CN1570090A - Cell suspension culture and anchorage culture serum free medium and its preparation method - Google Patents
Cell suspension culture and anchorage culture serum free medium and its preparation method Download PDFInfo
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- CN1570090A CN1570090A CN 200410018407 CN200410018407A CN1570090A CN 1570090 A CN1570090 A CN 1570090A CN 200410018407 CN200410018407 CN 200410018407 CN 200410018407 A CN200410018407 A CN 200410018407A CN 1570090 A CN1570090 A CN 1570090A
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- 239000012679 serum free medium Substances 0.000 title claims abstract description 47
- 238000002360 preparation method Methods 0.000 title claims abstract description 39
- 238000004114 suspension culture Methods 0.000 title claims abstract description 28
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims abstract description 32
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 claims abstract description 26
- 235000015921 sodium selenite Nutrition 0.000 claims abstract description 26
- 239000011781 sodium selenite Substances 0.000 claims abstract description 26
- 229960001471 sodium selenite Drugs 0.000 claims abstract description 26
- 210000002966 serum Anatomy 0.000 claims abstract description 20
- 102000004877 Insulin Human genes 0.000 claims abstract description 17
- 108090001061 Insulin Proteins 0.000 claims abstract description 17
- 239000000843 powder Substances 0.000 claims abstract description 17
- 229940125396 insulin Drugs 0.000 claims abstract description 16
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 10
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 10
- 239000000243 solution Substances 0.000 claims description 123
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 62
- 210000004027 cell Anatomy 0.000 claims description 60
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 37
- 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 claims description 32
- 239000011780 sodium chloride Substances 0.000 claims description 31
- 239000011734 sodium Substances 0.000 claims description 30
- 238000010828 elution Methods 0.000 claims description 24
- FRGKKTITADJNOE-UHFFFAOYSA-N sulfanyloxyethane Chemical compound CCOS FRGKKTITADJNOE-UHFFFAOYSA-N 0.000 claims description 23
- 229920000936 Agarose Polymers 0.000 claims description 22
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 22
- 229940098773 bovine serum albumin Drugs 0.000 claims description 22
- 238000004873 anchoring Methods 0.000 claims description 20
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 18
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 18
- 102000016359 Fibronectins Human genes 0.000 claims description 16
- 108010067306 Fibronectins Proteins 0.000 claims description 16
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- 235000012000 cholesterol Nutrition 0.000 claims description 16
- XNSAINXGIQZQOO-SRVKXCTJSA-N protirelin Chemical compound NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@H]1NC(=O)CC1)CC1=CN=CN1 XNSAINXGIQZQOO-SRVKXCTJSA-N 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 239000012228 culture supernatant Substances 0.000 claims description 15
- 210000002590 marrow fibroblast Anatomy 0.000 claims description 15
- 102000004190 Enzymes Human genes 0.000 claims description 14
- 108090000790 Enzymes Proteins 0.000 claims description 14
- 239000012153 distilled water Substances 0.000 claims description 14
- 238000004587 chromatography analysis Methods 0.000 claims description 12
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 238000004115 adherent culture Methods 0.000 claims description 9
- 239000011521 glass Substances 0.000 claims description 9
- 238000004321 preservation Methods 0.000 claims description 9
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 9
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 9
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- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 9
- 238000007796 conventional method Methods 0.000 claims description 8
- 150000001875 compounds Chemical class 0.000 claims description 7
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- 239000002609 medium Substances 0.000 claims description 7
- 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 claims description 6
- 108010062580 Concanavalin A Proteins 0.000 claims description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 6
- 108010039918 Polylysine Proteins 0.000 claims description 6
- 230000001464 adherent effect Effects 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 6
- 238000013016 damping Methods 0.000 claims description 6
- 238000000432 density-gradient centrifugation Methods 0.000 claims description 6
- 238000010790 dilution Methods 0.000 claims description 6
- 239000012895 dilution Substances 0.000 claims description 6
- 239000012530 fluid Substances 0.000 claims description 6
- 229960000890 hydrocortisone Drugs 0.000 claims description 6
- 239000008141 laxative Substances 0.000 claims description 6
- 230000002475 laxative effect Effects 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 6
- 229920000656 polylysine Polymers 0.000 claims description 6
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- KCXFHTAICRTXLI-UHFFFAOYSA-N propane-1-sulfonic acid Chemical compound CCCS(O)(=O)=O KCXFHTAICRTXLI-UHFFFAOYSA-N 0.000 claims description 4
- 239000011550 stock solution Substances 0.000 claims description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 3
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 claims description 3
- 229910021380 Manganese Chloride Inorganic materials 0.000 claims description 3
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 claims description 3
- 229920002684 Sepharose Polymers 0.000 claims description 3
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 3
- 238000001042 affinity chromatography Methods 0.000 claims description 3
- WQZGKKKJIJFFOK-PQMKYFCFSA-N alpha-D-mannose Chemical compound OC[C@H]1O[C@H](O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-PQMKYFCFSA-N 0.000 claims description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 3
- 239000011324 bead Substances 0.000 claims description 3
- 239000001110 calcium chloride Substances 0.000 claims description 3
- 235000011148 calcium chloride Nutrition 0.000 claims description 3
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 3
- 239000004202 carbamide Substances 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 238000004440 column chromatography Methods 0.000 claims description 3
- PAFZNILMFXTMIY-UHFFFAOYSA-N cyclohexylamine Chemical compound NC1CCCCC1 PAFZNILMFXTMIY-UHFFFAOYSA-N 0.000 claims description 3
- 238000000502 dialysis Methods 0.000 claims description 3
