Pelodiscus sinensis heart cell continuous cell line and establishing method and ultra-low-temperature cryopreservation method thereof

A heart cell and construction method technology, applied in artificial cell constructs, animal cells, vertebrate cells, etc., can solve problems such as restricting in-depth research on Chinese soft-shelled turtle virus diseases, and achieve the effect of avoiding the difficulty of cell migration.

Active Publication Date: 2015-11-11
ZHEJIANG WANLI UNIV
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  • Abstract
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Problems solved by technology

It can be seen that the heart cell line of Chinese soft-shelled turtle is an ideal carrier for the study of viral diseases of Chinese soft-shelled turtle. The continuous cell line of cells has not been reported so far. Therefore, the research on Chinese soft-shelled turtle cells has become a bottleneck restricting the in-depth research of Chinese soft-shelled turtle viral diseases. The study of continuous cell lines of Chinese soft-shelled turtle heart cells is very important to break the research bottleneck of Chinese soft-shelled turtle viral diseases. significance

Method used

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  • Pelodiscus sinensis heart cell continuous cell line and establishing method and ultra-low-temperature cryopreservation method thereof
  • Pelodiscus sinensis heart cell continuous cell line and establishing method and ultra-low-temperature cryopreservation method thereof
  • Pelodiscus sinensis heart cell continuous cell line and establishing method and ultra-low-temperature cryopreservation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Example 1: Construction of a continuous cell line of soft-shelled turtle heart cells

[0029] 1.1 Preparation of PBS disinfectant and cell culture medium

[0030] PBS disinfectant solution: Add antibiotics to PBS so that the penicillin concentration is 100 IU / ml, the streptomycin concentration is 100 μg / ml, and the amphotericin B concentration is 20 μg / ml.

[0031] Primary culture medium: add fetal bovine serum, cell growth factor EGF and antibiotics to the MEM medium, so that the volume of fetal bovine serum accounts for 15% of the total volume, the concentration of EGF is 10ng / ml, the concentration of penicillin is 100IU / ml, streptomycin The concentration of acetaminophen was 100 μg / ml, the concentration of amphotericin B was 20 μg / ml, the pH value of the culture medium was adjusted to 7.0-7.2, and it was stored in a 4-degree refrigerator for later use.

[0032] Subculture medium: add fetal bovine serum and antibiotics to the MEM medium, so that the volume of fetal b...

Embodiment 2

[0037] Example 2: Construction of a continuous cell line of soft-shelled turtle heart cells

[0038] 2.1 Primary culture

[0039] Use 0.1% potassium permanganate solution to soak and disinfect the soft-shelled turtle body for 30 minutes. After cleaning, wipe the surface of the soft-shelled turtle body with 75% alcohol, and place it in an ultra-clean workbench. The heart was taken with sterile dissecting instruments, placed in a sterile petri dish, and washed with PBS disinfectant to remove blood clot tissue and connective tissue. Next, cut the heart to 0.8mm with sterile instruments 3 Small and large tissue pieces were digested with 0.125% trypsin at room temperature for 25min, and then the digested tissue pieces were inoculated on 25cm 2 Add 2ml of the primary culture medium to the cell culture flask and place it upside down in a 30°C incubator to start the primary culture. After 24 hours, turn the cell culture flask over and add the primary culture medium to 10ml. Thereaf...

Embodiment 3

[0042] Example 3: Cell cryopreservation and recovery

[0043] 3.1 Cell cryopreservation

[0044] Mix fetal calf serum and DMSO at a volume ratio of 9:1, and mix well to obtain a cell cryopreservation protection solution. Take the cells in the logarithmic growth phase, digest them with 0.25% trypsin-EDTA solution, centrifuge the cell suspension at 1000rpm for 8min, discard the supernatant, add the prepared cell freezing solution to the cell pellet, resuspend, and make the cells The concentration is 1×10 6 cells / ml, transfer 1ml of the cell suspension to a 1.8ml cryopreservation tube and place the cryopreservation tube in a programmed cooling box, overnight at -80°C, and then store it in liquid nitrogen for long-term storage.

[0045] 3.2 Cell recovery

[0046] Take the frozen tube out of the liquid nitrogen, put it into a 37°C water bath and shake it quickly until it melts, then transfer the thawed cells to a 15ml centrifuge tube in a sterile operating table, and add an equa...

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Abstract

The invention relates to a pelodiscus sinensis heart cell continuous cell line which is preserved in the common microorganism center of China Committee for Culture Collection of Microorganisms and has a preservation number of CGMCC No.10597, and further relates to an establishing method and ultra-low-temperature cryopreservation method of the pelodiscus sinensis heart cell continuous cell line. The establishing method includes the following steps of firstly, conducting primary culture, wherein a pelodiscus sinensis heart is cut into tissue small pieces after being preprocessed, trypsin is inoculated into a culture bottle after being digested at the room temperature, and subculture is not started until cells are fully laid on 90% or more of the bottle bottom; secondly, conducting subculture, wherein air is blown on the bottle bottom through a trypsin digesting method to make adherent cells fall down so that subculture can be conducted. By means of the established pelodiscus sinensis heart cell continuous cell line, the research on viral diseases of pelodiscus sinensis and other turtle animals and the research on related therapy drugs for instance the analysis of chromosome and the research of iridescent virus can be conducted on the level of molecular cells, and the requirements for pelodiscus sinensis germplasm resource conservation and theoretical research and application can be met.

Description

technical field [0001] The invention relates to the technical field of biological cell culture, in particular to a continuous cell line of soft-shelled turtle heart cells, a construction method thereof, and a cryopreservation method at ultra-low temperature. Background technique [0002] Chinese soft-shelled turtle (Pelodiscus sinensis), also known as water fish, soft-shelled turtle, and turtle, belongs to Reptilia, Turtle order, and Turtle family. It is distributed wildly in China, Japan, northern Vietnam, South Korea, and eastern Russia. It is a precious species. , Aquatic animals with high economic value. In recent years, the breeding volume of soft-shelled soft-shelled turtles has been increasing year by year. However, due to artificial temperature control, high-density intensification and other breeding methods, the natural living habits of soft-shelled soft-shelled turtles have been changed. The soft-shelled turtle disease has seriously threatened my country's soft-she...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071A01N1/02
Inventor 钱国英郭海杰毛芝娟汪财生
Owner ZHEJIANG WANLI UNIV
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