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Hybridoma fluid nutrient medium free of feeder cells

A hybridoma cell and liquid culture medium technology is applied in the field of feeder-free hybridoma cell liquid culture medium, which can solve the problems of feeder cell blocking, influence observation, pollution, etc., and achieve the effect of simplified procedures

Active Publication Date: 2018-07-03
YUNNAN ANIMAL SCI & VETERINARY INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are the following bottlenecks in the preparation and application of feeder cells: first, the preparation conditions of feeder cells are harsh, and if you do not pay attention, it is easy to bring in other pollution; secondly, the preparation of feeder cells must be in line with the time of cell fusion. The best opportunity is lost; thirdly, feeder cells are not conducive to the growth of hybridoma cells
Because the feeder cells grow directly under the hybridoma cells, when observed with an inverted microscope, the feeder cells will block and refract light, affecting observation; fourth, the feeder cells are easily mistaken for hybridoma cells by inexperienced technicians

Method used

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  • Hybridoma fluid nutrient medium free of feeder cells
  • Hybridoma fluid nutrient medium free of feeder cells
  • Hybridoma fluid nutrient medium free of feeder cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Follow the steps below to prepare the medium for this application:

[0025] 1. Using T75 cell culture flasks (CORNING, USA), the ratio of 1 flask to 5 flasks was used to passage normally overgrown L929 cells (a kind of mouse fibroblast, purchased from Kunming Institute of Zoology) and C6 / 36 cells. Cells (a kind of mosquito cell, originally retained in this unit), in which L929 cells are passaged using RPMI1640 complete medium (Gibco, USA), and C6 / 36 cells are passaged using M199 complete medium (Gibco, USA);

[0026] 2. When the cells are full, that is, the gaps between the cells have been filled by their own cells, collect the culture fluids of the two types of cells separately, and use a speed of 1000 RPM to precipitate the cell debris, and then filter the supernatant with a 0.22 micron filter membrane. Filter, pack separately in sterile bottles, and store at 4°C after packing;

[0027] 3. Take a 100ml sterile reagent bottle and add 20ml each of the two cell culture ...

Embodiment 2

[0033] Follow the steps below to prepare the medium for this application:

[0034] 1. Use T75 cell culture flasks (CORNING, USA) respectively, according to the ratio of 1 vial to 4 vials, to normally passage the overgrown L929 cells (a kind of mouse fibroblast) and C6 / 36 cells (a kind of mosquito cells) , wherein L929 cells are subcultured using DMEM complete medium (Gibco, USA), and C6 / 36 cells are subcultured using M199 complete medium (Gibco, USA);

[0035] 2. After the cells are overgrown, collect the culture fluids of the two types of cells separately, precipitate the cell debris at a speed of 1000 RPM, and then filter the supernatant to sterilize with a 0.22 micron filter membrane;

[0036] 3. Take a 100ml sterile reagent bottle and add 20ml each of the two cell culture solutions prepared in the previous step;

[0037] 4. Add 10 μl of 10% ethyl ciprofloxacin solution (Bayer, Germany);

[0038] 5. Add 50ml 1×DMEM (Gibco, USA);

[0039] 6. Add fetal bovine serum (Gibco,...

Embodiment 3

[0043] Taking the preparation of bluetongue virus monoclonal antibody as an example, the culture medium of the present application was used to prepare hybridoma cells secreting the monoclonal antibody.

[0044] 1. According to the conventional method, immunize BABL / c mice (from Kunming Medical University) with purified bluetongue virus serum type 1 antigen (preserved by the unit) several times, and cultivate SP2 / 0 with RPMI1640 growth medium as required After the cells are mature, spleen lymphocytes and SP2 / 0 cells of mice are taken for fusion. Among them, feeder cells do not need to be prepared. Because it is not the key point, it will not be described here. For specific methods, refer to pages 940-946 of the first edition of "Principles and Techniques of In Vitro Culture" published by Science Press in February 2001;

[0045] 2. Add RPMI1640 growth medium to the cell suspension just after fusion to 40ml, shake well and centrifuge at 1000RPM for 10 minutes;

[0046] 3. With ...

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Abstract

The invention provides a hybridoma fluid nutrient medium free of feeder cells. The hybridoma fluid nutrient medium free of feeder cells contains a, 20-30% in volume of a culture fluid culturing L929 cells; b, 20-30% in volume of a culture fluid culturing L6 / 36 cells; and c, 8-20% in volume of fetal calf serum. Compared with the prior art, the tedious process of preparing feed cells is cancelled, and the synthesized hybridoma are more easily observed and can grow and breed normally.

Description

technical field [0001] The present application relates to the technical field of animal cell culture, specifically to the technical field of hybridoma cell culture, and more specifically to a feeder-free hybridoma liquid culture medium. Background technique [0002] During the synthetic process of hybridoma cells, the culture medium of hybridoma cells is very critical. If the nutrients in the culture medium are not enough, even if the hybridoma cells are successfully produced by cell fusion, they cannot proliferate normally, and even die quickly. So many researchers are studying the optimal culture medium for hybridoma cells. [0003] At present, even the best hybridoma cell culture medium is still insufficient in growth factors, and feeder cells must be added to the culture space, because feeder cells can secrete many growth factors that hybridoma cells need to grow. However, there are the following bottlenecks in the preparation and application of feeder cells: first, th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20
CPCC12N5/163
Inventor 高林李乐李华春何于雯廖德芳姚俊
Owner YUNNAN ANIMAL SCI & VETERINARY INST
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