CN102533634A - Serum-free protein-free chemical culture medium for Chinese hamster ovary (CHO) cells - Google Patents
Serum-free protein-free chemical culture medium for Chinese hamster ovary (CHO) cells Download PDFInfo
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Abstract
The invention relates to a serum-free protein-free chemical culture medium for Chinese hamster ovary (CHO) cells. The serum-free protein-free chemical culture medium is characterized by comprising inorganic salts with the concentration of 5.029-50.294 g/L, amino acids with the concentration of 0.554-5.536 g/L, vitamins with the concentration of 0.006-0.06 g/L, trace elements with the concentration of 2.612-26.123 g/L, a biological small peptide A with the concentration of 0.005-0.05 g/L and a biological small peptide B with the concentration of 0.25-2.5 g/L, wherein the sequence of the amino acids of the biological small peptide A is proline-glycine-lysine-cysteine-histidine-serine and the sequence of the amino acids of the biological small peptide B is lysine-cysteine-tyrosine-threonine-cysteine-leucine. According to the serum-free protein-free chemical culture medium, the pH value is 7.1-7.5, the osmotic pressure is 280-320 mOsm/kg, and no growth of bacteria and fungi exists, so that a serum-free culture environment clear solution for the cell growth and the cell protein expression and the like, of the CHO cells can be provided.
Description
Technical field
The present invention relates to a kind of biological technical field, is that a kind of special serum-free to Chinese hamster ovary celI does not have albumen chemistry substratum.
Background technology
It is a kind of synthetic pure chemistry composition substratum that does not need animal serum that serum-free does not have albumen chemistry substratum; Through increasing clear and definite microchemistry composition and biological small peptide component as serum-free cell culture medium; Replace the protein ingredient in the serum-free cell culture medium, support culturing cell not have the suspension growth in the albumen chemistry substratum at serum-free.Thereby pollution and the not clear composition of serum that can avoid animal serum in cell cultivation process, to bring are produced the uncertain influence that causes to downstream.
One Chinese patent application numbers 200610024459 discloses a kind of serum-free animal cell culture medium and preparation method thereof; One Chinese patent application numbers 200910265976 discloses a kind of serum-free animal cell culture medium dry powder, liquid nutrient medium and preparation method thereof; Though these two disclosed products of patented technology are serum-free animal cell culture medium, medium component is not no albumen, pure chemistry composition.
Chinese hamster ovary celI (Chinese hamster ovary cell) derives from Chinese hamster ovary.The CHO-K1 cell is incubated at nineteen sixty-eight [Kao.F.T and Park T.T .proc.Natl Acad.sci USA 60:1275-1281 (1968)] the earliest. existing at present inferior strain of multiple difference and mutant strain.This cell strain is widely used in biological protein medicament production.This cell strain can obtain from ATCC or other establishment.
Summary of the invention
The purpose of this invention is to provide a kind of Chinese hamster ovary celI serum-free and do not have albumen chemistry substratum; Its serum proteins composition is replaced by composition clear and definite trace element and biological small peptide; Can be under serum-free or other ESC condition the sustenticular cell suspension growth, have that component is clear and definite, the protein free advantage of serum-free.
The Chinese hamster ovary celI serum-free does not have albumen chemistry substratum; It is characterized in that comprising the inorganic salt of 5.029-50.294g/L, the amino acid of 0.554-5.536g/L, VITAMINs and the 2.612-26.123g/L trace element of 0.006-0.06g/L; And biological small peptide A of 0.005-0.05 mg/L and the biological small peptide B of 0.25-2.5mg/L, the amino acid ordering of wherein biological small peptide A is proline(Pro)-glycocoll-Methionin-halfcystine-Histidine-Serine; Biological small peptide B is Methionin-halfcystine-tyrosine-Threonine-halfcystine-leucine.
