High-throughput automatic continuous cell culture method and application thereof

A cell culture, high-throughput technology, applied in animal cells, biochemical equipment and methods, biomaterial sampling methods, etc., can solve problems such as target protein retention, blocking membrane pores, and continuous culture is not considered.

A cell culture, high-throughput technology, applied in animal cells, biochemical equipment and methods, biomaterial sampling methods, etc., can solve problems such as target protein retention, blocking membrane pores, and continuous culture is not considered.

CN111139216APending Publication Date: 2020-05-12SHANGHAI WUXI BIOLOGIC TECH CO LTD
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  • High-throughput automatic continuous cell culture method and application thereof
  • High-throughput automatic continuous cell culture method and application thereof
  • High-throughput automatic continuous cell culture method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] Taking the selection of recombinant CHO-K1 cells to produce a certain monoclonal antibody of IgG1 type as an example (the CHO-K1 is a proprietary cell line of Shanghai WuXi Biotechnology Company), the implementation process is described in detail.

[0109] (1) Resuscitate a CHO-K1 cell line required in this example: use CD CHO medium (purchased from ThermoFisher) for culture, and the culture container is a 125ml-1L shake flask (purchased from Corning). The cell line is inoculated in a culture vessel filled with CDCHO medium, and cultured in a Kuhner or INFORS Multitron constant temperature shaker. Shaker culture setting parameters are temperature 36.5°C, humidity 80%, CO 2 The concentration is 60%, and the rotation speed of the shaker is 125rpm.

[0110] After recovery, every 2-4 days, wait until the cell density grows to 1.0-3.0×10 6 cells / ml range to the next generation. When passing to the 4th to 6th generation, the Advanced CHO production medium (purchased from T...

Embodiment 2

[0121] Taking the perfusion medium screening of two CHO-K1 clones containing different foreign protein coding genes as an example, the implementation process is described in detail

[0122] (1) Resuscitate two different CHO-K1 cell line clone A and clone B required in this embodiment, wherein the protein type produced by clone A is Fc fusion protein, and the protein type produced by clone B is IgG4 monoclonal antibody: The clones A and B were respectively inoculated in culture containers containing CD CHO medium (purchased from Thermo Fisher), and the culture vessels were 125ml-1L shake flasks (purchased from Corning), and placed in a Kuhner or INFORS Multitron constant temperature shaker. The culture was carried out in the bed, and the shaker culture setting parameters were temperature 36.5°C, humidity 80%, CO 2 The concentration is 60%, and the rotation speed of the shaker is 125rpm.

[0123] After recovery, every 2-4 days, wait until the cell density grows to 1.0-3.0×10 6...

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Abstract

The invention discloses a high-throughput automatic continuous cell culture method and application thereof. The method comprises the following steps: (1) conducting inoculation and culture on a provided cell culture; (2) conducting cell sedimentation, and removing supernate in reactors; (3) adding a fresh culture medium; (4) calculating a release rate (B) according to the density of cells in the supernate, and maintaining the density of the cells; and (5) repeating the steps (2) to (4) until continuous cell culture is completed. According to the high-throughput automatic continuous cell culture method and application thereof, compared with a traditional perfusion reactor, a provided continuous cell culture model is high in throughput, high in automation degree, small in culture volume andlow in cost; and compared with culture by means of shaking, the high-throughput automatic continuous cell culture method has strong rector characteristics, strong culture result representation, low probability of personal errors and high reliability. Moreover, an additional cell retention device in the perfusion reactor does not need to be used in the high-throughput automatic continuous cell culture method, so that the problem that the cell retention device is liable to be blocked is solved.

Description

[0001] Priority and related applications [0002] This application claims the priority of the Chinese patent application 201811295263.4, filed on November 1, 2018, entitled High-throughput, automated continuous cell culture method and its application, the entire content of this application including the appendix is ​​incorporated by reference into this application . technical field [0003] The invention relates to the fields of biopharmaceuticals and biotechnology, in particular to the technical field of animal cell culture, and in particular to a high-throughput and automatic continuous cell culture method and its application in this field. Background technique [0004] After decades of development, cell culture technology has become a mature technology for the production of many important proteins (especially those glycosylated complex macromolecular proteins). These proteins are mostly used in the prevention and treatment of some serious human diseases, such as cancer, ...

Claims

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Application Information

Patent Timeline
12 May 2020
Publication
CN111139216A
IPC
C12N5/07; C12M1/36; C12M1/00
CPC
C12N5/06; C12M33/22; C12M41/48
Inventors
王振守; 李敏