High-density continous pouring culture of animal cell

A technology of perfusion culture and animal cells, applied in animal cells and other directions, can solve the problems of economic loss, cell death, limited increase in viable cell density and product concentration, etc.

Inactive Publication Date: 2006-05-31
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are still some deficiencies in the research work of these scholars. The main reason is that they often only consider glucose and/or glutamine. Limiting factors, in the end, often cause a large number of cells to die, the number of dead cells is greater than the number of living cells, and the increase in the density of living cells and product concentration is still limited
Although the medium continuous perfusion culture has more advantages than fed-bat

Method used

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  • High-density continous pouring culture of animal cell
  • High-density continous pouring culture of animal cell
  • High-density continous pouring culture of animal cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Are HB58 hybridoma cells (obtained from ATCC) employed in serum-free medium? ? ? After subculture adaptation according to the method disclosed in the literature, inoculate in a 50-liter bioreactor with an inoculation density of 2.0×10 5 cells / ml.

[0053] (2) Under the conditions of 37°C and pH 7.2, in the initial serum-free medium, after 60 hours of culture, the serum-free perfusion medium was cultured at 1.0v / v day -1 The perfusion rate is perfused into the bioreactor for perfusion culture, the culture time is 60 days, and the liquid is collected from the 10th day, and the cell density can reach 2.40×10 7 cells / ml, the product concentration can reach 624mg / ml; see the cell changes in the culture process figure 1 , in the figure, curve 1 is the living cell density, and curve 2 is the monoclonal antibody concentration.

[0054] Table 1 Continuous irrigation in 50L bioreactor

Embodiment 2

[0056] Recombinant CHO cells (rCHO SS3 A2, expressing human anticoagulant factor III) were inoculated in a B.BRAUN 2-liter bioreactor at a seeding density of 2.75×10 5 cells / ml.

[0057] (2) Under the conditions of 37°C and pH 7.2, in the serum-free initial medium, after 40 hours of culture, the serum-free perfusion medium was added at 0.58v / v day -1 The perfusion rate is perfused into the bioreactor for perfusion culture, the culture time is 28 days, and the liquid is collected from the 8th day, and the highest cell density can reach 1.1×10 7 cells / ml, the product concentration can reach 390U / ml; see the cell changes during the culture process figure 2 , among the figures, curve 3 is the viable cell density, curve 4 is the total cell density, and curve 5 is the expression level of the product (ATIII).

[0058] Through the optimization of the culture medium and the culture process of the present invention, various nutrients in the culture medium are controlled at lower appr...

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Abstract

The invention opened a high density filling culture method of the animal cells. It study the metabolic rule of the cell growth, the nutrient consumption and the metabolic byproduct accumulation by studying theirs dynamics to determine and control the optimal nutritive condition. It feed the nutrient quality of cell growth by the stoichiometric relationship and the filling culture. The process can control the nutrient in a proper low concentration, so we can get the high density of the cells and the product concentration.

Description

technical field [0001] The invention relates to a large-scale, high-density, serum-free continuous perfusion culture method for animal cells. technical background [0002] The cultivation process of animal cells in the bioreactor can have different operation modes, such as batch culture (Batch), continuous culture (Continuous), perfusion culture (Perfusion) and fed-batch culture (Fed-batch), etc., different operation modes has its different characteristics. [0003] Continuous perfusion culture is to add fresh nutrient solution at a certain rate during the culture process, and discharge the supernatant at the same rate. The fragments are separated, and the living cells are returned to the reactor to continue culturing. Continuous perfusion culture not only maintains a relatively constant culture state, avoids the accumulation of harmful metabolic by-products, but also enables the cells in the reactor to reach a relatively high density. [0004] In recent years, animal cel...

Claims

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Application Information

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IPC IPC(8): C12N5/07
Inventor 谭文松朱明龙牛红星周燕华平顾小华
Owner EAST CHINA UNIV OF SCI & TECH
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