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B-cell cultivation method

A cell culture, B cell technology, applied in the field of B cell culture

Active Publication Date: 2019-08-13
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But in hybridoma technology, only a portion of the B cells obtained from the immunized experimental animal can fuse and multiply

Method used

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  • B-cell cultivation method
  • B-cell cultivation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0938] Culture of EL-4 B5 cells

[0939] Thaw frozen EL-4B5 cells quickly in a 37°C water bath and dilute with 10 ml of EL-4B5 medium. After centrifugation at 300xg for 10 minutes, the supernatant was discarded and the pellet was resuspended in 1ml of culture medium.

[0940] EL-4 B5 cells were seeded in T175 culture flasks at a cell density of 8x 10 cells / ml. Cell density was measured every two days and adjusted to 8x 10 4 cells / ml. Cells have a doubling time of about 18 hours.

[0941] Cells were harvested and adjusted to 1x10 6 The cell density of cells / ml was followed by gamma-irradiation at 50Gy.

Embodiment 2

[0943] Co-cultivation of B cells and EL-4 B5 cells

[0944] In 200 μl / well EL-4 B5 medium (containing Pansorbin Cells (SAC) (Calbiochem (Merck), Darmstadt, Deutschland), EL-4 B5 cells (5×10 4 / well) and rabbit thymocyte supernatant (TSN) or cytokine mixture (CM) with PMA) in 96-well plates in 5% CO 2 Single-cell sorted B cells were cultured in an atmospheric incubator at 37°C for 7 days. B cell culture supernatants were removed for screening and cells were collected immediately for variable region gene cloning or frozen at -80°C in 100 μl of RLT buffer (Qiagen, Hilden, Germany).

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Abstract

Herein is reported a method for co-cultivating B-cells in the presence of phorbol myristate acetate, IL-1beta, TNFalpha, IL-2, IL-10 and IL-6.

Description

[0001] Here we report a method for the co-culture of B cells with feeder cells, in which a defined synthetic feeder mixture is applied. This method can be used, for example, to obtain the amino acid sequence of at least the variable domain of a monoclonal antibody secreted by co-cultured B cells, and for producing antibodies. Background technique [0002] To obtain monoclonal antibody-secreting cells, the hybridoma technique developed by Koehler and Milstein is widely used. But in hybridoma technology, only a portion of the B cells obtained from the immunized experimental animal can fuse and multiply. The source of B cells is usually an organ of the experimental animal being immunized, such as the spleen. [0003] In 1984 Zubler et al. started to develop different methods for obtaining monoclonal antibody secreting cells (see e.g. Eur. J. Immunol. 14 (1984) 357-63, J. Exp. Med. 160 (1984) 1170-1183) . Here, B cells are obtained from the blood of the immunized experimental a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0781C12N5/0786C12N5/0783C12N5/078C07K16/06C12N5/16C07K16/00
CPCC12N5/0635C12N2501/2301C12N2501/2302C12N2501/2306C12N2501/231C12N2501/25C12N2501/727C12N2501/999C12N2502/1107C12N2502/70C12P21/00
Inventor F·容格
Owner F HOFFMANN LA ROCHE & CO AG
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