High-throughput cell suspension culture method
A culture method and cell suspension technology, which is applied in the field of high-throughput cell suspension culture in 24-well culture plates, can solve the problems of low throughput and high cost of instrument consumables, and achieve the effects of good suspension, shortening the research and development cycle, and saving culture medium.
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Embodiment 1
[0048] Example 1: Screening of the main factors affecting the high-throughput cell suspension culture of the present invention
[0049] At a temperature of 36.5°C and a relative humidity of 80%, CO 2 Under the condition of 5% (v / v) concentration, fix the 24-well culture plate in the humidor, and reinforce the absorbent cotton or sponge soaked with water around to ensure the stability of the humidity environment of the humidor, and replace it every 2 to 3 days once to prevent the growth of microorganisms such as bacteria, mold, etc. Using CHO blank cells as cell lines, the key parameters affecting cell growth such as seeding density, culture volume, and shaker rotation speed were screened. Two parallel study groups were set up in each group of experiments, and the parameter levels were set as shown in Table 1:
[0050] Table 1 Influencing factors and level design
[0051]
[0052] (1) Research on culture volume
[0053] Take 0.3×10 6 Cells / mL Cell Density CHO cells were...
Embodiment 2
[0065] Example 2: Screening of recombinant anti-VEGFR2 antibody cell lines using the present invention
[0066] (1) Construction of recombinant plasmid of anti-VEGFR2 antibody
[0067] The full sequence of the antibody was directly synthesized by a gene synthesis company (reference: WO03075840A2, WHO DrugInformation Vol 23, No. 3, 2009), and the heavy chain and light chain genes were cloned to express In the plasmid pCHO 1.0, after the sequencing was verified to be correct, the plasmid was extracted by using the plasmid intermediate extraction kit NucleoBond Xtra Midi (purchased from Macherey-Nagel Company, Cat. No.: 740410.50), and quantified using the Denovix DS-11 Micro UV Nucleic Acid Detector,- Store at 80°C for later use.
[0068] The specific construction process of the recombinant plasmid is as follows: the DNA sequences of the heavy chain and light chain of the anti-VEGFR2 monoclonal antibody are synthesized, and the 5' and 3' ends contain EcoRV / PacI and AvrII / BstZ17...
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