- 229910052731 fluorine Inorganic materials 0.000 claims description 3
- 239000011737 fluorine Substances 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 239000011565 manganese chloride Substances 0.000 claims description 3
- 235000002867 manganese chloride Nutrition 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 235000015320 potassium carbonate Nutrition 0.000 claims description 3
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 3
- AYRVGWHSXIMRAB-UHFFFAOYSA-M sodium acetate trihydrate Chemical compound O.O.O.[Na+].CC([O-])=O AYRVGWHSXIMRAB-UHFFFAOYSA-M 0.000 claims description 3
- 239000001488 sodium phosphate Substances 0.000 claims description 3
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 3
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims 1
- 150000001412 amines Chemical class 0.000 claims 1
- 230000012010 growth Effects 0.000 abstract description 9
- 238000002474 experimental method Methods 0.000 abstract description 7
- 102000016938 Catalase Human genes 0.000 abstract description 5
- 108010053835 Catalase Proteins 0.000 abstract description 5
- 239000004017 serum-free culture medium Substances 0.000 abstract description 5
- 239000002994 raw material Substances 0.000 abstract description 4
- 238000000034 method Methods 0.000 abstract description 3
- 230000001737 promoting effect Effects 0.000 abstract description 2
- 102000007562 Serum Albumin Human genes 0.000 abstract 1
- 108010071390 Serum Albumin Proteins 0.000 abstract 1
- 230000002349 favourable effect Effects 0.000 abstract 1
- 241001465754 Metazoa Species 0.000 description 5
- 238000005303 weighing Methods 0.000 description 5
- 229940088597 hormone Drugs 0.000 description 4
- 239000005556 hormone Substances 0.000 description 4
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
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- 210000003725 endotheliocyte Anatomy 0.000 description 3
- 210000004920 epithelial cell of skin Anatomy 0.000 description 3
- 210000004881 tumor cell Anatomy 0.000 description 3
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 3
- DWGWCWDDNTVOGF-UHFFFAOYSA-N Cl.Cl.Cl.Cl.Cl.Cl Chemical compound Cl.Cl.Cl.Cl.Cl.Cl DWGWCWDDNTVOGF-UHFFFAOYSA-N 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- NUGRUDUZGYQLNE-UHFFFAOYSA-N cyclohexanamine;propane-1-sulfonic acid Chemical compound CCCS(O)(=O)=O.NC1CCCCC1 NUGRUDUZGYQLNE-UHFFFAOYSA-N 0.000 description 2
- 210000001671 embryonic stem cell Anatomy 0.000 description 2
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- UZOVYGYOLBIAJR-UHFFFAOYSA-N 4-isocyanato-4'-methyldiphenylmethane Chemical compound C1=CC(C)=CC=C1CC1=CC=C(N=C=O)C=C1 UZOVYGYOLBIAJR-UHFFFAOYSA-N 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- AUNGANRZJHBGPY-UHFFFAOYSA-N D-Lyxoflavin Natural products OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-UHFFFAOYSA-N 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- -1 Keratinocyte-SFM) Substances 0.000 description 1
- 150000000994 L-ascorbates Chemical class 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
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- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A serum-free medium of cell suspension culture and wall sticking culture and the preparation process thereof are disclosed. It makes cow serum albumin powder, insulin powder, catalase powder, sodium selenite powder and di-sulfhydryl alcohol solution as raw materials, and be prepared into serum free culture medium suitable for cell suspension culture by certain process. The serum free culture medium suitable for wall sticking culture is prepared by adding cell fibrous joints protein on the basis of said process, the wall sticking promoting factor for prespreading on the bottom of the culture bottom is also processed. The invention enables the cell to maintain normal growth by excluding the serum interference, therefore, the experiment result is more scientific and accurate, in addition, it has a favorable repeatability.
Description
Technical field
The present invention relates to the medical experiment articles for use, especially relate to a kind of being used at the cell suspension culture of vitro culture humans and animals tissue and cell and serum free medium of adherent culture and preparation method thereof.
Background technology
In medical experiment, the tissue of humans and animals and cell except that the basic medium that need be rich in amino acid, inorganic salt, VITAMIN, lipid material and energy matter (as carbohydrate), also need to provide the animal serum of other nutrition, cytokine and hormone when vitro culture.Traditional cell and tissue culture all will be used the animal serum of 5-20%, but because the composition of animal serum is very complicated, so far do not understand fully composition wherein as yet fully, for example contain 500 multiple proteins in serum, wherein some proteinic function also imperfectly understands.Thereby in the medical science experiment, often, be difficult for reaching stdn because of the serum difference that adds causes experimental result to produce some differences, influence the repeatability and the accuracy of experimental result.As the experiment of some research polypeptide factor effect, because of containing the indefinite polypeptide cytokines of concentration, hormone or other material in the serum, they may influence the function etc. of growth, immunity identification, the specific polypeptide factor and the cell surface receptor of cell.Also there is supressor in addition in the serum, and antigen and antibody, they can disturb external immunization experiment result.
The external research that has just begun to carry out the serum-free culture method as far back as 1979.Now external serum free medium kind of producing is a lot, be applicable to the serum free medium of insect cell, hybridoma, skin epithelial cells, hematopoietic cell, tumour cell, neurocyte, liver cell, endotheliocyte, embryonic stem cell respectively, there is following shortcoming jointly in they:
(1) is only applicable to the cell of a certain particular tissue type, as neurocyte, liver cell, epithelial cells, endotheliocyte, embryonic stem cell;
(2) be respectively applied for the adherent culture except that the specific serum free medium of minority, still do not have the adherent culture that a kind of cell attachment serum free medium is applicable to the broad variety cell.Have as can be used for the serum free medium that cell attachment cultivates abroad: skin epithelial cells serum free medium (Keratinocyte-SerumFree Media, Keratinocyte-SFM), scavenger cell serum free medium (Macrophage-SFM), endotheliocyte serum free medium (Endothelial-SFM).
