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Non-tumorigenic mdck cell line and screening method for amplifying influenza virus

An influenza virus, non-tumorigenic technology, applied in microorganism-based methods, animal cells, viruses/phages, etc., can solve the problems of non-tumorigenicity, 1-100 cells may form tumors, etc., and achieve cost savings , the effect of shortening the time

Active Publication Date: 2016-09-28
SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] However, there are also some controversies about the matrix of MDCK cells. For example, the original cell lines of MDCK are non-tumorigenic; some cell lines derived from MDCK cells are highly tumorigenic (it is possible that 1-100 cells may be form tumors); highly tumorigenic cell substrates have never been used to produce viral vaccines and highly tumorigenic cell substrates pose significant regulatory challenges [FDA. Use of MDCK Cells for Manufacture of Inactivated Influenza Vaccines: Introduction]

Method used

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  • Non-tumorigenic mdck cell line and screening method for amplifying influenza virus
  • Non-tumorigenic mdck cell line and screening method for amplifying influenza virus
  • Non-tumorigenic mdck cell line and screening method for amplifying influenza virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1, have the acquisition of non-tumorigenic MDCK-2B2 cell line

[0052] 1. Obtaining MDCK monoclonal cells

[0053] 1. Cell digestion and counting

[0054] Passage ATCC CCL-34 MDCK P55 in serum-containing medium (Minimum essential medium + 10% fetal bovine serum (FBS) + 2mM glutamine) for a limited number of passages, MDCK P63 generation cells, select a bottle of T-25 cells for Digestion and counting.

[0055] Take a bottle of MDCK P63 T-25 cells, discard the culture supernatant, wash twice with 1mL trypsin (purchased from Gibco), then add 1mL trypsin, put in CO 2 Incubate in an incubator, observe under the microscope that the cells become round after 15 minutes, add 9 mL of medium to stop digestion, mix the cell suspension, take 10 μL and drop it on a cell counting plate for counting.

[0056] 2. Cell density adjustment and plating

[0057]In order to ensure that there is no single-well polyclonal existence in the 96-well cell plate, reduce the total numb...

Embodiment 2

[0067] Growth morphology and biochemical indicators of embodiment 2, MDCK-2B2 cells

[0068] 1. MDCK-2B2 cells have epithelial-like morphology

[0069] P85 MDCK-2B2 was observed under a microscope (40×), and it was found to be fusiform with regular edges, adherent cells, and epithelial-like morphology.

[0070] 2. MDCK-2B2 has a higher growth density

[0071] Inoculate with the same initial density (2.5×10 5 cells / 5mL) of MDCK-2B2 and MDCK in T-25 cell culture flasks, 7 flasks for each, one of the flasks was extracted at intervals of 24 hours for cell counting, counting continuously for 7 days, MDCK-2B2 cells in 7 days The totals are: 1.8×10 5 cells, 2.9×10 5 cells, 11.6×10 5 cells, 22×10 5 cells, 20×10 5 cells, 20.8×10 5 cells and 10.5×10 5 cells; the total number of cells in MDCK in 7 days were: 2.3×10 5 cells, 4.8×10 5 cells, 19.1×10 5 cells, 21×10 5 cells, 23.5×10 5 cells, 24.87×10 5 cells and 14.5×10 5 cells. Such as image 3 As shown, MDCK-2B2 cells hav...

Embodiment 3

[0076] Embodiment 3, identification of MDCK-2B2 cell chromosome

[0077] 1. Harvest the cells, wash the cell wall twice with 0.85% NaCl solution;

[0078] 2. Digest with 0.25% Trypsin-EDTA for 10-15 minutes. When the cell surface appears wrinkled and changes visible to the naked eye, use a pipette to blow down the cells at 453g (1500rpm / min, eppendorf 5810R) and centrifuge at room temperature for 10 minutes. Remove the supernatant and leave about 0.5 ml;

[0079] 3. Hypotonicity: Add 4-6ml of 0.075M KCl at 37°C, blow with a pipette, and bathe in water at 37°C for 3-5 minutes

[0080] 4. Pre-fixation: add 1.5ml ± (methanol: glacial acetic acid = 3:1) fixative, mix gently in a 37°C water bath for 5 minutes, and centrifuge at 453g (1500rpm / min, eppendorf 5810R) for 10 minutes at room temperature. Remove the supernatant and leave about 0.5ml;

[0081] 5. Fixation: Add 8ml± of fixative, mix gently, bathe in 37℃ water for 10 minutes, and centrifuge at room temperature at 453g (15...

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Abstract

The invention discloses a non-tumor-causing MDCK cell line for amplifying influenza virus and a screening method thereof. The cell line is MDCK‑2B2, and the preservation number is CCTCC C201323. The present invention provides a non-tumorigenic cell line MDCK-2B2 and its subclone cell line, which grow into adherent cells and / or have epithelial-like morphology and / or can support the replication of various viruses, and can amplify Higher titer virus; In addition, the present invention has found a method for screening non-tumorigenic cell lines, which can assist the judgment of tumorigenicity through the growth rate of cells and the morphological changes of cells, that is, relatively slow-growing single-pore cells, which The tumorigenicity will be significantly reduced, and the appearance of giant cells will significantly increase the tumorigenicity. When screening non-tumorigenic cell lines on a large scale, such auxiliary judgment methods can shorten time and save costs.

Description

technical field [0001] The present invention relates to MDCK-derived cell lines, more specifically, to a non-tumorigenic MDCK cell line for amplifying influenza virus and a screening method thereof. Background technique [0002] The annual influenza epidemic will have a relatively large impact on both developed and developing countries. According to statistics, in the season when influenza is prevalent, about 5-15% of the people in the world will develop upper respiratory tract infection every year, and about 250,000-500,000 people will die [WHO. Influenza Fact Sheet www.who.int / mediacentre / factsheets / fs211 / en / ]. Influenza vaccination is one of the main measures to prevent and control influenza, which can significantly reduce morbidity and mortality, so it is particularly important to provide vaccines effectively and quickly. At present, most influenza vaccines still use chicken embryos to produce the strains announced by WHO. Due to the shortage of chicken embryos in the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12N7/00C12R1/91C12R1/93
Inventor 陈则杨梅
Owner SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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