Method for removing mycoplasma in PK15 cells

A mycoplasma and cell technology, applied in the field of cell biology, can solve the problems of infecting cells in the bottle and mycoplasma entering the cell bottle, etc., and achieve the effect of short time

Pending Publication Date: 2018-11-06
SHANDONG SINDER TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, conventional cell culture generally uses a gas-permeable cell bottle or a sealed cell bottle to loosen the bottle stopper a little to maintain cell permeability. However, in the case of mycoplasma in the environment, it is easy for mycoplasma to enter the cell bottle. Infection of flask cells

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  • Method for removing mycoplasma in PK15 cells
  • Method for removing mycoplasma in PK15 cells
  • Method for removing mycoplasma in PK15 cells

Examples

Experimental program
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Effect test

Embodiment 1

[0031] A method for removing mycoplasma in PK15 cells, specifically comprising the following steps:

[0032] (1) Configure DMEM medium according to the instructions of DMEM medium (gbico Dulbecco's Modified Eagle Medium high glucose medium, gbico, REF 12100-046), and prepare 0.25% trypsin (Soleibao , T8150), and then filtered through 0.1 μm bacteria filter (Millex, SLVV033RS). Bovine serum (Jinyuankang 20171228) was irradiated with cobalt 60, inactivated at 56°C for 30 minutes, and then distributed for use.

[0033] Then, the high-sugar DMEM medium, 0.25% trypsin, and bovine serum (Jinyuankang, 20171228) were all negative by PCR detection before use, and subsequent operations were performed.

[0034] (2) Add the bovine serum prepared in step (1) to DMEM medium to configure a cell growth solution with a bovine serum content of 8%, and use the 0.25% trypsin prepared in step (1) to cover the monolayer of PK15 cells Digest and disperse, subculture according to the ratio of 1:5, ...

Embodiment 2

[0038] The F6 generation PK15 cells obtained in the above-mentioned example 1 are cultured and subcultured to the F25 generation with the DMEM medium without mycoplasma, bovine serum and 0.25% trypsin prepared in the step (1) of the example 1, and need to be sealed and airtight Subculture was carried out in cell culture flasks, and all operations were carried out under sterile conditions. DNA was extracted from F12, F20, and F25 generation cells for PCR detection. The PCR detection kit was the Beijing Century Yuanheng Mycoplasma Detection Kit. Such as image 3 As shown, the PCR product electrophoresis results showed that, except for the positive control, the PK15 cells of the other three passages were all negative for mycoplasma. And the F25 generation PK15 cells were detected by electron microscopy, such as Figure 4 As shown, the mycoplasma-free PK15 cells prepared by the present invention are still negative for mycoplasma after 25 generations.

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Abstract

The invention discloses a method for removing mycoplasma in PK15 cells. The method comprises the following steps: (1) preparing a DMEM medium and 0.25% pancreatin separately, filtering the medium andthe pancreatin respectively through a bacteria filter, sterilizing bovine serum and conducting inactivation treatment for split charging and use; (2) digesting and dispersing PK15 cells fully growingon a monolayer with the 0.25% pancreatin prepared in the step (1), and adding a cell growth solution for passage; (3) adding a Plasmocin treatment reagent for continuous culture; (4) conducting passage after the PK15 cells grow into a dense monolayer, utilizing the cell growth solution prepared in the step (2) and containing 25 <Mug / ml of the Plasmocin treatment reagent as a passage medium, addingthe passage medium as per the ratio of 1:5 after the PK15 cells grow fully, then conducting passage consecutively for 4 generations to obtain the PK15 cells without the mycoplasma. By means of the method, the mycoplasma in the PK15 cells can be effectively removed within short time, and the obtained mycoplasma-free PK15 cells do not need to use the Plasmocin treatment reagent in the later passageprocess, and no secondary infection of the cells occurs.

Description

technical field [0001] The invention belongs to the technical field of cell biology, and in particular relates to a method for removing mycoplasma in PK15 cells. Background technique [0002] Mycoplasma is a kind of prokaryotic microorganism lacking cell wall, its size is generally between 0.3-0.5 μm, it is highly pleomorphic, and has various shapes such as spherical, rod-shaped, filamentous, and branched. At present, the mycoplasma removal methods for cells are mostly removed with different antibiotics, such as fluoroquinolones, tetracycline derivatives, and macrolide antibiotics. The treatment methods are: (1) obtain the cells to be treated; (2) use The culture medium containing 10 μg / ml ciprofloxacin cultivates the cells to be treated; (3) regularly renews the medium, and keeps the growth density of the cells to be treated at 50% to 80%; (4 ) stop using the culture medium after no mycoplasma contamination is detected. After these antibiotics were used for a period of ti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12M1/12
CPCC12M47/12C12N5/0686
Inventor 郭春丽王学波刘志亮王丹娜常娓娓罗济冠李晓林王相芹李朝阳
Owner SHANDONG SINDER TECH
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