The applicant has applied for a patent of invention " a kind of serum free medium and concentrated solution and preparation method thereof " (application number: 02157709.9) on December 17th, 2002, the main raw material of five kinds of compositions such as the bovine serum albumin solution in this patent of invention, insulin solutions, purifying human transferrin solution, cholesterol solution, hydrogen peroxide enzyme solution all is Powdered, can weighing, do not contain polypeptide factor and hormone substance, thereby both can avoid the unstable brought because of serum lot number difference, being unlikely again influences experimental result.The serum free medium of this patent of invention is to make cell keep normal growth under the situation of getting rid of serum interference, thereby makes experimental result science and accurate more, and better repeatability is arranged, and is applicable to the growth of polytype tissue and cell.But its shortcoming is to be not suitable for carrying out the cell attachment cell cultures.
Summary of the invention
The object of the present invention is to provide a kind of tissue and the cell suspension culture of cell and serum free medium of adherent culture and preparation method thereof that is applicable to than broad variety, it can make cell keep normal growth under the situation of getting rid of serum interference, thereby make experimental result science and accurate more, and better repeatability is arranged.
The object of the present invention is achieved like this: the prescription of serum free medium of the present invention comprises the bovine serum albumin solution in final concentration: 5-10mg/ml, insulin solutions: 8-20 μ g/ml, purifying human transferrin solution: 1-30 μ g/ml, cholesterol solution: 20-60 μ g/ml and hydrogen peroxide enzyme solution: 20-60 μ g/ml is characterized in that: also comprise sodium selenite solution: 17.3-34.6 μ g/ml and 3-mercaptoethanol solution (1%): 0.35-0.70ml.This serum free medium is suitable for cell suspension culture.
In the serum free medium that is suitable for cell suspension culture of the present invention, also can add cell Fibronectin: 25-50 μ g/ml and short anchoring factor (PAF) in final concentration.It is suitable for cell attachment and cultivates.
The preparation method who is suitable for the serum free medium of cell suspension culture of the present invention is:
(1), compound concentration is respectively bovine serum albumin (the BovineSerum Albumin of 50-100mg/ml according to a conventional method, abbreviation BSA) Regular Insulin (Insulin of solution, 80-400 μ g/ml, abbreviation Ins) purifying human transferrin (the Human Transferrin of solution, 10-300 μ g/ml, abbreviation TF) cholesterol (Cholesterol of solution, 200-600 μ g/ml, abbreviation Chol) catalase (Catalase of solution and 200-600 μ g/ml, abbreviation Cat) solution all is dissolved in Yi Sikefu improvement cutter than Ke Shi substratum;
(2), the preparation of Sodium Selenite (Sodium Selenite, abbreviation SS) solution: the 1.73mg Sodium Selenite powder with commercially available is dissolved in the 10ml triply distilled water Entkeimung, 4 ℃ of refrigerators preservations;
(3), the preparation of 3-mercaptoethanol (2-mercaptoethanol, abbreviation 2-ME) solution: commercially available 3-mercaptoethanol is added in the triply distilled water, make into 1% solution, be stock solution, Entkeimung is put 4 ℃ of refrigerators and is preserved;
(4), above-mentioned seven kinds of solution are mixed by formula concentration, again with Yi Sikefu improvement cutter than Ke Shi substratum (Iscove ' s modified Dulbecco ' s media, abbreviation IMDM) dilution is the 500ml/ bottle, promptly get the cell suspension culture serum free medium (Suspension-Serum Free Media, SP-SFM).
The preparation method who is suitable for the foster base of cell attachment cultivation serum-free wall of the present invention is:
(1), compound concentration is respectively the bovine serum albumin solution of 50-100mg/ml, the insulin solutions of 80-400 μ g/ml, the purifying human transferrin solution of 10-300 μ g/ml, the cholesterol solution of 200-600 μ g/ml and the hydrogen peroxide enzyme solution of 200-600 μ g/ml according to a conventional method, all is dissolved in Yi Sikefu improvement cutter than Ke Shi substratum;
(2), the preparation of sodium selenite solution: commercially available Sodium Selenite powder 1.73mg is dissolved in the 10ml triply distilled water Entkeimung, 4 ℃ of refrigerators preservations;
(3), the preparation of 3-mercaptoethanol solution: commercially available 3-mercaptoethanol is added in the triply distilled water, make into 1% solution, be stock solution, Entkeimung is put 4 ℃ of refrigerators and is preserved;
(4), the preparation of cell Fibronectin solution:
