
- C12N2500/14—Calcium; Ca chelators; Calcitonin
-
- 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
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/20—Transition metals
- C12N2500/22—Zinc; Zn chelators
-
- 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
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/20—Transition metals
- C12N2500/24—Iron; Fe chelators; Transferrin
-
- 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
- C12N2500/44—Thiols, e.g. mercaptoethanol
-
- 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
- C12N2500/70—Undefined extracts
- C12N2500/80—Undefined extracts from animals
- C12N2500/84—Undefined extracts from animals from mammals
-
- 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
- C12N2501/998—Proteins not provided for elsewhere
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明属于细胞培养基技术领域，公开了一种人外周血淋巴细胞培养基。本发明培养基主要包含以下成分：RPMI 1640培养基粉末，碳酸氢钠，HEPES游离酸，硫酸庆大霉素，肝素钠，巯基乙醇，谷氨酰胺，钙离子，铁离子，钛离子，植物血凝素。本发明的培养基可含血清或蛋白成分，也可无血清、蛋白成分，而且其主要利用某些特定金属离子促进细胞增殖。本发明培养基成分明确，性能稳定，成本低廉，能够有效促进人全血中淋巴母细胞增殖分裂，具有较高的淋巴细胞转化率和淋巴细胞分裂指数。
Description
技术领域
本发明属于细胞培养基技术领域，具体涉及一种人外周血淋巴细胞培养基的配方及其制备方法。
背景技术
人体外周血液中的淋巴细胞，几乎都处于GI期(G0期)，一般情况下是不再继续分裂的，如果在培养基中加入刀豆球蛋白(ConA)、脂多糖(LPS)、白细胞介素2(IL-2)、植物血凝素(PHA)等促分裂剂和抗原物质时，小淋巴细胞会受诱导剂刺激转化为淋巴母细胞，随后进入有丝分裂。经过短期的细胞培养，秋水仙碱溶液的处理，低渗和固定，就可获得部分有丝分裂中期的细胞。这种方法已为临床医学、病毒学、药理学、遗传毒性学等方面广泛应用。
已知的影响人外周血淋巴细胞生长的原因有：
1)病毒或微生物感染：当细胞置于体外培养时，病毒或微生物对细胞具有竞争性作用，一旦细胞被污染就会导致细胞死亡。因此，在培养过程中，要确保细胞生存环境无污染。
2)营养物质缺乏：细胞正常生长代谢的重要前提是充足的营养物质，包括维持细胞正常代谢所需的各种蛋白质、氨基酸、维生素、微量元素、碳水化合物等，以及促进细胞生长分裂的物质(生长因子)，如生长激素、胰岛素等含氮激素，睾酮、孕酮等固醇类激素等。在细胞培养过程中，营养物质会逐渐消耗，若培养基中的物质供应不足会导致细胞生长缓慢，影响细胞培养结果。因此，培养基中应包含足够的营养物质来满足细胞生长、增殖所需。除了常规的维生素、氨基酸、无机盐和微量元素外，通常还会在培养基中添加一定量的蛋白质、血清、生长因子等，满足细胞生长、增殖所需。
3)培养环境不稳定：培养环境包括培养温度、是否需要二氧化碳、稳定的pH值等。恒定适宜的温度可维持细胞的旺盛生长，若温度偏离最适温度，细胞的正常代谢会受到影响，严重时导致细胞死亡。氧气和二氧化碳是维持细胞生存的必要条件之一，氧气的功能是参与三羧酸循环，产生供给细胞生长增殖的能量和合成细胞生长所需要的各种成分；二氧化碳的主要功能是维持细胞培养体系的pH值。合适的pH值也是细胞生长增殖的重要前提，细胞培养过程中产生的代谢物会影响pH值的变化，从而影响细胞生长。