Wherein inorganic salt play the effect of osmotic pressure regulator, the inorganic salt that the present invention adopted with and following at the consumption of substratum:
| CaCl2 (anhydrous) | 58.3-582.9 |
| CuSO4·5H2O | 0.0006-0.006 |
| FeSO4·7H2O | 0.21-2.1 |
| Fe(NO3)2·9H2O | 0.025-0.25 |
| KCl | 155.3-1553 |
| MgSO4 (anhydrous) | 24.41-244.1 |
| MgCl2 (anhydrous) | 14.29-142.9 |
| NaCl | 3490.7-34907 |
| NaHCO3 | 1218.9-12189 |
| NaH2PO4·H2O | 31.6-316 |
| Na2HPO4 (anhydrous) | 35.5-354.9 |
| ZnSO4·7H2O | 0.22-2.2 |
Amino acid provides necessary nutrition component for institute's cultured cells, the amino acid that the present invention adopted with and following at the consumption of substratum:
| L-Ala | 2.225-22.25 |
| L-arginine HCl | 73.5-735 |
| Aspartic acid | 3.34-33.4 |
| L-asparagine H2O | 3.75-37.5 |
| Halfcystine HClH2O | 8.76-87.6 |
| Gelucystine 2HCl | 15.6-156 |
| L-glutamic acid | 3.67-36.7 |
| Stimulina | 182.1-1821 |
| Glycocoll | 9.17-91.7 |
| Histidine HClH2O | 15.7-157 |
| Isoleucine | 27.18-271.8 |
| Leucine | 29.5-295 |
| Methionin HCl | 45.6-456 |
| Methionine(Met) | 8.6-86 |
| Phenylalanine(Phe) | 17.7-177 |
| Proline(Pro) | 8.62-86.2 |
| Serine | 13.1-131 |
| Threonine | 26.7-267 |
| Tryptophane | 4.5-45 |
| Tyrosine 2Na2H2O | 27.9-279 |
| Xie Ansuan | 26.4-264 |
VITAMINs is that cellular metabolism is necessary, is one of main additive of serum free medium:
| Vitamin H | 0.002-0.02 |
| VA | 1.09-10.9 |
| Folic acid | 1.32-13.2 |
| Vitamin PP | 1.0-10 |
| Pyridoxal .Hcl | 1.01-10.1 |
| Pyridoxol .Hcl | 0.015-0.15 |
| Vitamin G | 0.11-1.1 |
| VitB1 .Hcl | 1.1-11 |
| Cobalamin | 0.35-3.5 |
Trace element is that cellular metabolism is necessary, is one of main additive of serum free medium, and the element that has need provide by serum, the trace element that the present invention adopted with and following at the consumption of substratum:
| Choline chloride 60 | 4.48-44.8 |
| Glucose | 1572.35-15723.5 |
| Xanthoglobulin (sodium salt) | 1.19-12 |
| The i-inositol | 6.25-62.5 |
| Linolic acid | 0.021-0.21 |
| Thioctic Acid | 0.053-0.53 |
| Putrescine 2HCl | 0.04-0.4 |
| Pyruvic acid (sodium salt) | 27.5-275 |
| Thymidine | 0.18-1.8 |
| pluronic F68 | 1000-10000 |
| Carbinolamine | 0.005-0.05 |
| Spermine | 0.005-0.05 |
| Thioglycerin | 0.001-0.1 |
| Single nickel salt | 0.001-0.01 |
| Manganous sulfate | 0.001-0.01 |
| Permanent white | 0.0010-0.01 |
| Silver Nitrate | 0.0010-0.01 |
| Aluminum chloride | 0.0010-0.01 |
Beneficial effectThe Chinese hamster ovary celI serum-free of the present invention's preparation does not have albumen chemistry substratum; PH7.1-7.5, osmotic pressure is 280-320 mOsm/kg, do not have bacterium and fungal growth; Can be CHO series cell, the settled solution of the serum-free culture environment of cell growth, expression of cellular proteins etc. can be provided.
Description of drawings
Fig. 1 is the cytological map after the Chinese hamster ovary celI serum-free of embodiment 1 preparation does not have albumen chemistry culture medium culturing CHO K1 cell 24h; Microscope amplifies 25 multiples, and visible from figure, it is mellow and full full that cell well-grown, cell keep.
Fig. 2 is CHO K1 cell does not have albumen chemistry substratum at the Chinese hamster ovary celI serum-free of embodiment 1 preparation a growth curve chart; Chinese hamster ovary celI was grown division well in 0 to 126 hour, explain that this substratum is fit to the Chinese hamster ovary celI growth.
Fig. 3 is CHO K1 cell does not have albumen chemistry substratum at the Chinese hamster ovary celI serum-free of embodiment 1 preparation a survival rate graphic representation.The survival rate of Chinese hamster ovary celI in 0 to 90 hour remains on more than 95%, and explaining that this substratum can not have under the albumen condition at serum-free provides good build environment for Chinese hamster ovary celI.
Embodiment
The raw material of all purchasing of raw materials Simgma cell cultures levels of the present invention, and press the related request storage.Biological small peptide A and biological small peptide B be solid polypeptide synthetic (R. B. Merrifield (1963). " Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide ".
J. Am. Chem. Soc. 85(14): 2149 – 2154.), its purity is no less than 99.9%.
Embodiment 1
The Chinese hamster ovary celI serum-free does not have albumen chemistry culture medium dry powder, and the component of substratum is following:
Inorganic salt: CaCl
2(anhydrous) 116.6mg, CuSO
45H
2O 0.0013mg, FeSO
47H
2O 0.417mg, Fe (NO
3)
29H
2O 0.05mg, KCl 311.8mg, MgSO
4(anhydrous) 48.84mg, MgCl
2(anhydrous) 28.64mg, NaCl 6995.5mg, NaHCO
32438mg, NaH
2PO
4H
2O 62.5mg, Na
2HPO
4(anhydrous) 71.02mg, ZnSO
47H
2O 0.43mg;
Amino acid: L-Ala 4.45mg, l-arginine HCl 147.5mg, aspartic acid 6.65mg, l-asparagine H
2O 7.5mg, halfcystine HClH
2O 17.56mg, Gelucystine 2HCl 31.29mg, L-glutamic acid 7.35mg, Stimulina 365mg, glycocoll 18.75mg, Histidine HClH
2O 31.48mg, Isoleucine 54.47mg, leucine 59.05mg, Methionin HCl 91.25mg; Methionine(Met) 17.24mg, phenylalanine-3,4-quinone 5.48mg, proline(Pro) 17.25mg, Serine 26.25mg; Threonine 53.45mg, tryptophane 9.02mg, tyrosine 2Na2H
2O 55.79mg, Xie Ansuan 52.85mg;
VITAMINs: vitamin H 0.0035mg, VA 2.24mg, folic acid 2.65mg, vitamin PP 2.02mg, pyridoxal HCl 2mg, pyridoxol HCl 0.03mg, vitamin G 0.22mg, VitB1 HCl 2.17mg, vitamins B
120.68mg,
Trace element: choline chloride 60 8.98mg, glucose 3151mg, xanthoglobulin (sodium salt) 2.39mg, i-inositol 12.6mg, linolic acid 0.042mg; Thioctic Acid 0.105mg, putrescine 2HCl 0.081mg, pyruvic acid (sodium salt) 55mg, thymidine 0.365mg, pluronic F68 2000mg; Carbinolamine 0.01mg, spermine 0.01mg, thioglycerin 0.02mg, single nickel salt 0.002mg; Manganous sulfate 0.002mg, permanent white 0.002mg, Silver Nitrate 0.002mg, aluminum chloride 0.002mg;
Biological small peptide: biological small peptide A (proline(Pro)-glycocoll-Methionin-halfcystine-Histidine-Serine) 0.01mg, biological small peptide B (Methionin-halfcystine-tyrosine-Threonine-halfcystine-leucine) 0.5mg;
Add dissolving of 950ml pure water and mixing, transfer pH to 7.5 with HCL or NaOH, constant volume is to 1000ml.Use the 0.22um negative pressure filtration, aseptic preservation.
