Vesicle centrifugal protective agent, application of protective agent and method for centrifugally extracting vesicles

A protective agent and vesicle technology, applied in the field of centrifugal extraction of vesicles and vesicle centrifugal protective agent, can solve the problems of low separation efficiency and yield of cell vesicles, and improve the yield and separation efficiency of vesicles, and the volume change The effect of small degree and shortening of extraction time

Active Publication Date: 2021-04-20
HUBEI SOUNDNY BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the above problems of low cell vesicle separation efficiency and yield in the prior art, the present invention provides a vesicle centrifugal protection agent and the vesicle centrifugal protection Application of agent in centrifugal extraction of vesicles, and method of centrifugal extraction of vesicles

Method used

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  • Vesicle centrifugal protective agent, application of protective agent and method for centrifugally extracting vesicles
  • Vesicle centrifugal protective agent, application of protective agent and method for centrifugally extracting vesicles
  • Vesicle centrifugal protective agent, application of protective agent and method for centrifugally extracting vesicles

Examples

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Embodiment 1

[0058] Example 1 Glycerin and sodium carboxymethyl cellulose are used in combination as a vesicle centrifugal protector

[0059] Preparation of cell suspension containing cell vesicles: Inoculate purified tumor cells in RPMI 1640 medium containing 10% fetal bovine serum (v / v), 100 U / mL penicillin, 100 mg / mL streptomycin, Place the medium inoculated with tumor cells in a carbon dioxide incubator at a temperature of 37°C and a gas atmosphere of 5% CO 2 cultured under the conditions until the cell concentration reached 2×10 7 cells / mL, then collect tumor cells and transfer them to a culture dish containing fresh medium, place the culture dish under ultraviolet radiation for 1 hour to induce cell apoptosis, and then culture in a carbon dioxide incubator for 16-20 hours to form stable cell vesicles , to obtain a cell suspension containing cell vesicles.

[0060] Differential centrifugation: The cell suspension containing cell vesicles is subjected to differential centrifugation t...

Embodiment 2

[0066] The optimization of embodiment 2 carboxymethyl cellulose sodium concentration

[0067]The cell suspension was prepared by the method of Example 1, and then the cell suspension was divided into different groups. First, 5% wt of glycerol was added, and then 0.25%, 0.5%, 1%, 2%, 4%, and 8% were added to each group respectively. %wt of sodium carboxymethylcellulose, mixed to obtain a suspension; the suspension was subjected to differential centrifugation according to step S2 of Example 1, and the cell vesicles obtained by differential centrifugation were analyzed using Malvern NS300 particle tracking The instrument was used to characterize the number of cell vesicles, and the characterization results are shown in figure 2 .

[0068] Depend on figure 2 It can be seen that the vesicle production depends on the concentration of sodium carboxymethyl cellulose within a certain range. As the concentration of sodium carboxymethyl cellulose increases, the number of cell vesicle...

Embodiment 3

[0069] The optimization of embodiment 3 glycerol concentration

[0070] Adopt the method for embodiment 1 to prepare cell suspension, cell suspension is divided into two big groups afterwards, every big group is divided into different subgroups again, and two big groups add the sodium carboxymethyl cellulose of 0.5%wt and 4%wt earlier respectively, Afterwards, each group added 0, 1%, 2%, 3%, 4%, 5%, 6%, 7% wt of glycerin and mixed to obtain a suspension; the suspension was prepared according to step S2 of Example 1 Perform differential centrifugation, and use the Malvern NS300 particle tracking analyzer to characterize the number of cell vesicles obtained by differential centrifugation. The characterization results are shown in image 3 .

[0071] Depend on image 3 It can be seen that glycerol within a certain concentration range can improve the protective effect of carboxymethylcellulose sodium on cell vesicles. During high-speed centrifugation, the appropriate concentrati...

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Abstract

The invention provides a vesicle centrifugal protective agent, application of the protective agent and a method for centrifugally extracting vesicles, and belongs to the technical field of biology. The vesicle centrifugal protective agent comprises glycerol and sodium carboxymethyl cellulose. On the basis of differential centrifugation, the glycerin and the sodium carboxymethyl cellulose are adopted as the centrifugal protective agent, the glycerin can permeate into cells to increase the solute concentration of an intracellular solution, and the sodium carboxymethyl cellulose is located outside the cells to increase the solute concentration of an extracellular solution, so that the osmotic pressure balance inside and outside the vesicles is achieved, the extrusion deformation resistance is improved, the vesicles are protected from being damaged in the ultra-high-speed centrifugation process to the maximum extent, and the yield of the vesicles is improved. Moreover, the addition of the sodium carboxymethyl cellulose can change the surface charge of the vesicles, destroy the electrostatic repulsion of the vesicles, cause the aggregation and precipitation of the adjacent vesicles, improve the precipitation speed of the vesicles, shorten the time required for extraction and further improve the yield and separation efficiency of the vesicles.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a vesicle centrifugal protectant and the application of the vesicle centrifugal protector in centrifugal extraction of vesicles, and also to a method for centrifugal extraction of vesicles. Background technique [0002] The cytoskeleton is an important structure for eukaryotic cells to maintain their basic shape. After the cells are exposed to exogenous or endogenous stimuli, the cytoskeleton will be rearranged, resulting in uneven local stress on the cell membrane. The abnormally stressed cytoplasm will expand to the outside of the cell, and then randomly wrap part of the cell content and release it to the outside of the cell in the form of vesicles. This special subcellular structure with a diameter of about 0.1-1 μm is called "cell vesicles" . Cell vesicles can be specifically recognized by target cells because they carry biomarker molecules of the original cells. More and more r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/00
Inventor 童雅琪陈彬张一
Owner HUBEI SOUNDNY BIOLOGICAL TECH
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