以上三个因素中，尤以第二点最难实现。因此，开发一种适宜人外周血淋巴细胞生长的培养基，是解决上述问题的关键，传统的培养基一般是在合成培养基中添加足量的血清(体积百分比5％-10％)，因为血清中除含各种蛋白质、多肽以外，还含有多种生长因子、微量元素等支持细胞生长，促进细胞增殖。但由于血清来自动物血液，动物的年龄、饮食差异、健康状况等对血清成分存在较大的影响，不同批次的血清质量存在较大波动；而且由于动物个体差异、饲养环境及血清提取加工条件等因素的影响，血清中可能含有病毒、热源等对细胞生长有害的成分。并且这些有害因子无法在培养基加工环节或培养过程中予以去除。虽然目前规模化的血清加工厂对动物、采集、加工、储存等环节进行了严格的质量控制，但血清的批间差异仍然明显，是导致细胞培养基培养效果存在批间差异的重要因素。
已有专利CN106520694A公开了一种人体外周血淋巴细胞培养基，其主要成分及浓度为：RPMI 1640培养基55g/L，小牛血清1.5g/L，胶原蛋白4.76g/L，透明质酸钠3.75g/L，胰高血糖素1.43g/L，促甲状腺释放激素6.8μg/L，白芍总苷22.56mg/L，亚油酸2.75g/L，丁二胺0.32g/L，水和葡萄糖铁盐3.5mg/L，谷胱甘肽4.25μg/L，多聚赖氨酸3.15mg/L。专利CN106754694A公开了一种人体外周血淋巴细胞培养基，其主要成分及浓度为：RPMI 1640培养基55g/L，Ham’s12培养基27g/L，小牛血清1.68g/L，透明质酸3.75g/L，酪蛋白磷酸肽4.12g/L，植物凝血素5.32g/L，枸杞多糖89.3μg/mL,黄芩苷4.2g/L，乳酸链球菌素0.58g/L。这些培养基具有淋巴细胞转化率与分裂指数高等优势，但其培养基中添加有牛血清、各种蛋白、多肽及其他生物提取物(如白芍总苷、枸杞多糖、黄芩苷)，由于此类成分多数通过血液、组织等进行提取，动物来源与喂养条件不同，动物个体差异，动物的年龄与健康状况等因素均会影响血清中各种蛋白、生长因子、多肽等有效成分的含量与比例；提取工艺、提取试剂等因素会导致提取物的纯度、生物活性出现波动；加工环节控制不当，则会带来病毒、热源等有害因子污染、蛋白变性、重金属污染等风险；以上影响因素难以准确控制，导致培养效果不稳定，存在较大的批间差异。另外，因为血清、蛋白含有较多的营养物质，适合微生物生长，极易导致细菌、真菌等生长繁殖，导致培养基失效，因此，此类培养基中通常需要添加较高浓度的抗生素并且需要在冷冻条件下保存。另外，血清、蛋白以及其他生物提取物的价格普遍较高，也使得培养基的成本居高不下。
因此，如果能开发一种不含血清、蛋白或其他食物提取物，成分简单明确、效果优良、性能稳定、成本低廉的人外周血淋巴细胞培养基，将有较高的经济前景和社会效益。
发明内容
针对现有细胞培养基含血清、蛋白及其他生物提取物，导致成分复杂不易控制的问题，本发明提供了一种人外周血淋巴细胞培养基及其制备方法。本发明培养基成分简单，原料易得，试剂稳定性好，培养淋巴细胞转化率与分裂指数高，成本低廉。
本发明通过以下技术方案实现：
一种人外周血淋巴细胞培养基，主要包含以下成分及其浓度：RPMI 1640培养基8-12g/L，碳酸氢钠1.0-3.0g/L，HEPES游离酸2.0-3.0g/L，牛血清0％-10％(V/V)，牛血清白蛋白0-0.01g/ml，硫酸庆大霉素25-75μg/mL，肝素钠6-8U/mL，巯基乙醇5-10μmol/L，谷氨酰胺0.1-0.5mmol/L，钙离子0.6-1.0mmol/L，铁离子1-3μmol/L，10-30μmol/L，植物血凝素10-100mg/L，溶剂为超纯水。
优选的，所述人外周血淋巴细胞培养基由以下成分及其浓度组成：RPMI 1640培养基10.4g/L，碳酸氢钠2.0g/L，HEPES游离酸2.38g/L，牛血清0％或5％，硫酸庆大霉素50μg/mL，肝素钠6.9U/mL，巯基乙醇7.15μmol/L，谷氨酰胺0.25mmol/L，钙离子0.8mmol/L，铁离子1.86μmol/L，钛离子20.8μmol/L，植物血凝素54mg/L。
优选地，所述人外周血淋巴细胞培养基制备方法，分为以下步骤：
1)称取10.4g RPMI 1640培养基粉末溶于1L超纯水中，加入2.0g碳酸氢钠，2.38gHEPES游离酸，混匀。
2)加入总体积的5％的牛血清，或不加入牛血清，混匀。
3)加入下列物质，使其终浓度达到下列要求：硫酸庆大霉素50μg/mL，肝素钠6.9U/mL，巯基乙醇7.15μmol/L，谷氨酰胺0.25mmol/L，钙离子0.8mmol/L，铁离子1.86μmol/L，20.8μmol/L，混匀。
4)将上述溶液混匀后用0.22μm的滤膜过滤，然后于无菌条件下加入植物血凝素，使其终浓度达到54mg/L，混匀即得。
本发明培养基与现有培养基相比具有以下优点：
