CN112779226A - Culture process of lentivirus adherent cells - Google Patents
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- 241000713666 Lentivirus Species 0.000 title claims abstract description 44
- 230000008569 process Effects 0.000 title claims abstract description 36
- 230000001464 adherent effect Effects 0.000 title claims abstract description 17
- 238000001890 transfection Methods 0.000 claims abstract description 32
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 26
- 238000004113 cell culture Methods 0.000 claims abstract description 12
- 239000012894 fetal calf serum Substances 0.000 claims abstract description 9
- 238000010899 nucleation Methods 0.000 claims abstract description 8
- 230000003833 cell viability Effects 0.000 claims abstract description 7
- 230000003698 anagen phase Effects 0.000 claims abstract description 6
- 239000012228 culture supernatant Substances 0.000 claims abstract description 6
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- 238000003306 harvesting Methods 0.000 claims abstract description 6
- 239000013612 plasmid Substances 0.000 claims abstract description 6
- 239000006228 supernatant Substances 0.000 claims abstract description 6
- 230000003044 adaptive effect Effects 0.000 claims abstract description 3
- 238000004115 adherent culture Methods 0.000 claims description 19
- 238000012258 culturing Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 12
- 210000004027 cell Anatomy 0.000 description 73
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- 229920002873 Polyethylenimine Polymers 0.000 description 5
- 229910000389 calcium phosphate Inorganic materials 0.000 description 4
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- 235000011010 calcium phosphates Nutrition 0.000 description 4
- 238000011194 good manufacturing practice Methods 0.000 description 4
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- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 4
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- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
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Abstract
The invention discloses a culture process of lentivirus adherent cells, which comprises the following steps: s1, HEK293T cells are subjected to adaptive acclimation culture in a culture device filled with VP-SFM culture medium and low-concentration fetal calf serum; s2 step S1 domestication of well-cultured HEK293T cells to establish a cell bank; s3 seeding HEK293T cells in logarithmic growth phase into the cell factory; s4 HEK293T cells were transfected in a four plasmid system; s5 transfection for 50-100 h, harvesting cell culture supernatant, and obtaining cell viability of more than 85% and transfection supernatant titer of 5 × 106Tu/mL or more of lentivirus cells. The culture process of the lentivirus adherent cells adopts low-concentration fetal calf serum, so that the production cost is reduced; the PEIpro method is used for transfection, so that the operation flow is simplified; the culture medium does not need to be replaced before and after cell transfection, so that the operation is greatly simplified, the risk of cell pollution is reduced, and the method has great advantages for GMP production; reduce the production facilityThe cost of the spare investment.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a culture process of lentivirus adherent cells.
Background
Lentiviruses are commonly used in Chimeric Antigen Receptor-T Cell therapy (CAR-T), which has the advantages of high transduction efficiency, integration of T Cell genome and sustained expression of target proteins. Today CAR-T production is mostly based on lentiviral vector mediated CAR gene transfer.
Currently, the processes for producing lentiviruses are mostly based on adherent cell culture processes (e.g. HEK 293T) relying on Fetal Bovine Serum (FBS), and the specific processes are as follows: in an incubator, the HEK293T cells were acclimatized and cultured in a DMEM high-glucose medium (gibco) +10% fetal bovine serum, the cultured cells were subjected to fluid exchange, and then transfected with a transfection reagent, usually by a calcium phosphate method, a liposome method (e.g., Lipofectamine2000 of Thermo corporation) or a polyethyleneimine method, and after the transfection was completed, the cells were again subjected to fluid exchange. The existing adherent cell culture process for producing the lentivirus has the defects that the fetal bovine serum with the concentration of 10% is adopted in the prior art, and the high-concentration fetal bovine serum FBS means high cost; moreover, because of the difference of batches and production places of Fetal Bovine Serum (FBS), the FBS with high concentration dependence means that the risk of the product with the difference between batches is higher, and thus the stable quality and the stable shape of the product are influenced; secondly, the cultured cells need to be subjected to fluid exchange treatment before and after transfection, so as to improve the transfection efficiency and reduce the cytotoxicity after transfection, which not only increases the complexity of operation, but also increases the risk of pollution, which poses a greater challenge to the scale-up production of medicines. In addition, the transfection reagent used for the transfection of adherent cells is usually a calcium phosphate method, a liposome method (e.g., Lipofectamine2000 from Thermo), or a polyethyleneimine method (e.g., PEI or PEImax), calcium phosphate and PEI or PEImax usually need to be self-prepared, which increases the time for passing quality detection, and the liposome in the transfection reagent has no permission of Good Manufacturing Practice (GMP), so that the subsequent application is difficult.
