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Lentivirus adherent cell culture process

A technology of adherent cells and culture process, applied in the direction of virus, animal cells, culture process, etc., can solve the problems of increasing the risk of cell contamination, difficulty in quality inspection of transfection reagents, large batch differences, etc., to reduce cell contamination risk, shorten the time to pass, and simplify the operation

Pending Publication Date: 2021-05-11
苏州博腾生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to aim at the existing lentivirus adherent cell culture process described in the background technology, which has high cost, large batch difference, needs to change the liquid before and after transfection, complicated operation, and increases the risk of cell contamination. Difficulty in quality inspection of dyeing reagents, etc., providing a lentiviral adherent cell culture process that can solve the aforementioned problems

Method used

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  • Lentivirus adherent cell culture process
  • Lentivirus adherent cell culture process
  • Lentivirus adherent cell culture process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] A lentivirus adherent cell culture process, which comprises the following steps:

[0023] S1 HEK293T cells were adaptively cultured in a culture device equipped with VP-SFM medium and 0.5% fetal bovine serum.

[0024] S2 Step S1 Acclimate well-cultured HEK293T cells to establish a cell bank.

[0025] In S3, HEK293T cells in the logarithmic growth phase were inoculated into the cell factory, and the cell inoculation density was 2×10 4 / cm 2 , the cell factory is Nunc's CF-2 cell factory.

[0026] S4 In the four-plasmid system, HEK293T cells were transfected, PEIpro method was used to transfect HEK293T cells, and PEIpro: DNA was transfected at a ratio of 1:1.

[0027] After 50 hours of S5 transfection, the cell culture supernatant was harvested, and the cell viability was above 85%, and the titer of the transfection supernatant was 5×10 6 Lentiviral cells above TU / mL.

Embodiment 2

[0029] A lentivirus adherent cell culture process, which comprises the following steps:

[0030] S1 HEK293T cells were adaptively cultured in a culture device containing VP-SFM medium and 1% fetal bovine serum.

[0031] S2 Step S1 Acclimate well-cultured HEK293T cells to establish a cell bank.

[0032] In S3, HEK293T cells in the logarithmic growth phase were inoculated into the cell factory, and the cell inoculation density was 3×10 4 / cm 2 , the cell factory is Nunc's CF-2 cell factory.

[0033] S4 In the four-plasmid system, HEK293T cells were transfected, PEIpro method was used to transfect HEK293T cells, and PEIpro: DNA was transfected at a ratio of 1:1.

[0034] After 100 hours of S5 transfection, the cell culture supernatant was harvested, and the cell viability was over 90%, and the titer of the transfection supernatant was 5×10 6 Lentiviral cells above TU / mL.

Embodiment 3

[0036] A lentivirus adherent cell culture process, which comprises the following steps:

[0037] S1 HEK293T cells were adaptively cultured in a culture device containing VP-SFM medium and 1.5% fetal bovine serum.

[0038] S2 Step S1 Acclimate well-cultured HEK293T cells to establish a cell bank.

[0039] In S3, HEK293T cells in the logarithmic growth phase were inoculated into the cell factory, and the cell inoculation density was 4×10 4 / cm 2 , the cell factory is Nunc's CF-2 cell factory.

[0040] S4 In the four-plasmid system, HEK293T cells were transfected, PEIpro method was used to transfect HEK293T cells, and PEIpro: DNA was transfected at a ratio of 1:1.

[0041] After 100 hours of S5 transfection, the cell culture supernatant was harvested, and the cell viability was over 90%, and the titer of the transfection supernatant was 5×10 6 Lentiviral cells above TU / mL.

[0042] In the above schemes of Examples 1-3, in addition to using the CF-2 cell factory, six-well pl...

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Abstract

The invention discloses a lentivirus adherent cell culture process. The process comprises the following steps: S1, carrying out adaptive domestication culture on HEK293T cells in a culture device filled with a VP-SFM culture medium and low-concentration fetal bovine serum; S2, establishing a cell bank for the HEK293T cells which are well domesticated and cultured in the step S1; S3, inoculating the HEK293T cells in the logarithmic phase into a cell factory; S4, in a four-plasmid system, carrying out transfection on the HEK293T cell; and S5, after 50-100 hours of transfection, harvesting a cell culture supernatant, so as to obtain the lentiviral cells with the cell viability of more than 85% and the transfection supernatant titer of more than 5*10 <6> TU / mL. According to the lentivirus adherent cell culture process disclosed by the invention, low-concentration fetal calf serum is adopted, so that the production cost is reduced; a PEIpro method is used for transfection, so that the operation process 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 that the cells are contaminated is reduced, and the process has great advantages for GMP production; and the investment cost of production equipment is reduced.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a lentivirus adherent cell culture process. Background technique [0002] Lentivirus is commonly used in Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T), which has the advantages of high transduction efficiency, integration of T cell genome and continuous expression of target proteins. Nowadays, CAR-T production is mostly based on lentiviral vector-mediated CAR gene transfer. [0003] At present, most of the processes used to produce lentivirus are based on the adherent cell culture process (such as HEK293T) that relies on fetal bovine serum (FBS). The specific process is: in the incubator, use DMEM high glucose medium (gibco) +10 The HEK293T cells were domesticated and cultured with % fetal bovine serum, and the cultured cells were exchanged for medium treatment, and then transfected with a transfection reagent, usually using calcium phosphate method, liposome method (such a...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N5/071
CPCC12N7/00C12N5/0686C12N2740/10051C12N2500/84
Inventor 胡迪超马红隋礼丽孔令洁
Owner 苏州博腾生物制药有限公司
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