1)成分明确，易于精确控制添加量，培养效果稳定。本发明的培养基可以不添加牛血清或蛋白成分，即使在添加牛血清或蛋白成分的方案中，其添加量也显著低于现有技术中的添加量水平。本发明除RPMI1640中含有基础维生素、氨基酸、无机盐、碳水化合物等成分之外，主要依靠特定的金属离子(钙离子，铁离子和钛离子)促进淋巴细胞的增殖。与血清，蛋白或其他生物提取物相比，无机盐金属离子的成分明确，可以精确掌握添加量来控制培养基的培养效果。而传统的培养基中多依靠添加血清、蛋白等生物提取物来促进细胞生长增殖，此类物质来自生物组织，其中包含成分复杂，有效成分不明确，而且提取过程中还可能引入不利于细胞生长的成分或微生物，不同批次的血清、蛋白的成分差异很大，导致培养基的培养效果不稳定。
2)培养基成本低。本方法利用钙、铁、钛等特定的无机盐来部分或完全取代常规培养基中的血清、蛋白及各种生长因子。与单位质量的钙、铁、钛等无机盐相比，用于细胞培养的胎牛血清、小牛血清、蛋白等的价格要高10倍以上，而提取自生物组织的各类生长因子价格更高；因此本方法制备的培养基成本较传统培养基有明显优势。
3)试剂稳定性好。常规培养基中添加了大量的血清、蛋白质等，其中血清中的有效成分主要是各种蛋白质和一些生长因子。蛋白质是由多种氨基酸通过肽键连接成的高分子化合物，在水溶液中以胶体的形式存在。当溶液pH值发生变化、冻融、微生物生长等情况下，蛋白质分子的电荷及空间结构发生改变，引起蛋白质的生物活性改变；蛋白质表面的电荷变化会导致胶体被破坏，逐渐引发分子之间互相凝聚而出现沉淀。因此，含血清或蛋白质的培养基通常需要在-18℃以下冷冻，来延缓蛋白发生改变，方能长期保存，维持其培养效果。本培养基中不添加蛋白类的高分子化合物，各种有效成分在水中均以溶液形式稳定存在，可以在4℃条件下长期保存，培养效果持续稳定。
4)本发明培养基培养效果好。与常规淋巴细胞培养基相比，本培养基在不添加血清、蛋白、生长因子及其他食物提取物的情况下，仅通过几种特定的金属离子即可实现良好的培养效果，淋巴细胞转化率与淋巴细胞分裂指数高于常规淋巴细胞培养基。
具体实施案例
下面结合实施案例对本发明做进一步详细说明，但这并非是对本发明的限制，本领域技术人员根据本发明的基本思想，可以做出各种修改或改进，但是只要不脱离本发明的基本思想，均在本发明的范围之内。
实施例1一种人外周血淋巴细胞培养基
RPMI 1640培养基10.4g/L，碳酸氢钠2.0g/L，HEPES游离酸2.38g/L，牛血清5％，硫酸庆大霉素50μg/mL，肝素钠6.9U/mL，巯基乙醇7.15μmol/L，谷氨酰胺0.25mmol/L，钙离子0.8mmol/L，铁离子1.86μmol/L，钛离子15μmol/L，植物血凝素54mg/L。
制备方法如下：
1)称取10.4g RPMI 1640培养基粉末溶于1L超纯水中，加入2.0g碳酸氢钠，2.38gHEPES游离酸，混匀。
2)加入总体积的5％的牛血清，混匀。
3)加入下列物质，使其终浓度达到配方要求：硫酸庆大霉素，肝素钠，巯基乙醇，谷氨酰胺，钙离子，铁离子，钛离子，混匀。
4)将上述溶液混匀后用0.22μm的滤膜过滤，然后于无菌条件下加入配方要求的植物血凝素，混匀即得。
实施例2一种人外周血淋巴细胞培养基
RPMI 1640培养基10.4g/L，碳酸氢钠2.0g/L，HEPES游离酸2.38g/L，牛血清白蛋白0.5％，硫酸庆大霉素50μg/mL，肝素钠7.2U/mL，巯基乙醇7.5μmol/L，谷氨酰胺0.2mmol/L，钙离子0.9mmol/L，铁离子1.9μmol/L，钛离子15μmol/L，植物血凝素72mg/L。
配制方法与实施例1类似。
实施例3一种人外周血淋巴细胞培养基
RPMI 1640培养基10.4g/L，碳酸氢钠2.0g/L，HEPES游离酸2.38g/L，硫酸庆大霉素50μg/mL，肝素钠6.9U/mL，巯基乙醇7.15μmol/L，谷氨酰胺0.25mmol/L，钙离子0.8mmol/L，铁离子1.86μmol/L，钛离子20.8μmol/L，植物血凝素54mg/L。
配制方法与实施例1类似。
实施例4人外周血淋巴细胞培养基的使用
用肝素钠抗凝管抽取人外周血。取市场上某品牌培养基为对照，与以上三种培养基一起测试。先将培养基置于37℃复温。在超净台或者酒精灯旁，颠倒混匀人抗凝血，接种0.5ml血至含5ml培养基的培养瓶内(7号针头种血28-35滴)，混匀。将培养瓶静置于37℃培养箱中培养。
培养至70-72小时后，向培养瓶中加入浓度为10μg/ml的秋水仙素50μl，继续培养1.5小时。开启培养瓶，用吸管略吹打后将培养液移至15ml的离心管内，1500rpm离心10分钟，吸去上清，留约0.5ml上清和沉淀。加入5ml 37℃预热的0.075M氯化钾溶液，用吸管充分打匀，于37℃水浴低渗30分钟。加入现配的固定液(甲醇：冰醋酸＝3:1)1ml，轻轻混匀，室温放置10分钟，1500rpm离心10分钟，吸去上清，留约0.5ml上清和沉淀。沿管壁缓慢加入新鲜固定液5ml，轻打混匀，室温放置30分钟，1500rpm离心10分钟，吸去上清。重复固定一次，离心去上清。加适量新鲜固定液调至合适的细胞浓度，在染色体分散仪中滴片。分散条件：温度25℃，湿度50％，待玻片完全干燥后取出。80℃烤箱烤片3小时，随后进行染色体分带染色处理。