Embodiment 2
It is as shown in table 1 that the Chinese hamster ovary celI serum-free that the embodiment of the invention 1 obtains does not have the physico-chemical property parameter and the detection method of albumen chemistry culture medium culturing, and detected result is as shown in table 2.
Table 1
| Test item | Detection method |
| Outward appearance | Visual inspection |
| The pH value | Get media samples 5ml to be detected, wait to rise again and to 25 ℃ of room temperatures, detect data with pH meter |
| Osmotic pressure | Get |
| Bacterium and fungi | Get media samples 10ml to be detected and in the 100mm petridish, place 37 ° of CO2gas incubators, continue to examine under a microscope situation such as sample clarity every day |
Table 2
It is as shown in table 3 that the Chinese hamster ovary celI serum-free that the present invention obtains does not have the cell cultures project and the cultural method of albumen chemistry culture medium culturing, and it is as shown in table 4 to have cultivated the cell stand density behind the 24h.
Table 3
| The cultivation project | Cultural method |
| Cell | Suspension CHO K1 cell shakes bottle, 20ml cell suspension/bottle at logarithmic phase with the density of the 2 * 105cells/ml 100ml that goes down to posterity |
| Nutrient solution | The Chinese hamster ovary celI serum-free does not have albumen chemistry substratum |
| Culture condition | 36.5 shaking table wave and culture in ℃ CO2gas incubator, rotating speed: 140 r/min |
Table 4
| Time (h) |
0 | 26 | 48 | 72 | 96 | 120 | 144 |
| Cell density (* 105cells/ml) | 3 | 7.9 | 8.5 | 13.8 | 15.8 | 17 | 9.8 |
| Cell survival rate (%) | 99.8 | 100 | 99.8 | 99.9 | 61 | 0 | 0 |
It is thus clear that; The Chinese hamster ovary celI serum-free of embodiment 1 preparation does not have albumen chemistry substratum; For pH7.5, osmotic pressure are 320 mOsm/kg, do not have bacterium and a fungal growth, can be CHO series cell, provide the serum-free of cell growth, expression of cellular proteins etc. not have the settled solution of albumen culture environment.
Embodiment 3
The Chinese hamster ovary celI serum-free does not have albumen chemistry substratum, the inorganic salt quality (unit: milligram) as follows:
CaCl2 (anhydrous) 58.3
CuSO4·5H2O 0.0006
FeSO4·7H2O 0.21
Fe(NO3)2·9H2O 0.025
KCl 155.3
MgSO4 (anhydrous) 24.41
MgCl2 (anhydrous) 14.29
NaCl 3490.7
NaHCO3 1218.9
NaH2PO4·H2O 31.6
Na2HPO4 (anhydrous) 35.5
ZnSO4·7H2O 0.22
Amino acid whose quality (unit: milligram) as follows:
L-Ala 2.225
L-arginine HCl 73.5
Aspartic acid 3.34
L-asparagine H2O 3.75
Halfcystine HClH2O 8.76
Gelucystine 2HCl 15.6
L-glutamic acid 3.67
Stimulina 182.1
Glycocoll 9.17
Histidine HClH2O 15.7
Isoleucine 27.18
Leucine 29.5
Methionin HCl 45.6
Methionine(Met) 8.6
Phenylalanine(Phe) 17.7
Proline(Pro) 8.62
Serine 13.1
Threonine 26.7
Tryptophane 4.5
Tyrosine 2Na2H2O 27.9
Xie Ansuan 26.4
The VITAMINs quality that is adopted (unit: milligram) as follows:
Vitamin H 0.002
VA 1.09
Folic acid 1.32
Vitamin PP 1.01
Pyridoxal .Hcl 1.009
Pyridoxol .Hcl 0.015
Vitamin G 0.11
VitB1 .Hcl 1.09
Cobalamin 0.35
The micro-quality that is adopted (unit: milligram) as follows:
Choline chloride 60 4.48
Glucose 1572.35
Xanthoglobulin (sodium salt) 1.19
I-inositol 6.25
Linolic acid 0.021
Thioctic Acid 0.053
Putrescine 2HCl 0.04
Pyruvic acid (sodium salt) 27.5
Thymidine 0.18
pluronic?F68 999.9
Carbinolamine 0.005
Spermine 0.005
Thioglycerin 0.0099
Single nickel salt 0.001
Manganous sulfate 0.001
Permanent white 0.0010
Silver Nitrate 0.0010
Aluminum chloride 0.0010
Biological small peptide A 0.005
Biological small peptide B 0. 25,
Above component is added dissolving of 950ml pure water and mixing, transfer pH to 7.5 with HCL or NaOH, constant volume is to 1000ml.Use the 0.22um negative pressure filtration, aseptic preservation.The Chinese hamster ovary celI serum-free of preparation does not have albumen chemistry substratum; For pH7.1, osmotic pressure are 280 mOsm/kg, do not have bacterium and a fungal growth; Can be CHO series cell, provide the serum-free of cell growth, expression of cellular proteins etc. not have the settled solution of albumen culture environment.
Embodiment 3
The Chinese hamster ovary celI serum-free does not have albumen chemistry substratum, the inorganic salt quality of employing (unit: milligram) as follows:
CaCl2 (anhydrous) 582.9
CuSO4·5H2O 0.006
FeSO4·7H2O 2.1
Fe(NO3)2·9H2O 0.25
KCl 1553
MgSO4 (anhydrous) 244.1
MgCl2 (anhydrous) 142.9
NaCl 34907
NaHCO3 12189
NaH2PO4·H2O 316
Na2HPO4 (anhydrous) 354.9
ZnSO4·7H2O 2.2；
The amino acid masses that adopts (unit: milligram) as follows:
L-Ala 22.25
L-arginine HCl 735
Aspartic acid 33.4
L-asparagine H2O 37.5
Halfcystine HClH2O 87.6
Gelucystine 2HCl 156
L-glutamic acid 36.7
Stimulina 1821
Glycocoll 91.7
Histidine HClH2O 57
Isoleucine 271.8
Leucine 295
Methionin HCl 456
Methionine(Met) 86
Phenylalanine(Phe) 177
Proline(Pro) 86.2
Serine 131
Threonine 267
Tryptophane 45
Tyrosine 2Na2H2O 279
Xie Ansuan 264;
The VITAMINs quality that the present invention adopted (unit: milligram) as follows:
Vitamin H 0.02
VA 10.9
Folic acid 13.2
Vitamin PP 10.1
Pyridoxal .Hcl 10.1
Pyridoxol .Hcl 0.15
Vitamin G 1.1
VitB1 .Hcl 11
Cobalamin 3.5;
The micro-quality that the present invention adopted (unit: milligram) as follows:
Choline chloride 60 44.8
Glucose 15723.5
Xanthoglobulin (sodium salt) 12
I-inositol 62.5
Linolic acid 0.21
Thioctic Acid 0.53
Putrescine 2HCl 0.4
Pyruvic acid (sodium salt) 275
Thymidine 1.8
pluronic?F68 10000
Carbinolamine 0.05
Spermine 0.05
Thioglycerin 0.1
Single nickel salt 0.01
Manganous sulfate 0.01
Permanent white 0.01
Silver Nitrate 0.01
Aluminum chloride 0.01
Biological small peptide A 0.05
Biological small peptide B 2.5
Above component is added dissolving of 950ml pure water and mixing, transfer pH to 7.5 with HCL or NaOH, constant volume is to 1000ml.Use the 0.22um negative pressure filtration, aseptic preservation.The Chinese hamster ovary celI serum-free of preparation does not have albumen chemistry substratum; For pH7.2, osmotic pressure are 300 mOsm/kg, do not have bacterium and a fungal growth; Can be CHO series cell, provide the serum-free of cell growth, expression of cellular proteins etc. not have the settled solution of albumen culture environment.
Claims (2)
1.CHO cell non-serum does not have albumen chemistry substratum; It is characterized in that comprising the inorganic salt of 5.029-50.294g/L, the amino acid of 0.554-5.536g/L, VITAMINs and the 2.612-26.123g/L trace element of 0.006-0.06g/L; And biological small peptide A of 0.005-0.05 mg/L and the biological small peptide B of 0.25-2.5mg/L, the amino acid ordering of wherein biological small peptide A is proline(Pro)-glycocoll-Methionin-halfcystine-Histidine-Serine; Biological small peptide B is Methionin-halfcystine-tyrosine-Threonine-halfcystine-leucine.
2. Chinese hamster ovary celI serum-free according to claim 1 does not have albumen chemistry substratum, it is characterized in that said inorganic salt with and following at the milligram number of every liter of substratum:
CaCl2 (anhydrous) 58.3-582.9
CuSO4·5H2O 0.0006-0.006
FeSO4·7H2O 0.21-2.1
Fe(NO3)2·9H2O 0.025-0.25
KCl?155.3-1553
MgSO4 (anhydrous) 24.41-244.1
MgCl2 (anhydrous) 14.29-142.9
NaCl 3490.7-34907
NaHCO3?1218.9-12189
NaH2PO4·H2O 31.6-316
Na2HPO4 (anhydrous) 35.5-354.9
ZnSO4·7H2O 0.22-2.2；
The amino acid that the present invention adopted with and every liter of substratum the milligram number following:
L-Ala 2.225-22.25
L-arginine HCl 73.5-735
Aspartic acid 3.34-33.4
L-asparagine H2O 3.75-37.5
Halfcystine HClH2O 8.76-87.6
Gelucystine 2HCl 15.6-156
L-glutamic acid 3.67-36.7
Stimulina 182.1-1821
Glycocoll 9.17-91.7
Histidine HClH2O 15.7-157
Isoleucine 27.18-271.8
Leucine 29.5-295
Methionin HCl 45.6-456
Methionine(Met) 8.6-86
Phenylalanine(Phe) 17.7-177
Proline(Pro) 8.62-86.2
Serine 13.1-131
Threonine 26.7-267
Tryptophane 4.5-45
Tyrosine 2Na2H2O 27.9-279
Xie Ansuan 26.4-264;
The VITAMINs that the present invention adopted with and every liter of substratum the milligram number following:
Vitamin H 0.002-0.02
VA 1.09-10.9
Folic acid 1.32-13.2
Vitamin PP 1.01-10.1
Pyridoxal .Hcl 1.009-10.1
Pyridoxol .Hcl 0.015-0.15
Vitamin G 0.11-1.1
VitB1 .Hcl 1.09-11
Cobalamin 0.35-3.5;
The trace element that the present invention adopted with and every liter of substratum the milligram number following:
Choline chloride 60 4.48-44.8
Glucose 1572.35-15723.5
Xanthoglobulin (sodium salt) 1.19-12
I-inositol 6.25-62.5
Linolic acid 0.021-0.21
Thioctic Acid 0.053-0.53
Putrescine 2HCl 0.04-0.4
Pyruvic acid (sodium salt) 27.5-275
Thymidine 0.18-1.8
pluronic?F68 999.9-10000
Carbinolamine 0.005-0.05
Spermine 0.005-0.05
Thioglycerin 0.0099-0.1
Single nickel salt 0.001-0.01
Manganous sulfate 0.001-0.01
Permanent white 0.0010-0.01
Silver Nitrate 0.0010-0.01
Aluminum chloride 0.0010-0.01.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106282090A (en) * | 2015-06-08 | 2017-01-04 | 齐鲁制药有限公司 | CHO-S cell line after a kind of domestication and cultural method and application |
| CN106399224A (en) * | 2016-12-13 | 2017-02-15 | 昆明润什生物科技有限公司 | Serum-free and protein-free cell culture medium |
| CN112410280A (en) * | 2020-11-12 | 2021-02-26 | 上海奥浦迈生物科技股份有限公司 | Serum-free medium for PK15 cell culture and application thereof |
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| CN112410280B (en) * | 2020-11-12 | 2022-06-03 | 上海奥浦迈生物科技股份有限公司 | Serum-free medium for PK15 cell culture and application thereof |
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