CN101892192A - Cell culture method capable of reducing heterologous protein residue in cell products - Google Patents
Cell culture method capable of reducing heterologous protein residue in cell products Download PDFInfo
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- CN101892192A CN101892192A CN 201010223763 CN201010223763A CN101892192A CN 101892192 A CN101892192 A CN 101892192A CN 201010223763 CN201010223763 CN 201010223763 CN 201010223763 A CN201010223763 A CN 201010223763A CN 101892192 A CN101892192 A CN 101892192A
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Abstract
The invention discloses a cell culture method capable of reducing heterologous protein residue in cell products, which comprises the following steps: inoculating cells onto a culture bottle which contains a blood serum medium; placing the culture bottle in an carbon dioxide gas incubator for culture; and at the beginning of the logarithmic growth period of the cells, changing the blood serum medium for a serum free medium. The finished products of adherent cells obtained by the culture method provided by the invention have effectively reduced heterologous protein content without being influenced in final culture effect due to the change of the blood serum medium for the serum free medium in the last step; and when the cell culture method is used for culturing human stem cells, the obtained final products are suitable to be used directly in clinic and have far fewer adverse effects than products cultured by using the blood serum medium all the time.
Description
Technical field
The present invention relates to a kind of cell culture processes, especially a kind of cell culture processes that reduces heterologous protein residual quantity in the cellular product.
Background technology
Under lab at present, the general cell culture medium that contains animal-origin serum that uses carries out the attached cell training of short run, but, if in the middle of the industrialization large-scale application, especially the attached cell of turning out uses the cell culture medium that contains animal-origin serum that many unfavorable factors are arranged when medicinal:
(1) being used for the serum of cell cultures generally will be through overheated deactivation, and heat-inactivated process can prevent the solvency action of serum pair cell, but this process also can make the many important component inactivations in the serum.
(2) because the organism difference that different batches serum is originated, also contain different compositions between the different batches serum inevitably, wherein some becomes branch to bring variation for institute's cultured cells, causes bringing unforeseen influence to finished product, makes its quality product be difficult to reach stdn.
(3) composition in the serum can not illustrated at short notice fully yet, and therefore, they are unknown to the influence of institute's culturing cell, also are out of contior.
In cell therapy product, remove the animal component in the cell culture medium, will improve security of products, weaken or reduce immunological rejection.In cell therapy product, use serum free medium also to be more conducive to the stdn of producing in theory.
But, up to the present also really be not suitable for the serum free medium of mass preparation cellular product:
(1) is used for the serum free medium of cell therapy product owing to will add various cytokines, causes cost to be higher than far away and contain blood serum medium.
(2) up to the present, we also do not understand which composition on cell proliferation, differentiation, gene stability or the like generation respective action in the serum; So, when using serum free medium mass preparation cell therapy product, be difficult to improvement by prescription and adjustment and reach and contain the same effect of blood serum medium.
(3) up to the present, various countries also do not make corresponding standard to being used to cultivate treatment with the serum free medium of cell; But to bovine serum etc. is that corresponding requirement is arranged.
Therefore at present, for faster in commercial application, better amplifying cells, we still need add serum when cultivating.Can there be more foreign protein in the middle of the product that finally obtains,, have then increased the hidden danger when using greatly, so need a kind of cell culture processes that reduces foreign protein content in the middle of the finished product badly if be used for clinically.
Summary of the invention
The purpose of this invention is to provide under a kind of prerequisite that guarantees other Physiology and biochemistry parameter constant of cell, reduce the cell culture processes of foreign protein residual quantity, make the cell of turning out be more suitable for clinical use.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is: a kind of cell culture processes that reduces heterologous protein residual quantity in the cellular product, may further comprise the steps: seed cells in the culturing bottle that contains blood serum medium, place CO2gas incubator to cultivate, when cell enters increased logarithmic phase, will contain blood serum medium and be replaced by serum free medium.
Described cell culture processes is an adherent culture.
The cell of described cell for when cultivating, having adherent characteristic.