于显微镜下观察均匀分布的4个视野下的总细胞数及分裂相细胞数，计算分裂指数。分裂指数＝分裂相细胞数/总细胞数*100％。结果如下：
根据本发明三个案例配制的外周血淋巴细胞培养基，培养外周血淋巴细胞，分裂指数达到3.5-4.5以上，而作为对照的某品牌培养基分裂指数仅为2.8，说明本发明的培养基培养效果明显优于常规培养基。
实施例5人外周血淋巴细胞培养基的保存稳定性
将按照实施例3配制的不含血清、蛋白的外周血淋巴细胞培养基，置4℃保存12个月后取出，以市场上某品牌培养基(-18℃保存，有效期内)为对照，按照实施例4的方法进行实验。于显微镜下观察均匀分布的4个视野下的总细胞数及分裂相细胞数，计算分裂指数。分裂指数＝分裂相细胞数/总细胞数*100％。
结果如下：
从以上数据可以看出，实施例3的培养基在4℃保存12个月后，其培养效果依然优于常规培养基，说明本发明的培养基可以在4℃下长期稳定保存。
Claims (3)
1.一种人外周血淋巴细胞培养基，其特征在于包含以下成分及其浓度：RPMI1640培养基8-12g/L，碳酸氢钠1.0-3.0g/L，HEPES游离酸2.0-3.0g/L，牛血清0％-10(V/V)％，牛血清白蛋白0-0.01g/ml，硫酸庆大霉素25-75μg/mL，肝素钠6-8U/mL，巯基乙醇5-10μmol/L，谷氨酰胺0.1-0.5mmol/L，钙离子0.6-1.0mmol/L，铁离子1-3μmol/L，钛离子10-30μmol/L，植物血凝素10-100mg/L，溶剂为超纯水。
2.根据权利要求1所述人外周血淋巴细胞培养基，其特征在于，由以下成分及浓度组成：RPMI 1640培养基10.4g/L，碳酸氢钠2.0g/L，HEPES游离酸2.38g/L，牛血清0％，硫酸庆大霉素50μg/mL，肝素钠6.9U/mL，巯基乙醇7.15μmol/L，谷氨酰胺0.25mmol/L，钙离子0.8mmol/L，铁离子1.86μmol/L，钛离子20.8μmol/L，植物血凝素54mg/L，溶剂为超纯水。
3.根据权利要求1所述人外周血淋巴细胞培养基制备方法，其特征在于，分为以下步骤：
1)称取RPMI 1640培养基粉末溶于1L超纯水中，加入配方量的碳酸氢钠、HEPES游离酸，混匀；
2)当人外周血淋巴细胞培养基不含血清或牛血清白蛋白成分时，直接进入步骤3)；当含牛血清或牛血清白蛋白成分时，加入配方量的牛血清或牛血清白蛋白，混匀；
3)加入硫酸庆大霉素、肝素钠、巯基乙醇、谷氨酰胺、钙离子、铁离子、钛离子，并使其终浓度达到配方要求，混匀；
4)将上述溶液混匀后用0.22μm的滤膜过滤，然后于无菌条件下加入配方量的植物血凝素，即得。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911011883.5A CN110669730B (zh) | 2019-10-23 | 2019-10-23 | 一种人外周血淋巴细胞培养基 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911011883.5A CN110669730B (zh) | 2019-10-23 | 2019-10-23 | 一种人外周血淋巴细胞培养基 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110669730A true CN110669730A (zh) | 2020-01-10 |
| CN110669730B CN110669730B (zh) | 2022-03-29 |
Family
ID=69083802
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911011883.5A Active CN110669730B (zh) | 2019-10-23 | 2019-10-23 | 一种人外周血淋巴细胞培养基 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110669730B (zh) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111349600A (zh) * | 2020-03-27 | 2020-06-30 | 郑州鲲鹏健康科技有限公司 | 一种pbmc体外培养基及其制备方法 |
