Non-tumorigenic MDCK cell line used for amplifying influenza viruses and screening method thereof

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

Active Publication Date: 2014-09-24
SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 t

Method used

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  • Non-tumorigenic MDCK cell line used for amplifying influenza viruses and screening method thereof
  • Non-tumorigenic MDCK cell line used for amplifying influenza viruses and screening method thereof
  • Non-tumorigenic MDCK cell line used for amplifying influenza viruses and screening method thereof

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 times, 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 number ...

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-tumorigenic MDCK cell line used for amplifying influenza viruses and a screening method thereof. The cell line is MDCK-2B2 with an accession number of CCTCC 201323. The invention provides the non-tumorigenic MDCK-2B2 cell line and a subclone cell line thereof; and the cell line can grow into adherent cells, and/or has an epithelioid form and/or can support duplication of a variety of viruses, and is capable of amplifying viruses with high titers. The invention also provides the screening method for the non-tumorigenic cell line. The growth speed and morphologic changes of cells can be used to assist in determination of tumorigenicity; i.e., simple-pore cells with a slow growth speed have substantially reduced tumorigenicity, and occurrence of giant cells enables tumorigenicity to be substantially increased; during large-scale screening of non-tumorigenic cell lines, such an auxiliary determination method can shorten time and save cost.

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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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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