1., people's marrow fibroblast culture supernatant: obtain BMNC with lymphocyte separation medium (Ficoll-Hypague) density gradient centrifugation, be incubated in the plastic culture bottle that is covered with 4% poly-lysine in advance with 1 * 106 cell/ml concentration, add 10% people AB serum and 10-6M hydrocortisone (final concentration), cultivated 5 days, form adherent individual layer, inhale and remove supernatant, change the cell suspension culture serum free medium and continue to cultivate 2 days, the results supernatant liquor is further purified;
2., 1 liter of people's marrow fibroblast culture supernatant being added gelatin-agarose (Sepharose) that the 1ml bovine serum albumin handles hatched 24 hours for 4 ℃, separate gelatin-agarose (Agarosc) pearl with the centrifugation method in conjunction with the cell Fibronectin, with the can of 10ml pearl in the chromatography column of 1.5cm diameter; This post is at room temperature used the 0.15M NaCl of 4 times of post bed capacity, 10mM Tutofusin tris-hydrochloric acid (Tris-HCl), the pH7.0 washing is with damping fluid C[4M urea, 0.5MNaCl, 50mM hexahydroaniline propane sulfonic acid (CAPS)], the pH11.0 elution; Determine protein peak, the each several part that will contain the cell Fibronectin mixes, and with buffer A (0.15M NaCl, 1mM CaCl2,10mM hexahydroaniline propane sulfonic acid, readjustment pH to 11.0) dialysis, detects protein concn, makes into 0.125g/ml concentration, is stored in-70 ℃;
(5), above-mentioned eight kinds of solution pressed formula rate mix, be diluted to the 500ml/ bottle with Yi Sikefu improvement cutter than Ke Shi substratum again, promptly get cell attachment cultivation serum free medium (Adhesion-SerumFree Media, AD-SFM);
(6), the preparation of short anchoring factor:
1., people's marrow fibroblast culture supernatant: obtain BMNC with the lymphocyte separation medium density gradient centrifugation, be incubated in the plastic culture bottle that is covered with 4% poly-lysine in advance with 1 * 106 cell/ml concentration, add 10% people AB serum and 10-6M hydrocortisone (final concentration), cultivated 5 days, form adherent individual layer, inhale and remove supernatant, change the cell suspension culture serum free medium and continue to cultivate 2 days, the results supernatant liquor is further purified;
2., granulated glass sphere affinity chromatography: glass microballon with 0.6M NaHCO3 (pH8.0) washing, is adorned post (5cm * 50cm), use 0.6M NaHCO3 equilibrate overnight, flow velocity 100ml/ hour.People's marrow fibroblast culture supernatant is adjusted to pH8.0 with 0.1N NaOH, filter, with 100ml people's marrow fibroblast culture supernatant with sample on the 100ml/ speed at one hour rating, elution peak (behind 65kd and the 75kd) appears, with following damping fluid (all containing tolylsulfonyl fluorine (PMSF)) wash-out (100ml/ hour):
A.200ml?0.6M?NaHCO3·pH8.0
B.100ml?0.6M?NaHCO3-0.2M?Na2CO3，pH9.3-9.5
C.100ml?H2O
D.500ml 0.2M K2CO3, pH11.0 will mix with the each several part of 0.6M NaHCO3-0.2M Na2CO3 elution, carry out next step purifying;
3. concanavalin A-agarose column/diethylaminoethyl-(DEAE) chromatography:
(2.5cm * 20cm) (2.5cm * 3cm), the 2.5cm of can thereon * 16cm concanavalin A-agarose column layer uses the phosphoric acid buffer balance, 30ml/ hour to chromatography column then with diethylaminoethyl--agarose can bottom;
Urge each part (about 200ml) of anchoring factor with 0.6M NaHCO3-0.2M Na2CO3 from containing of glass bead column elution and be applied to concanavalin A/diethylaminoethyl-post, 30ml/ hour, press the laxative remedy elution:
1) 30ml 0.25M sodium phosphate (Na/pi), pH6.0,30ml/ hour;
2) 200ml 25mM Na/Pi, pH6.0,15ml/ hour;
3) 250ml contains the 25mM Na/Pi of 50mM seminose, pH6.0,30ml/ hour;
4) 30ml 25mM Na/Pi, pH6.0,30ml/ hour;
5) 60ml contains the 25mM Na/Pi of 100mM NaCl, pH6.0,30ml/ hour;
6) 100ml contains the 25mM Na/Pi of 0.5M Alpha-Methyl mannoside and 1M NaCl, pH6.0,30ml/ hour;
7) 100ml contains the 0.15M NaCl 20mM Tutofusin tris of (containing 1mM MgC12,1mM CaCl2 and 1mM MnCl2), pH7.3.Contain the elution peak 25mM Na/Pi elution that contains 100mM NaCl of the material of PAF;
4. heparin-agarose column chromatography:
Heparin-agarose chromatography column (the dress of 0.7cm * 10cm) post, and with the 25mMNa/Pi that contains 100mM NaCl, pH6.0 balance (15ml/ hour), the part difference (not mixing) that contains short anchoring factor of concanavalin A/diethylaminoethyl-purifying goes up sample to the heparin-agarose post, 7.5ml/ hour, by the laxative remedy elution, promptly get and urge anchoring factor:
1) 20ml contains the 25mM Na/Pi of 100mM NaCl, pH6.0 (15ml/ hour);
2) 20ml contains among the 50mM Na/Pi of 100mM NaCl, pH7.0 (4ml/ hour);
3) 20ml contains among the 50mM Na/Pi of 1M NaCl, pH7.4 (15ml/ hour);
At the bottom of overlaying the culturing bottle bottle with short anchoring factor, cultivate with cell attachment cultivation serum free medium mixed culture cell injection culturing bottle again (7), earlier.