Described cell will contain blood serum medium again and be replaced by serum free medium after cultivating through going down to posterity.
Described cell is a stem cell.
Described stem cell is a human stem cell.
Described stem cell is inferior myeloid-lymphoid stem cell.
Described stem cell is a human mesenchymal stem cell.
Described stem cell is the human mesenchymal stem cell from external umbilical cord and placenta source.
The described blood serum medium that contains is one or more combination among DMEM/F12, DMEM, MCDB-201 or the M199, wherein contains the serum of volume ratio 2%～20%.
Described serum is bovine serum or horse serum.
Also add in the described substratum cytokine is arranged.
Described cytokine is the one or more combination among EGF, bFGF or the VEGF.
The content of described EGF, bFGF or VEGF is 1～100ng/ml.
Described carbonic acid gas culture condition is 37 ℃, saturated humidity, gas concentration lwevel 5%, and oxygen concn is 1%～20%.
The density of described cell inoculation when culturing bottle is (0.1～2) * 10
4/ cm
2
The invention has the beneficial effects as follows: the attached cell that obtains with cultural method provided by the present invention, replace with serum free medium owing in the end will contain blood serum medium in the step, so can be on the basis that does not influence final culture effect, reduce the content of foreign protein in the finished product effectively, if be applied to the cultivation of human stem cell, the finished product that then obtain are suitable for clinical direct use, and untoward reaction obviously is less than all the time to use and contains the product that blood serum medium is turned out.
Description of drawings
Fig. 1 is the different culture condition MTT results of the inferior myeloid-lymphoid stem cell of human chorionic;
Fig. 2 is the residual ELISA detected result of the inferior myeloid-lymphoid stem cell bovine serum albumin of human chorionic;
Fig. 3 is the residual ELISA detected result of human marrow mesenchymal stem cell bovine serum albumin;
Fig. 4 is the residual ELISA detected result of human cord blood derived cell bovine serum albumin;
Fig. 5 is that difference is changed the residual ELISA detected result of the inferior myeloid-lymphoid stem cell preparation bovine serum albumin of liquid time human chorionic;
Fig. 6 is the residual ELISA detected result of rat placenta derived mesenchymal stem cell bovine serum albumin.
Embodiment
Following embodiment is described in further detail the present invention:
(1) takes out freeze-stored cell from qualified cell bank, at 37 ℃ of recovery cells that thaw; (2) press 2 * 10 with the DMEM/F12 substratum that contains volume by volume concentration 10% (down together) serum
4/ cm
2Density be inoculated in the culturing bottle, place 37 ℃, saturated humidity, 5% CO2 incubator to cultivate, when cell merges to 80-90%, use the digestive ferment peptic cell, counting; (3) respectively with preparing the DMEM/F12 substratum that contains 10% serum and DMEM/F12 substratum by the suitable concn re-suspended cell; (4) be divided into three groups: change the liquid group with the DMEM/F12 substratum again after containing DMEM/F12 substratum group, the DMEM/F12 substratum group of 10% serum and using the DMEM/F12 culture medium culturing to 70% that contains 10% serum to merge earlier, each group cell is cultivated every group 6 multiple hole with 96 orifice plates; (5) cultivate end and added 10 microlitre MTT in preceding four hours, detect every group of OD value, the high viable count height of representing of OD value to cultivating the MTT method that finishes to know with professional occurrences in human life.
The result: serum-free culture group OD value is minimum; Change liquid group OD value a little less than containing serologic group, but the P value is greater than 0.05, statistics does not have significant difference (Fig. 1)
The amplification and the bovine serum albumin residue detection of the inferior myeloid-lymphoid stem cell of embodiment 2 human chorionics
(1) takes out freeze-stored cell from qualified cell bank, at 37 ℃ of recovery cells that thaw; (2) press 2 * 10 with the DMEM/F12 substratum that contains volume by volume concentration 10% (down together) serum
4/ cm
2Density be inoculated in the culturing bottle, place 37 ℃, saturated humidity, 5% CO2 incubator to cultivate, cell went down to posterity by 1: 3 when 80-90% merges, and went down to posterity through 3 times; (3) reach the DMEM/F12 nutrient solution that changed complete serum-free at 70% o'clock at passage cell degrees of fusion for the third time and cultivated 12 hours, control group does not carry out this and changes the liquid step; (4) use the digestive ferment peptic cell, counting with preparing cytoprotective liquid by the suitable concn re-suspended cell, divides in the sterile chamber of packing into, after the sealing, is placed on proper environment and preserves; (5) getting ELISA method that the part preparation knows with professional occurrences in human life carries out the bovine serum albumin residue and detects.
The result: cell reaches 80-90% and merges changing liquid after 12 hours.The preparation for preparing, bovine serum albumin is residual to be 6.2ng/1 * 10
7Cell control group 37.3ng/1 * 10
7Cell (Fig. 2).
The amplification of embodiment 3 human marrow mesenchymal stem cells and bovine serum albumin residue detection
(1) takes out freeze-stored cell from qualified cell bank, at 37 ℃ of recovery cells that thaw; (2) mix composition, the serum of volume by volume concentration 2-10%, 10 with containing 60%DMEM/F12 nutrient solution and 40%MCDB-201 nutrient solution
-8M dexamethasone (dexamethasone), 0.2-5 * ITS,, 0.1-10mML-glutamine (L-glutaminate), the substratum of 1-100ng/ml human epidermal growth factor and 1-100ng/ml Prostatropin etc. is by 2 * 10
4/ cm
2Density be inoculated in the culturing bottle, place 37 ℃, saturated humidity, 5% CO2 incubator to cultivate, cell went down to posterity by 1: 3 when 80-90% merges, and went down to posterity through 3 times; (3) reach the 60%DMEM/F12 nutrient solution and the 40%MCDB-201 nutrient solution mixed culture medium that changed complete serum-free at 70% o'clock at passage cell degrees of fusion for the third time and cultivated 24 hours, control group does not carry out this and changes the liquid step; (4) use the digestive ferment peptic cell, counting with preparing cytoprotective liquid by the suitable concn re-suspended cell, divides in the sterile chamber of packing into, after the sealing, is placed on proper environment and preserves; (5) getting ELISA method that the part preparation knows with professional occurrences in human life carries out the bovine serum albumin residue and detects.
The result: cell reaches 80-90% and merges changing liquid after 24 hours.The preparation for preparing, bovine serum albumin is residual to be 2.7ng/1 * 10
7Cell, control group 32.7ng/1 * 10
7Cell (Fig. 3).
The amplification and the bovine serum albumin residue detection of embodiment 4 human cord bloods source endothelial progenitor cells
(1) takes out freeze-stored cell from qualified cell bank, at 37 ℃ of recovery cells that thaw; (2) with the M199 substratum of the bFGF of the heparin of VEGF, 10U/ml that contains 20% serum, 10ng/ml and 4ng/ml by 2 * 10
4/ cm
2Density be inoculated in the culturing bottle, place 37 ℃, saturated humidity, 5% CO2 incubator to cultivate, cell went down to posterity by 1: 3 when 80-90% merges, (3) reach at 80% o'clock at the passage cell degrees of fusion and change complete serum-free, the M199 nutrient solution that contains the bFGF of the heparin of VEGF, 10U/ml of 10ng/ml and 4ng/ml was cultivated 24 hours, and control group does not carry out this and changes the liquid step; (4) use the digestive ferment peptic cell, counting with preparing cytoprotective liquid by the suitable concn re-suspended cell, divides in the sterile chamber of packing into, after the sealing, is placed on proper environment and preserves; (5) getting ELISA method that the part preparation knows with professional occurrences in human life carries out the bovine serum albumin residue and detects.
The result: cell reaches 80-90% and merges changing liquid after 24 hours.The preparation for preparing, bovine serum albumin is residual to be 8.3ng/1 * 10
7Cell, control group 52.8ng/1 * 10
7Cell (Fig. 4).
(1) takes out freeze-stored cell from qualified cell bank, at 37 ℃ of recovery cells that thaw; (2) press 2 * 10 with the DMEM/F12 substratum that contains volume by volume concentration 10% (down together) serum
4/ cm
2Density be inoculated in the culturing bottle, place 37 ℃, saturated humidity, 5% CO2 incubator to cultivate, cell went down to posterity by 1: 3 when 80-90% merges, and went down to posterity through 2 times; (3) when the 2nd passage cell enters increased logarithmic phase, divide and to change liquid 4 hours, 12 hours, 24 hours and 4 groups of control groups, control group does not carry out this and changes the liquid step; (4) use the digestive ferment peptic cell, counting with preparing cytoprotective liquid by the suitable concn re-suspended cell, divides in the sterile chamber of packing into, after the sealing, is placed on proper environment and preserves; (5) getting ELISA method that the part preparation knows with professional occurrences in human life carries out the bovine serum albumin residue and detects.
Result: while harvested cell, the preparation for preparing; The detection bovine serum albumin is residual: 4 hours, 12 hours, 24 hours and control group are respectively 24.1ng/1 * 10
7Cell, 6.9ng/1 * 10
7Cell, 3.2ng/1 * 10
7Cell, 35.1ng/1 * 10
7Cell (Fig. 5).
Amplification and the bovine serum albumin residue detection of embodiment 6 rat placenta derived mesenchymal stem cells
(1) takes out frozen rat placenta derived mesenchymal stem cell, at 37 ℃ of recovery cells that thaw; (2) press 2 * 10 with the DMEM/F12 substratum that contains volume by volume concentration (down together) 10% serum
4/ cm
2Density be inoculated in the culturing bottle, place 37 ℃, saturated humidity, 5% CO2 incubator to cultivate, cell went down to posterity by 1: 3 when 80-90% merges, and went down to posterity through 2 times; (3) when the 2nd passage cell entered increased logarithmic phase, experimental group was changed liquid, and control group does not carry out this and changes the liquid step; Use the digestive ferment peptic cell after (4) 12 hours, counting with preparing cytoprotective liquid by the suitable concn re-suspended cell, divides in the sterile chamber of packing into, after the sealing, is placed on proper environment and preserves; (5) getting ELISA method that the part preparation knows with professional occurrences in human life carries out the bovine serum albumin residue and detects.
The result: cell reaches 80-90% and merges changing liquid after 12 hours.The preparation for preparing, bovine serum albumin is residual to be 4.7ng/1 * 107 cell control group 27.7ng/1 * 107 cells (Fig. 6).
What use in the foregoing description is bovine serum, with horse serum also can, no longer give an example.
Though method of the present invention is changed liquid and removed the bovine serum support, the increased logarithmic phase cell is the cytokine of secretory cell growth needs in a large number, can guarantee that cell normally increases; Utilize this cultural method so both to guarantee well amplifying cells of cell, also can significantly reduce the heterologous protein content in the final harvested cell.
In sum, content of the present invention is not limited in the above-described embodiment, and the knowledgeable people in the same area can propose other embodiment easily within technical director's thought of the present invention, but this embodiment comprises within the scope of the present invention.
Claims (16)
1. cell culture processes that reduces heterologous protein residual quantity in the cellular product, it is characterized in that, may further comprise the steps: seed cells in the culturing bottle that contains blood serum medium, place CO2gas incubator to cultivate, when cell enters increased logarithmic phase, will contain blood serum medium and be replaced by serum free medium.
2. the cell culture processes of heterologous protein residual quantity is characterized in that in the minimizing cellular product according to claim 1, and described cell culture processes is an adherent culture.
3. the cell culture processes of heterologous protein residual quantity is characterized in that in the minimizing cellular product according to claim 1, the cell of described cell for have adherent characteristic when cultivating.
4. the cell culture processes of heterologous protein residual quantity is characterized in that in the minimizing cellular product according to claim 1, and described cell will contain blood serum medium again and be replaced by serum free medium after cultivating through going down to posterity.
5. according to the cell culture processes of heterologous protein residual quantity in claim 1,2, the 3 or 4 described minimizing cellular product, it is characterized in that described cell is a stem cell.
6. the cell culture processes of heterologous protein residual quantity is characterized in that in the minimizing cellular product according to claim 5, and described stem cell is a human stem cell.
7. the cell culture processes of heterologous protein residual quantity is characterized in that in the minimizing cellular product according to claim 5, and described stem cell is inferior myeloid-lymphoid stem cell.
8. the cell culture processes of heterologous protein residual quantity is characterized in that in the minimizing cellular product according to claim 6, and described stem cell is a human mesenchymal stem cell.
9. cell culture processes according to claim 8 is characterized in that, described stem cell is the human mesenchymal stem cell from external umbilical cord and placenta source.
10. the cell culture processes of heterologous protein residual quantity in the minimizing cellular product according to claim 1, it is characterized in that, the described blood serum medium that contains is one or more combination among DMEM/F12, DMEM, MCDB-201 or the M199, wherein contains the serum of volume ratio 2%～20%.
11. the cell culture processes of heterologous protein residual quantity is characterized in that in the minimizing cellular product according to claim 10, described serum is bovine serum or horse serum.
12. the cell culture processes of heterologous protein residual quantity is characterized in that in the minimizing cellular product according to claim 10, also adding in the described substratum has cytokine.
13. the cell culture processes of heterologous protein residual quantity is characterized in that in the minimizing cellular product according to claim 12, described cytokine is the one or more combination among EGF, bFGF or the VEGF.
14. cell culture processes according to claim 13 is characterized in that, the content of described EGF, bFGF or VEGF is 1～100ng/ml.
15. the cell culture processes of heterologous protein residual quantity is characterized in that in the minimizing cellular product according to claim 1, described carbonic acid gas culture condition is 37 ℃, saturated humidity, gas concentration lwevel 5%, and oxygen concn is 1%～20%.
16. the cell culture processes of heterologous protein residual quantity is characterized in that in the minimizing cellular product according to claim 1, the density of described cell inoculation when culturing bottle is (0.1～2) * 10
4/ cm
2
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103421739A (en) * | 2013-05-24 | 2013-12-04 | 北京汉氏联合生物技术有限公司 | Method for separating umbilical cord mesenchymal stem cell effectively |
| CN108753736A (en) * | 2018-05-04 | 2018-11-06 | 上海荣盛生物药业有限公司 | Produce the method and its application of varicella virus stoste |
| CN109536443A (en) * | 2018-12-10 | 2019-03-29 | 天津长和生物技术有限公司 | Mescenchymal stem cell, cell culture medium and cultural method and application |
| CN111893155A (en) * | 2020-07-10 | 2020-11-06 | 深圳市华启生物科技有限公司 | Method for producing recombinant protein |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103421739A (en) * | 2013-05-24 | 2013-12-04 | 北京汉氏联合生物技术有限公司 | Method for separating umbilical cord mesenchymal stem cell effectively |
| CN103421739B (en) * | 2013-05-24 | 2015-09-09 | 北京汉氏联合生物技术有限公司 | A kind of method of high efficiency separation umbilical cord mesenchymal stem cells |
| CN108753736A (en) * | 2018-05-04 | 2018-11-06 | 上海荣盛生物药业有限公司 | Produce the method and its application of varicella virus stoste |
| CN108753736B (en) * | 2018-05-04 | 2022-03-01 | 上海荣盛生物药业股份有限公司 | Method for preparing varicella virus stock solution and application thereof |
| CN109536443A (en) * | 2018-12-10 | 2019-03-29 | 天津长和生物技术有限公司 | Mescenchymal stem cell, cell culture medium and cultural method and application |
| CN111893155A (en) * | 2020-07-10 | 2020-11-06 | 深圳市华启生物科技有限公司 | Method for producing recombinant protein |
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Open date: 20101124 |