| CN114480275A (zh) * | 2022-01-13 | 2022-05-13 | 力因精准医疗产品（上海）有限公司 | 一种外周血淋巴细胞培养基 |
| CN116333983A (zh) * | 2023-04-19 | 2023-06-27 | 中信湘雅生殖与遗传专科医院有限公司 | 外周血样本的培养方法及其应用 |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997048821A1 (en) * | 1996-06-19 | 1997-12-24 | Research Development Foundation | Biochemical analysis of antioxidant function |
| CN1275618A (zh) * | 2000-06-21 | 2000-12-06 | 张国庆 | 一种t－淋巴细胞培养方法 |
| CN1280194A (zh) * | 2000-06-21 | 2001-01-17 | 张国庆 | 一种t－淋巴细胞培养液 |
| CN102352343A (zh) * | 2011-09-28 | 2012-02-15 | 上海柯莱逊生物技术有限公司 | 一种淋巴细胞培养基及其使用方法 |
| CN109055309A (zh) * | 2018-08-02 | 2018-12-21 | 广州白云山拜迪生物医药有限公司 | 一种无血清细胞培养基及其用途 |
| CN109294986A (zh) * | 2018-10-26 | 2019-02-01 | 深圳市润科生物科技有限公司 | 一种t淋巴细胞无血清培养基及其制备方法 |
-
2019
- 2019-10-23 CN CN201911011883.5A patent/CN110669730B/zh active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997048821A1 (en) * | 1996-06-19 | 1997-12-24 | Research Development Foundation | Biochemical analysis of antioxidant function |
| CN1275618A (zh) * | 2000-06-21 | 2000-12-06 | 张国庆 | 一种t－淋巴细胞培养方法 |
| CN1280194A (zh) * | 2000-06-21 | 2001-01-17 | 张国庆 | 一种t－淋巴细胞培养液 |
| CN102352343A (zh) * | 2011-09-28 | 2012-02-15 | 上海柯莱逊生物技术有限公司 | 一种淋巴细胞培养基及其使用方法 |
| CN109055309A (zh) * | 2018-08-02 | 2018-12-21 | 广州白云山拜迪生物医药有限公司 | 一种无血清细胞培养基及其用途 |
| CN109294986A (zh) * | 2018-10-26 | 2019-02-01 | 深圳市润科生物科技有限公司 | 一种t淋巴细胞无血清培养基及其制备方法 |
Non-Patent Citations (2)
| Title |
|---|
| 李鸣等: "淋巴细胞经TCR-CD3活化增殖作用的分析", 《免疫学杂志》 * |
| 陈富军等: "钛离子对T淋巴细胞体外增殖及活化的影响", 《中国组织工程研究》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111349600A (zh) * | 2020-03-27 | 2020-06-30 | 郑州鲲鹏健康科技有限公司 | 一种pbmc体外培养基及其制备方法 |
| CN114480275A (zh) * | 2022-01-13 | 2022-05-13 | 力因精准医疗产品（上海）有限公司 | 一种外周血淋巴细胞培养基 |
| CN116333983A (zh) * | 2023-04-19 | 2023-06-27 | 中信湘雅生殖与遗传专科医院有限公司 | 外周血样本的培养方法及其应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110669730B (zh) | 2022-03-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yao et al. | Animal‐cell culture media: History, characteristics, and current issues | |