The present invention has two kinds of preparations: cell suspension culture serum free medium and cell attachment are cultivated serum free medium.The former is to be raw material with bovine serum albumin powder, insulin powder, purifying human transferrin powder, cholesterol powder, catalase powder, Sodium Selenite powder and 3-mercaptoethanol solution, forms by certain prepared.The latter is divided into two portions, and a part is the substratum part, is based on the cell suspension culture base, adds the cell Fibronectin and makes; Another part is an extention, is the short anchoring factor that is prepared from the gel-filtration method.These raw materials or be commercially available, or self-control purifying all can weighings, do not contain polypeptide factor and hormone substance, thereby both can avoid the unstable brought because of serum lot number difference, are unlikely to influence experimental result again.Therefore cell suspension culture and cell attachment cultivation serum free medium is to make cell keep normal growth under the situation of getting rid of serum interference, thereby makes experimental result science and accurate more, and better repeatability is arranged.It also has following characteristics:
(1) is added with the hydrogen peroxide enzyme solution in the prescription: in cell suspension culture and cell attachment cultivation serum free medium, add the hydrogen peroxide enzyme solution and can promote the long term growth and the clone of cell, and can replace bovine serum albumin and tyrosine.Studies have shown that hydrogen peroxide is cell growth and clone's a main toxicant.Tyrosine in the substratum, tryptophane, riboflavin, ascorbate salt etc. all can cause the formation of hydrogen peroxide, hydrogen peroxide can be by the damage of the oxidation of the oxidation of unsaturated fatty acids on the cytolemma, cell protein and/or DNA and is caused the death of cell, and catalase then can decomposition of hydrogen peroxide;
(2) be added with Sodium Selenite and 3-mercaptoethanol in the prescription, the former is Wheat Protein also, and the latter can promote lymphocytic growth;
(3) two kinds of formulations are arranged, can be respectively applied for cell suspension culture and cell attachment and cultivate, particularly the latter is divided into two portions.A part is a substratum, contains purified cell Fibronectin; Another part is an extention, is purified short anchoring factor, can be used for overlaying at the bottom of the culturing bottle to promote cell attachment.
(4) concentration of various compositions is different with analogous products in the prescription: is 5-10mg/ml as bovine serum albumin in 1 times of serum free medium, and external like product majority only is 0.4mg/ml.Experiment confirm, the bovine serum albumin of 5-10mg/ml is promoting to be much better than 0.4mg/ml aspect the cell growth.
(5) the present invention is applicable to the growth of polytype tissue and cell: the present invention has been applied to the vitro culture as hematopoietic cell, lymphocyte, skin epithelial cells, tumour cell, stem cell, dendritic cell, monkey-kidney cells, immunologically competent cell.The applicant has also studied the apoptosis of tumour cell with the present invention and since got rid of serum existence itself can apoptosis-induced material interference, can reflect the apoptosis situation more truly.
Embodiment
Below in conjunction with embodiment the present invention is described in further detail.
Embodiment 1: the preparation of cell suspension culture serum free medium
(1), compound concentration is respectively the bovine serum albumin solution of 100mg/ml, the insulin solutions of 80 μ g/ml, the purifying human transferrin solution of 10 μ g/ml, the cholesterol solution of 200 μ g/ml and the hydrogen peroxide enzyme solution of 200 μ g/ml according to a conventional method, all is dissolved in Yi Sikefu improvement cutter than Ke Shi substratum;
(2), the preparation of sodium selenite solution: commercially available Sodium Selenite powder is taken by weighing 1.73mg, make it to be dissolved in the 10ml triply distilled water, Entkeimung, 4 ℃ of refrigerators preservations;
(3), the preparation of 3-mercaptoethanol solution: commercially available 3-mercaptoethanol 1ml is added in the 99ml triply distilled water, make it into 1% solution, Entkeimung, 4 ℃ of refrigerators preservations;
(4), measure the 1% 3-mercaptoethanol solution of 50.0ml bovine serum albumin solution, 50.0ml insulin solutions, 50.0ml purifying human transferrin solution, 50.0ml cholesterol solution, 50.0ml hydrogen peroxide enzyme solution, 0.05ml sodium selenite solution and 0.35ml respectively, above-mentioned seven kinds of solution are mixed, be the 500ml/ bottle with Yi Sikefu improvement cutter than the dilution of Ke Shi substratum again, get product.
Embodiment 2: the preparation of cell suspension culture serum free medium
(1), according to a conventional method prepare bovine serum albumin solution, the insulin solutions of 400 μ g/ml, the purifying human transferrin solution of 300 μ g/ml, the cholesterol solution of 600 μ g/ml and the hydrogen peroxide enzyme solution of 600 μ g/ml that final concentration is respectively 50mg/ml, all be dissolved in Yi Sikefu improvement cutter than Ke Shi substratum;
(2), the preparation of sodium selenite solution: commercially available Sodium Selenite powder is taken by weighing 1.73mg, make it to be dissolved in the 10ml triply distilled water, Entkeimung, 4 ℃ of refrigerators preservations;
(3), the preparation of 3-mercaptoethanol solution: commercially available 3-mercaptoethanol 1ml is added in the 99ml triply distilled water, make it into 1% solution, Entkeimung, 4 ℃ of refrigerators preservations;
(4), measure the 1% 3-mercaptoethanol solution of 50.0ml bovine serum albumin solution, 50.0ml insulin solutions, 50.0ml purifying human transferrin solution, 50.0ml cholesterol solution, 50.0ml hydrogen peroxide enzyme solution, 0.1ml sodium selenite solution and 0.35ml respectively, above-mentioned seven kinds of solution are mixed, be the 500m/ bottle with Yi Sikefu improvement cutter than the dilution of Ke Shi substratum again, get product.
Embodiment 3: cell attachment is cultivated the preparation of serum free medium
(1), compound concentration is respectively the bovine serum albumin solution of 50-100mg/ml, the insulin solutions of 80-400 μ g/ml, the purifying human transferrin solution of 10-300 μ g/ml, the cholesterol solution of 200-600 μ g/ml and the hydrogen peroxide enzyme solution of 200-600 μ g/ml according to a conventional method;
(2), the preparation of sodium selenite solution: commercially available Sodium Selenite powder is taken by weighing 1.73mg, makes it to be dissolved in the 10ml triply distilled water, Entkeimung, 4 ℃ of refrigerators are preserved, and make the sodium selenite solution that final concentration is 17.3 μ g/ml;
(3), the preparation of 3-mercaptoethanol solution: commercially available 3-mercaptoethanol 1ml is added in the 99ml triply distilled water, make it into 1% solution, Entkeimung, 4 ℃ of refrigerators preservations;
(4), the preparation of cell Fibronectin solution:
1., people's marrow fibroblast culture supernatant: obtain BMNC with the lymphocyte separation medium density gradient centrifugation, be incubated in the plastic culture bottle that is covered with 4% poly-lysine in advance with 1 * 106 cell/ml concentration, add 10% people AB serum and 10-6M hydrocortisone (final concentration), cultivated 5 days, form adherent individual layer, inhale and remove supernatant, change the cell suspension culture serum free medium and continue to cultivate 2 days, the results supernatant liquor is further purified;
2., 1 liter of people's marrow fibroblast culture supernatant being added gelatin-agarose that the 1ml bovine serum albumin handles hatched 24 hours for 4 ℃, separate gelatin-sepharose 4B with the centrifugation method in conjunction with the cell Fibronectin, with the can of 10ml pearl in the chromatography column of 1.5cm diameter; This post is at room temperature used the 0.15M NaCl of 4 times of post bed capacity, 10mM Tutofusin tris-hydrochloric acid (Tris-HCl), the pH7.0 washing is with damping fluid C[4M urea, 0.5M NaCl, 50mM hexahydroaniline propane sulfonic acid], the pH11.0 elution; Determine protein peak, the each several part that will contain the cell Fibronectin mixes, and with buffer A (0.15M NaCl, 1mM CaCl2,10mM hexahydroaniline propane sulfonic acid, readjustment pH to 11.0) dialysis, detects protein concn, makes into 0.125g/ml concentration, is stored in-70 ℃;
(5), measure 1% 3-mercaptoethanol solution and the 0.1ml Fibronectin solution of 50.0ml bovine serum albumin solution, 50.0ml insulin solutions, 50.0ml purifying human transferrin solution, 50.0ml cholesterol solution, 50.0ml superoxol, 0.05ml sodium selenite solution, 0.175ml respectively, above-mentioned eight kinds of solution are mixed, be the 500ml/ bottle with Yi Sikefu improvement cutter than the dilution of Ke Shi substratum again, promptly get cell attachment and cultivate serum free medium;
(6), the preparation of short anchoring factor:
1., people's marrow fibroblast culture supernatant: obtain BMNC with the lymphocyte separation medium density gradient centrifugation, be incubated in the plastic culture bottle that is covered with 4% poly-lysine in advance with 1 * 106 cell/ml concentration, add 10% people AB serum and 10-6M hydrocortisone (final concentration), cultivated 5 days, form adherent individual layer, inhale and remove supernatant, change the cell suspension culture serum free medium and continue to cultivate 2 days, the results supernatant liquor is further purified;
2., granulated glass sphere affinity chromatography: glass microballon with 0.6M NaHCO3 (pH8.0) washing, is adorned post (5cm * 50cm), use 0.6M NaHCO3 equilibrate overnight, flow velocity 100ml/ hour.People's marrow fibroblast culture supernatant is adjusted to pH8.0 with 0.1N NaOH, filter, with 100ml people's marrow fibroblast culture supernatant with sample on the 100ml/ speed at one hour rating, elution peak (behind 65kd and the 75kd) appears, with following damping fluid (all containing tolylsulfonyl fluorine (PMSF)) wash-out (100ml/ hour):
A.200ml?0.6M?NaHCO3·pH8.0
B.100ml?0.6M?NaHCO3-0.2M?Na2CO3，pH9.3-9.5
C.100ml H2OD.500ml 0.2M K2CO3, pH11.0 will mix with the each several part of 0.6M NaHCO3-0.2M Na2CO3 elution, carry out next step purifying;
3. concanavalin A-agarose column/diethylaminoethyl-(DEAE) chromatography:
(2.5cm * 20cm) (2.5cm * 3cm), the 2.5cm of can thereon * 16cm concanavalin A-agarose column layer uses the phosphoric acid buffer balance, 30ml/ hour to chromatography column then with diethylaminoethyl--agarose can bottom;
Urge each part (about 200ml) of anchoring factor with 0.6M NaHCO3-0.2M Na2CO3 from containing of glass bead column elution and be applied to concanavalin A/diethylaminoethyl-post, 30ml/ hour, press the laxative remedy elution:
1) 30ml 0.25M sodium phosphate (Na/pi), pH6.0,30ml/ hour;
2) 200ml 25mM Na/Pi, pH6.0,15ml/ hour;
3) 250ml contains the 25mM Na/Pi of 50mM seminose, pH6.0,30ml/ hour;
4) 30ml 25mM Na/Pi, pH6.0,30ml/ hour;
5) 60ml contains the 25mM Na/Pi of 100mM NaCl, pH6.0,30ml/ hour;
6) 100ml contains the 25mM Na/Pi of 0.5M Alpha-Methyl mannoside and 1M NaCl, pH6.0,30ml/ hour;
7) 100ml contains the 0.15M NaCl 20mM Tutofusin tris of (containing 1mM MgCl2,1mM CaCl2 and 1mM MnCl2), and pH7.3 contains the elution peak 25mM Na/Pi elution that contains 100mM NaCl of the material of PAF;
4. heparin-agarose column chromatography:
Heparin-agarose chromatography column (the dress of 0.7cm * 10cm) post, and with the 25mMNa/Pi that contains 100mM NaCl, pH6.0 balance (15ml/ hour), the part difference (not mixing) that contains short anchoring factor of concanavalin A/diethylaminoethyl-purifying goes up sample to the heparin-agarose post, 7.5ml/ hour, by the laxative remedy elution, promptly get and urge anchoring factor:
1) 20ml contains the 25mM Na/Pi of 100mM NaCl, pH6.0 (15ml/ hour);
2) 20ml contains among the 50mM Na/Pi of 100mM NaCl, pH7.0 (4ml/ hour);
3) 20ml contains among the 50mM Na/Pi of 1M NaCl, pH7.4 (15ml/ hour);
At the bottom of overlaying the culturing bottle bottle with short anchoring factor, cultivate with cell attachment cultivation serum free medium mixed culture cell injection culturing bottle again (7), earlier.