Another cell culture process for lentivirus is a suspension cell culture process, lentivirus can grow in a medium (CD media) with specific chemical compositions independent of Fetal Bovine Serum (FBS), which has the advantages of easy scale-up production, and especially has greater advantages for the requirements of 50L, 200L and 1000L scale, but the titer is usually not as high as that of an adherent cell process, the investment of hardware equipment (cell culture shaker, bioreactor, biochemical detector and the like) is usually more expensive, a downstream purification process usually uses deep filtration, the cost of the used membrane material is also higher, and the recovery rate of lentivirus is usually not as high as that of the adherent cell, so that only a few companies develop mature processes.
Disclosure of Invention
The invention aims to provide a culture process of lentivirus adherent cells, which can solve the problems that the existing culture process of the lentivirus adherent cells in the background art has high cost and large batch difference, needs to change liquid before and after transfection, has complex operation, increases the risk of cell pollution, is difficult to detect the quality of a transfection reagent and the like.
In order to achieve the purpose, the invention is realized by the following technical scheme: the culture process of lentivirus adherent cells is characterized in that: which comprises the following steps:
S1, HEK293T cells are subjected to adaptive acclimation culture in a culture device filled with VP-SFM culture medium and low-concentration fetal calf serum;
s2 step S1 domestication of well-cultured HEK293T cells to establish a cell bank;
s3 seeding HEK293T cells in logarithmic growth phase into the cell factory;
s4 HEK293T cells were transfected in a four plasmid system;
s5 transfection for 50-100 h, harvesting cell culture supernatant, and obtaining cell viability of more than 90% and transfection supernatant titer of 5 × 106Tu/mL or more of lentivirus cells.
In a further embodiment, in step S1, the concentration of fetal bovine serum is 0.5% to 1.5%.
Further, in the step S3, the cell concentration of the HEK293T cells inoculated into the cell factory is 2X 104～6×104/cm2。
In a further embodiment, the cell factory in step S3 is the CF-2 cell factory of Nunc.
Further, in step S4, HEK293T cells were transfected using the PEipro method, DNA 1: 1, transfection is performed.
The culture process of lentivirus adherent cells has the following advantages: 1) compared with the scheme of domesticating and culturing 10% of fetal calf serum in a culture medium in the prior art, the culture process of the lentivirus adherent cells adopts the fetal calf serum with low concentration, usually 0.5-1.5%, reduces the dependence of cells on the fetal calf serum, can reduce the production cost, and also reduces the risk of influence of batch difference and production place difference of the fetal calf serum on the product quality; 2) compared with the scheme of performing cell transfection by using a calcium phosphate method, a liposome method or a polyethyleneimine method in the prior art, the lentivirus adherent cell culture process of the invention has the advantages that the PEIpro method is used for transfection, so that the operation flow is simplified, the GMP specification is met, and the passing time of quality detection can be shortened; 3) according to the lentivirus adherent cell culture process, a culture medium does not need to be replaced before and after cell transfection, so that the operation is greatly simplified, the risk of cell contamination is reduced, and the lentivirus adherent cell culture process has great advantages for GMP production; 4) the culture process of the slow virus adherent cells saves the necessary hardware equipment such as a peristaltic pump, a liquid storage tank, a liquid distribution tank, a laminar flow hood and the like for liquid exchange before and after transfection, so compared with the existing adherent production process, the used hardware equipment is less than that of the traditional adherent process, and the input cost of the production equipment is reduced.
Drawings
FIG. 1 is a schematic view of the lentivirus titer obtained by selecting different fetal bovine serum concentrations in step S1 using the lentivirus adherent cell culture process of the present invention.
FIG. 2 is a bar graph showing the titer of lentiviruses obtained by setting different cell seeding densities in step S3 using the lentivirus adherent cell culture process of the present invention.
FIG. 3 is a bar graph showing the viability of lentiviruses obtained in step S3 using different cell factories using the lentivirus-adherent cell culture process of the present invention.
Detailed Description
The technical solutions of the present invention are described clearly and completely by the following embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A lentivirus adherent cell culture process, comprising the following steps:
s1 HEK293T cells were acclimatized in culture devices containing VP-SFM medium and 0.5% fetal bovine serum.
S2 step S1 domesticates well-cultured HEK293T cells to establish cell banks.
S3 HEK293T cells in logarithmic growth phase were seeded at a cell factory density of 2X 104/cm2The cell factory was Nunc's CF-2 cell factory.
S4 HEK293T cells were transfected in a four plasmid system using the PEIpro method for transfection of HEK293T cells, PEIpro: DNA at 1: 1, transfection is performed.
S5 transfection for 50h, harvesting cell culture supernatant to obtain cell activity more than 85%, transfection supernatant titer 5 × 106Tu/mL or more of lentivirus cells.