| JP3329387B2 (ja) | 培地濃縮物技術 | |
| CN110669730B (zh) | 一种人外周血淋巴细胞培养基 | |
| Golde et al. | Thyroid hormones stimulate erythropoiesis in vitro | |
| CN102115729B (zh) | 无血清培养bhk21细胞的方法和制备疫苗的方法 | |
| EP0872180B1 (en) | Medium composition for external fertilization | |
| US20080286862A1 (en) | Physiochemical Culture Conditions for Embryonic Stem Cells | |
| Stubblefield et al. | Effects of sodium chloride concentration on growth, biochemical composition, and metabolism of HeLa cells | |
| CN104073463A (zh) | 一种支持cho高密度悬浮培养的无血清无蛋白培养基 | |
| CN104293729A (zh) | 一种高效的无血清培养基 | |
| CN116083350B (zh) | 一种培养df-1细胞的完全培养基 | |
| CN111748527B (zh) | 化学成分限定高效补料培养基及其制备方法和应用 | |
| CN106148269A (zh) | 一种哺乳动物细胞培养基及其添加剂 | |
| CN105039251B (zh) | 一种pH值稳定的淋巴细胞无血清培养基及其制备方法和应用 | |
| CN119162099A (zh) | 免疫细胞用培养基，其制备方法及其应用 | |
| CN100432218C (zh) | 无血清配方的动物细胞培养基胎牛血清替代剂 | |
| Phillips et al. | Oxygen uptake and lactate formation of HeLa cells. | |
| CN106520694A (zh) | 一种人体外周血淋巴细胞培养基及其制备方法 | |
| Okabe et al. | Growth factor (s) produced by a human leukemic cell line growing in a protein-free chemically defined medium | |
| EP1516045B1 (de) | Verwendung von glutaminfreien medium | |
| US10837042B2 (en) | Feed mixing device and its use | |
| CN111849863A (zh) | 支持cho细胞高效生产单克隆抗体的培养基添加剂及其制备方法和应用 | |
| CN116004517A (zh) | 一种化学成分限定培养基及应用 | |
| CN112795532A (zh) | 一种人牙周膜干细胞无血清培养基 | |
| RU2236457C2 (ru) | Способ получения питательной среды "лимфокар" для культивирования лимфоцитов периферической крови человека и животных |
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 | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20220310 Address after: 310000 floors 4 and 5, building 2, No. 208, Zhenzhong Road, Sandun Town, Xihu District, Hangzhou City, Zhejiang Province Applicant after: HANGZHOU GENE-META MEDICAL DEVICE Co.,Ltd. Address before: 310030 floor 6, building 5, No. 1, Xiyuan Road, Xihu District, Hangzhou City, Zhejiang Province Applicant before: HANGZHOU BAORONG TECHNOLOGY Co.,Ltd. |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |