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Method for improving electroporation efficiency of electric energy induced living cell membrane

A technology of electroporation and cell membrane, applied to other methods of inserting foreign genetic materials, electric/wave energy processing enzymes, biochemical equipment and methods, etc., can solve the problems of irreversible electroporation of cell membranes, etc.

Inactive Publication Date: 2019-04-09
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] So far, there is no report showing that the use of nanosecond-scale pulsed electric fields can cause irreversible electroporation of cell membranes

Method used

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  • Method for improving electroporation efficiency of electric energy induced living cell membrane
  • Method for improving electroporation efficiency of electric energy induced living cell membrane
  • Method for improving electroporation efficiency of electric energy induced living cell membrane

Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1

[0030] Implementation case 1: The perforation effect on SMMC7721 cells when the capacitor is charged at 10,000 volts and released into 13 ohm, 25 ohm and 50 ohm isotonic saline solutions

[0031] Hepatocellular carcinoma SMMC7721 was established by the Second Military Medical University in 1980 from a 50-year-old male patient with primary liver cancer, and has been widely used in the diagnosis and treatment of liver cancer.

[0032] Propidium iodide (PI) cannot pass through the complete cell membrane. PI enters the cytoplasm of the cell and can combine with DNA or RNA in the cell membrane to emit a red fluorescent signal. It is a commonly used detection method for perforation on the cell membrane. Fluorescent dyes.

[0033] Field emission scanning electron microscope (FESEM) has ultra-high resolution, and can observe biological samples such as tissues, cells, microorganisms, and biological macromolecules at the nanometer scale, and obtain a faithful three-dimensional surface o...

Embodiment example 2

[0038] Implementation Case 2 The perforation effect on SK-Hep1 cells when the capacitor is charged at 10,000 volts and released into 13 ohm, 25 ohm and 50 ohm isotonic saline solutions

[0039] SK-Hep1 cells are tumor cells isolated and identified from the postoperative sample of a 52-year-old male adenoma patient by Memorial Sloan-Kettering Cancer Center in the United States in the 1970s. The cell type is endothelial, but morphologically it is epithelial , can form tumors consistent with the characteristics of liver cancer cells in model animals. It is a tumor cell commonly used in basic medical research.

[0040] Figure 4 Medium, 2×10 6 SK-Hep1 cells were treated with nanosecond pulsed electric field in 1ml of physiological balanced salt solution, and the results showed that with the increase of the impedance of isotonic physiological saline solution, the percentage of PI-positive cells in SK-Hep1 cells treated with 200 pulsed electric field A significant increase. In t...

Embodiment example 3

[0043] Example 3 Irreversible electroporation of SMMC7721, HCCLM3 and SK-Hep1 cells induced by releasing 200 and 500 pulses when the capacitor was charged at 10000 volts

[0044] Hepatocellular carcinoma HCCLM3 is a hepatocellular carcinoma cell line with high metastatic potential isolated and established from human liver cancer samples by the Liver Cancer Institute of Zhongshan Hospital affiliated to Fudan University. It is widely used in the diagnosis and treatment of liver cancer.

[0045] Annexin V-FITC is a fluorescent detection reagent that can specifically bind to phosphatidylserine. Phosphatidylserine usually appears on the inner side of the living cell membrane. If Annexin V-FITC can be detected in the cell, it indicates that the cell membrane is perforated or the phosphatidylserine in the inner membrane of the cell appears outside the cell membrane. If PI can enter the cell membrane, the ability of Annexin V-FITC to bind to the cell further proves that the cell membr...

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Abstract

The invention discloses a method for improving electroporation efficiency of an electric energy induced living cell membrane. Electric energy stored in a capacitor is released into an iso-osmia normalsaline solution in a mode of index waveform nanosecond electric pulse through a conduction wire, and then cell membranes of living cells in the normal saline solution have electroporation. When the impedance of the normal saline solution is greater than the characteristic impedance of the conduction wire, as charges are reflected in the conduction wire, the voltage released into the loaded electric pulse is higher than that loaded at a capacitor discharge start point, then when same single pulse energy stored in the capacitor is released into living cells suspended in the normal saline solution, the voltage of the pulse has a higher highest peak value, and the efficiency that induced living cells have functional electroporation is improved. Living cells of a greater ratio can be induced to have cell membrane electroporation by outputting less total energy.

Description

technical field [0001] The invention relates to a method for improving the electroporation efficiency of living cell membranes induced by electric energy. technical background [0002] Living cells in biological tissues are isolated from the microenvironment by a cell membrane composed of a layer of phospholipid bimolecules, which is the basis for cells to survive and proliferate in the environment. Previous studies have believed that when a square wave or trapezoidal electrical pulse is released into biological tissue, when the pulse width of the pulsed electric field exceeds 30 microseconds (us), the field strength exceeds 800 volts / centimeter (V / cm), and more than 8 electrical pulses Inducing cell membrane cell membrane perforation, this is the current application of high-voltage pulsed electric fields in the biomedical field, and the use of high-intensity pulsed electric fields can cause reversible or irreversible electroporation of cell membranes. Reversible cell membr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N13/00C12N15/87
CPCC12N13/00C12N15/87
Inventor 殷胜勇刘振闫克平郑树森
Owner ZHEJIANG UNIV
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