Example 2
A lentivirus adherent cell culture process, comprising the following steps:
s1 HEK293T cells were acclimatized in culture devices containing VP-SFM medium and 1% fetal bovine serum.
S2 step S1 domesticates well-cultured HEK293T cells to establish cell banks.
S3 HEK293T cells in logarithmic growth phase were seeded at a cell factory density of 3X 104/cm2The cell factory was Nunc's CF-2 cell factory.
S4 HEK293T cells were transfected in a four plasmid system using the PEIpro method for transfection of HEK293T cells, PEIpro: DNA at 1: 1, transfection is performed.
S5 transfection for 100h, harvesting cell culture supernatant to obtain cell activity more than 90%, transfection supernatant titer 5 × 10 6Tu/mL or more of lentivirus cells.
Example 3
A lentivirus adherent cell culture process, comprising the following steps:
s1 HEK293T cells were acclimatized in culture devices containing VP-SFM medium and 1.5% fetal bovine serum.
S2 step S1 domesticates well-cultured HEK293T cells to establish cell banks.
S3 HEK293T cells in logarithmic growth phase were seeded at 4X 10 cell seeding density in cell factory4/cm2The cell factory was Nunc's CF-2 cell factory.
S4 HEK293T cells were transfected in a four plasmid system using the PEIpro method for transfection of HEK293T cells, PEIpro: DNA at 1: 1, transfection is performed.
S5 transfection for 100h, harvesting cell culture supernatant to obtain cell activity more than 90%, transfection supernatant titer 5 × 106Tu/mL or more of lentivirus cells.
In the protocols of examples 1 to 3 described above, six-well plates and T75 cell culture vessels can be used in addition to the CF-2 cell factory.
As shown in fig. 1, in the lentivirus adherent cell culture process of the present invention, different fetal bovine serum concentrations are selected in step S1, and finally the titers of the obtained lentiviruses are different, and it can be seen that the titer of the obtained lentivirus is the highest when the fetal bovine serum concentration is 1%.
As shown in FIG. 2, in the lentivirus adherent cell culture process of the present invention, different cell seeding densities are set in step S3, and the titers of the finally obtained lentiviruses are different, and it can be seen that the cell seeding density is 4X 104/cm2At that time, the titer of the obtained lentivirus reached the highest.
As shown in fig. 3, in the lentivirus adherent cell culture process of the present invention, in step S3, different cell factories are used, and finally obtained lentiviruses have different cell viability rates, and it can be seen that, when a T75 cell culture container is used, the cell viability rate is the highest and can reach more than 90%, when a six-well plate cell culture container is used, the cell viability rate can reach more than 89%, and when a CF-2 cell factory is used, the cell viability rate can reach more than 86.
Experimental data show that the lentivirus adherent cell culture process has high cell survival rate of over 86 percent, and the obtained lentivirus has high titer of 5 multiplied by 106 TU/mL。
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (5)
1. A process for culturing lentivirus adherent cells, which is characterized in that: which comprises the following steps:
s1, HEK293T cells are subjected to adaptive acclimation culture in a culture device filled with VP-SFM culture medium and low-concentration fetal calf serum;
s2 step S1 domestication of well-cultured HEK293T cells to establish a cell bank;
s3 seeding HEK293T cells in logarithmic growth phase into the cell factory;
s4 HEK293T cells were transfected in a four plasmid system;
s5 transfection for 50-100 h, harvesting cell culture supernatant, and obtaining cell viability of more than 85% and transfection supernatant titer of 5 × 106Tu/mL or more of lentivirus cells.
2. The lentiviral adherent cell culture process of claim 1, wherein: in step S1, the concentration of fetal calf serum is 0.5% -1.5%.
3. The lentiviral adherent cell culture process of claim 1,the method is characterized in that: in step S3, the seeding density of HEK293T cells in the cell factory is 2 × 104～6×104/cm2。
4. The lentiviral adherent cell culture process of claim 1, wherein: the cell factory in step S3 is the CF-2 cell factory of Nunc.
5. The lentiviral adherent cell culture process of claim 1, wherein: in step S4, HEK293T cells were transfected using PEIpro method with DNA at 1: 1, transfection is performed.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114214287A (en) * | 2021-12-28 | 2022-03-22 | 广州泛恩生物科技有限公司 | Liquid exchange medium and application thereof |
| WO2023072178A1 (en) * | 2021-10-27 | 2023-05-04 | Porton Advanced Solutions Ltd. | Methods for developing a cell line for producing virus in suspension cell culture |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2023072178A1 (en) * | 2021-10-27 | 2023-05-04 | Porton Advanced Solutions Ltd. | Methods for developing a cell line for producing virus in suspension cell culture |
| CN114214287A (en) * | 2021-12-28 | 2022-03-22 | 广州泛恩生物科技有限公司 | Liquid exchange medium and application thereof |
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