Embodiment 4: cell attachment is cultivated the preparation of serum free medium
The preparation of sodium selenite solution among the embodiment 4, the preparation of 3-mercaptoethanol solution, the preparation of cell Fibronectin solution is identical with embodiment 3 with the preparation of short anchoring factor, and difference is:
(8), compound concentration is respectively the bovine serum albumin solution of 50mg/ml, the insulin solutions of 400 μ g/ml, the purifying human transferrin solution of 300 μ g/ml, the cholesterol solution of 600 μ g/ml and the hydrogen peroxide enzyme solution of 600 μ g/ml according to a conventional method, all is dissolved in Yi Sikefu improvement cutter than Ke Shi substratum.0.1ml the 1% 3-mercaptoethanol solution of sodium selenite solution, 0.35ml, 0.2ml Fibronectin solution, above-mentioned eight kinds of solution are mixed, be the 500ml/ bottle with Yi Sikefu improvement cutter than the dilution of Ke Shi substratum again, promptly get cell attachment and cultivate serum free medium.
Claims (4)
1, the serum free medium of a kind of cell suspension culture and adherent culture, prescription comprises the bovine serum albumin solution in final concentration: 5-10mg/ml, insulin solutions: 8-20 μ g/ml, purifying human transferrin solution: 1-30 μ g/ml, cholesterol solution: 20-60 μ g/ml and hydrogen peroxide enzyme solution: 20-50 μ g/ml is characterized in that: also comprise sodium selenite solution: 17.3-35.0 μ g/ml and 1% 3-mercaptoethanol solution: 0.35-1.0ml.
2, the serum free medium of cell suspension culture as claimed in claim 1 and adherent culture is characterized in that: also can add the cell Fibronectin in final concentration in the prescription: 25-50 μ g/ml and short anchoring factor.
3, the preparation method of the serum free medium of cell suspension culture as claimed in claim 1 and adherent culture is characterized in that:
(1), compound concentration is respectively the bovine serum albumin solution of 50-100mg/ml, the insulin solutions of 80-400 μ g/ml, the purifying human transferrin solution of 10-300 μ g/ml, the cholesterol solution of 200-600 μ g/ml and the hydrogen peroxide enzyme solution of 200-600 μ g/ml according to a conventional method, all is dissolved in Yi Sikefu improvement cutter than Ke Shi substratum;
(2), the preparation of sodium selenite solution: commercially available Sodium Selenite powder 1.73mg is dissolved in the 10ml triply distilled water Entkeimung, 4 ℃ of refrigerators preservations;
(3), the preparation of 3-mercaptoethanol solution: commercially available 3-mercaptoethanol is added in the triply distilled water, make into 1% solution, be stock solution, Entkeimung is put 4 ℃ of refrigerators and is preserved;
(4), above-mentioned seven kinds of solution pressed formula rate mix, be the 500ml/ bottle with Yi Sikefu improvement cutter than the dilution of Ke Shi substratum again, promptly get the cell suspension culture serum free medium.
4, the preparation method of the serum free medium of cell suspension culture as claimed in claim 2 and adherent culture is characterized in that:
(1), compound concentration is respectively the bovine serum albumin solution of 50-100mg/ml, the insulin solutions of 80-400 μ g/ml, the purifying human transferrin solution of 10-300 μ g/ml, the cholesterol solution of 200-600 μ g/ml and the hydrogen peroxide enzyme solution of 200-600 μ g/ml according to a conventional method, all is dissolved in Yi Sikefu improvement cutter than Ke Shi substratum;
(2), the preparation of sodium selenite solution: commercially available Sodium Selenite powder 1.73mg is dissolved in the 10ml triply distilled water Entkeimung, 4 ℃ of refrigerators preservations;
(3), the preparation of 3-mercaptoethanol solution: commercially available 3-mercaptoethanol is added in the triply distilled water, make into 1% solution, be stock solution, Entkeimung is put 4 ℃ of refrigerators and is preserved;
(4), the preparation of cell Fibronectin solution:
1., people's marrow fibroblast culture supernatant: obtain BMNC with the lymphocyte separation medium density gradient centrifugation, be incubated in the plastic culture bottle that is covered with 4% poly-lysine in advance with 1 * 106 cell/ml concentration, add 10% people AB serum and 10-6M hydrocortisone (final concentration), cultivated 5 days, form adherent individual layer, inhale and remove supernatant, change the cell suspension culture serum free medium and continue to cultivate 2 days, the results supernatant liquor is further purified;
2., 1 liter of people's marrow fibroblast culture supernatant being added gelatin-agarose that the 1ml bovine serum albumin handles hatched 24 hours for 4 ℃, separate gelatin-sepharose 4B with the centrifugation method in conjunction with the cell Fibronectin, with the can of 10ml pearl in the chromatography column of 1.5cm diameter; This post is at room temperature used the 0.15M NaCl of 4 times of post bed capacity, 10mM Tutofusin tris-hydrochloric acid, the pH7.0 washing is with damping fluid C[4M urea, 0.5M NaCl, 50mM hexahydroaniline propane sulfonic acid], the pH11.0 elution; Determine protein peak, the each several part that will contain the cell Fibronectin mixes, and with buffer A (0.15MNaCl, 1mM CaCl2,10mM encircle amine propane sulfonic acid, readjustment pH to 11.0) dialysis, detects protein concn, makes into 0.125g/ml concentration, is stored in-70 ℃;
