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A serum-free medium adapted to pk-15 full suspension growth, a preparation method thereof, and a full suspension acclimation method applied to pk-15 cells

A serum-free medium, PK-15 technology, applied in the field of cell engineering, can solve the problems of inability to support full suspension culture of PK-15 cells, no growth, cell aggregation, etc., to avoid costs and digestion processes, and enhance stability. The effect of reducing the production of ammonia

Active Publication Date: 2021-03-05
SICHUAN BAINUOJI TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the medium used in the expansion stage of PK-15 cell culture is a conventional medium such as MEM and DMEM with a serum concentration of 5%-10%, or a low-serum or serum-free medium, but these mediums are all It is only suitable for adherent culture and cannot support the full suspension culture of PK-15 cells; this is because the cells will be seriously agglomerated and not grow or grow slowly when using ordinary conventional medium or serum-free medium in the process of culturing PK-15 cells And other issues

Method used

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  • A serum-free medium adapted to pk-15 full suspension growth, a preparation method thereof, and a full suspension acclimation method applied to pk-15 cells
  • A serum-free medium adapted to pk-15 full suspension growth, a preparation method thereof, and a full suspension acclimation method applied to pk-15 cells
  • A serum-free medium adapted to pk-15 full suspension growth, a preparation method thereof, and a full suspension acclimation method applied to pk-15 cells

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Experimental program
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Effect test

Embodiment 1-3

[0035]Example 1-3 The amount of each component of the serum-free medium adapted to PK-15 full suspension growth refers to the data given in Table 1. The specific preparation process of this medium is: first dissolve Tween 80 in 50 ml of water for injection , then add linoleic acid, vitamin E, myristic acid, stearic acid, and vitamin A in sequence, and wait until they are fully dissolved, rehydration with water for injection to 500 ml, and then add β-mercaptoethanol, sodium chloride, Dextrose, stirred until clear, supplemented with water for injection to 900ml, then added the remaining components, after the addition, continued to stir until completely dissolved, then supplemented with water for injection to 1000mL, filtered through a 0.22μm filter, sterilized and stored at 4°C Keep away from light.

[0036] Table 1 embodiment 1-3 adapts to the serum-free medium component and consumption of PK-15 full suspension growth

[0037]

[0038]

[0039]

Embodiment 4

[0041] The process applied to the full suspension acclimation of PK-15 cells is: based on the well-grown PK-15 adherent cells purchased from ATCC, the PK-15 cells were digested with 0.2% trypsin for 8 minutes by conventional methods After that, resuspend with 20ml of the culture medium provided in Example 2, and resuspend the cells in 3×10 6 The density of cells / mL was inoculated into 125ml shaking flask (shaking flask was pre-treated with siliconizing agent to make it more difficult for cells to adhere to the wall), then added 30ml of the medium provided in Example 2, and then placed the medicine bottle in a place containing 5% CO 2 Cultured on a constant temperature shaker with a culture temperature of 37°C and a shaker speed of 120rpm; samples were taken every 24 hours to measure the cell density and viability with a cell counter, and the state of the cells was observed with an inverted microscope, and the medium was replaced every 48 hours. By observing the cells every day ...

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Abstract

The present invention relates to a serum-free medium adapted to PK-15 full-suspension growth, a preparation method thereof and a full-suspension acclimation method applied to pK-15 cells. The medium contains amino acids, Tween, linoleic acid, vitamin E, Myristic acid, stearic acid, vitamin A, β‑mercaptoethanol, sodium chloride, glucose, cadmium chloride, linoleic acid, choline chloride, inositol, and ammonium metavanadate. The serum-free medium provided by the present invention can realize the full suspension growth of PK‑15 cells, thereby omitting the cost of microcarriers and the digestion process, and solving the technical problem that it is difficult to digest and enlarge the PK‑15 cell microcarrier suspension culture process.

Description

technical field [0001] The invention belongs to the field of cell engineering, and relates to a protein-free, hydrolyzate-free serum-free medium, in particular to a serum-free medium suitable for PK-15 cell suspension culture and its preparation method and its application to pK-15 cells. Whole-suspension acclimatization method for 15 cells. Background technique [0002] Cell culture is the core and basic technology in the production of proteins, polypeptide drugs, vaccines and other biological products. Cell culture medium provides the nutrients and suitable environment for cell growth in vitro, which is the basis and key factor of in vitro cell culture. . General cell culture medium is composed of inorganic salts, amino acids, vitamins, carbon sources, trace elements and other components. [0003] PK-15 cell is a kind of cell commonly used in the production of animal vaccine, which is mainly used to produce porcine circovirus type 2 (PCV2) and porcine parvovirus (PPV). P...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12N5/02
CPCC12N5/0686C12N2500/10C12N2500/12C12N2500/14C12N2500/16C12N2500/20C12N2500/22C12N2500/24C12N2500/30C12N2500/32C12N2500/33C12N2500/34C12N2500/35C12N2500/36C12N2500/38C12N2500/40C12N2500/44C12N2500/46C12N2500/60C12N2500/95C12N2501/39C12N2501/999
Inventor 扶星星吴福文王永胜马海强
Owner SICHUAN BAINUOJI TECH CO LTD
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