(5), above-mentioned eight kinds of solution pressed formula rate mix, be diluted to the 500ml/ bottle with Yi Sikefu improvement cutter than Ke Shi substratum again, promptly get cell attachment cultivation serum free medium;
(6), the preparation of short anchoring factor:
1., people's marrow fibroblast culture supernatant: obtain BMNC with the lymphocyte separation medium density gradient centrifugation, be incubated in the plastic culture bottle that is covered with 4% poly-lysine in advance with 1 * 106 cell/ml concentration, add 10% people AB serum and 10-6M hydrocortisone (final concentration), cultivated 5 days, form adherent individual layer, inhale and remove supernatant, change the cell suspension culture serum free medium and continue to cultivate 2 days, the results supernatant liquor is further purified;
2., granulated glass sphere affinity chromatography: glass microballon with 0.6M NaHCO3 (pH8.0) washing, is adorned post (5cm * 50cm), use 0.6M NaHCO3 equilibrate overnight, flow velocity 100ml/ hour.People's marrow fibroblast culture supernatant is adjusted to pH8.0 with 0.1N NaOH, filter, with 100ml people's marrow fibroblast culture supernatant with sample on the 100ml/ speed at one hour rating, elution peak (behind 65kd and the 75kd) appears, with following damping fluid (all containing the tolylsulfonyl fluorine) wash-out (100ml/ hour):
A.200ml?0.6M?NaHCO3·pH8.0
B.100ml?0.6M?NaHCO3-?0.2M?Na2CO3，pH9.3-9.5
C.100ml?H2O
D.500ml?0.2M?K2CO3，pH11.0
To mix with the each several part of 0.6M NaHCO3-0.2M Na2CO3 elution, carry out next step purifying;
3. concanavalin A-agarose column/diethylaminoethyl-chromatography:
(2.5cm * 20cm) (2.5cm * 3cm), the 2.5cm of can thereon * 16cm concanavalin A-agarose column layer uses the phosphoric acid buffer balance, 30ml/ hour to chromatography column then with diethylaminoethyl--agarose can bottom;
Urge each part (about 200ml) of anchoring factor with 0.6M NaHCO3-0.2M Na2CO3 from containing of glass bead column elution and be applied to concanavalin A/diethylaminoethyl-post, 30ml/ hour, press the laxative remedy elution:
1) 30ml 0.25M sodium phosphate (Na/pi), pH6.0,30ml/ hour;
2) 200ml 25mM Na/Pi, pH6.0,15ml/ hour;
3) 250ml contains the 25mM Na/Pi of 50mM seminose, pH6.0,30ml/ hour;
4) 30ml 25mM Na/Pi, pH6.0,30ml/ hour;
5) 60ml contains the 25mM Na/Pi of 100mM NaCl, pH6.0,30ml/ hour;
6) 100ml contains the 25mM Na/Pi of 0.5M Alpha-Methyl mannoside and 1M NaCl, pH6.0,30ml/ hour;
7) 100ml contains the 0.15M NaCl 20mM Tutofusin tris of (containing 1mM MgCl2,1mM CaCl2 and 1mM MnCl2), and pH7.3 contains the elution peak 25mM Na/Pi elution that contains 100mM NaCl of the material of PAF;
4. heparin-agarose column chromatography:
Heparin-agarose chromatography column (the dress of 0.7cm * 10cm) post, and with the 25mMNa/Pi that contains 100mM NaCl, pH6.0 balance (15ml/ hour), the part difference (not mixing) that contains short anchoring factor of concanavalin A/diethylaminoethyl-purifying goes up sample to the heparin-agarose post, 7.5ml/ hour, by the laxative remedy elution, promptly get and urge anchoring factor:
1) 20ml contains the 25mM Na/Pi of 100mM NaCl, pH6.0 (15ml/ hour);
2) 20ml contains among the 50mM Na/Pi of 100mM NaCl, pH7.0 (4ml/ hour);
3) 20ml contains among the 50mM Na/Pi of 1M NaCl, pH7.4 (15ml/ hour);
At the bottom of overlaying the culturing bottle bottle with short anchoring factor, cultivate with cell attachment cultivation serum free medium mixed culture cell injection culturing bottle again (7), earlier.
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| CN100348718C (en) * | 2005-10-18 | 2007-11-14 | 中国人民解放军军事医学科学院生物工程研究所 | Culture medium without animal originating component and serum for HEK293 cell adhesion culture |
| CN101412985B (en) * | 2007-10-15 | 2012-06-13 | 华东理工大学 | Serum-free medium for in vitro cultivation and amplification of mesenchymal stem cells |
| CN101597633B (en) * | 2008-06-03 | 2012-08-22 | 哈药集团生物工程有限公司 | New method for producing recombinant human hematopoietin by cell suspension culture |
| CN101914490A (en) * | 2010-08-13 | 2010-12-15 | 中国医科大学 | Human amnion mesenchymal stem cell serum-free culture medium and culture method thereof |
| CN102719480A (en) * | 2012-07-08 | 2012-10-10 | 大连医科大学 | Pre-processing method of cell culture plate for suspension cell liposome transfection |
| CN103555665A (en) * | 2013-08-12 | 2014-02-05 | 北京东方华辉生物医药科技有限公司 | SFM (serum-free medium) for culturing MSCs (mesenchymal stem cells) |
| CN105039251A (en) * | 2015-07-27 | 2015-11-11 | 广州市达晖生物技术有限公司 | Lymphocyte serum-free medium with stable pH value as well as preparation method and application of lymphocyte serum-free medium |
| CN105039251B (en) * | 2015-07-27 | 2018-12-18 | 广州达晖生物技术股份有限公司 | A kind of lymphocyte serum of pH stable and its preparation method and application |
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