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Method and kit for separating exosome

A technology of reagents and body fluids, which is applied in the field of biomedicine, can solve the problems of long time consumption, large centrifugal force, and high cost, and achieve the effects of simple operation, high RNA yield, and improved yield

Pending Publication Date: 2017-02-15
广州锐博生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The ultracentrifugation method mainly uses the difference in size, density, etc. of exosomes and the surrounding environmental substances to separate. Its disadvantages are high requirements for centrifugation equipment and high cost; repeated centrifugation is required, and the operation steps are complicated; the centrifugal force may destroy the structure of exosomes , so the yield of exosomes obtained is not high
The ultrafiltration method uses the size characteristics of exosomes to separate them from the surrounding environment, which solves the shortcomings of the ultracentrifugation method, which takes a long time and requires special equipment, but because there may be other substances in the same size range in the surrounding environment, Therefore, the purity of isolated exosomes is not high

Method used

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  • Method and kit for separating exosome
  • Method and kit for separating exosome
  • Method and kit for separating exosome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1, Molecular Weight Optimization of Hydrophilic Polymer Used in Isolation of Exosomes

[0087] 1. Sample preparation

[0088] 1. Plasma

[0089] Human peripheral blood samples were collected using EDTA anticoagulated blood collection tubes, left to stand at 4°C for 3 hours, centrifuged at 4000×g for 10 minutes, and the supernatant was taken as a plasma sample.

[0090] 2. Cell supernatant

[0091] Using DMEM medium (containing 10% exosome-depleted FBS), at 37 °C and 5% CO 2 The 3T3 cells were cultured under culture conditions, and after 2 days of normal culture, the cell supernatant samples were collected (centrifuged at 1000 rpm for 2 minutes).

[0092]2. Configure exosome separation solution

[0093] Use hydrophilic polymers PEG8000, PEG10000, PEG20000, dextran sulfate 50000, dextran sulfate 40000, PVP25000 and PVP40000 to dissolve in water to prepare exosome separation liquid storage solution as follows: PEG8000 aqueous solution with a concentration of 20...

Embodiment 2

[0125] Example 2, optimization of the concentration of hydrophilic polymer used for isolating exosomes

[0126] 1. Sample preparation

[0127] 1. Plasma: same as Example 1;

[0128] 2. Cell supernatant: the same method as in Example 1, except that the cells are A549;

[0129] 2. Configure exosome separation solution

[0130] Since Example 1 found out that hydrophilic polymers PEG8000, PEG20000, dextran sulfate 40000, PVP25000 and PVP40000 are effective exosome separation liquids, the exosome separation liquid storage liquid prepared by dissolving in water is as follows:

[0131] Exosome separation fluid for plasma separation: PEG8000 aqueous solution with a concentration of 400mg / ml, dextran sulfate 40000 with a concentration of 400mg / ml, and PVP25000 with a concentration of 400mg / ml;

[0132] Exosome separation fluid for cell supernatant separation: PEG20000 at a concentration of 400mg / ml, dextran sulfate 40000 at a concentration of 400mg / ml, and PVP40000 at a concentratio...

Embodiment 3

[0152] Example 3, the use of hydrophilic polymers in combination to separate exosomes

[0153] 1. Sample preparation

[0154] 1. Plasma: same as Example 1;

[0155] 2. Cell supernatant: the same method as in Example 1, except that the cells are Hela;

[0156] 2. Configure exosome separation solution

[0157] Since Example 2 finds out that the best hydrophilic polymers for separating plasma exosomes are PEG8000 and PVP25000, and the best hydrophilic polymers for separating cell supernatant exosomes are PEG20000 and dextran sulfate 40000, therefore, it is possible Two kinds of hydrophilic polymers were mixed to prepare exosome separation fluid storage solution, as follows:

[0158] Exosome separation liquid for plasma separation: PEG8000 aqueous solution with a concentration of 300mg / ml, PVP25000 with a concentration of 300mg / ml;

[0159] Exosome separation fluid for cell supernatant separation: PEG20000 at a concentration of 300mg / ml, and dextran sulfate 40000 at a concentr...

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Abstract

The invention discloses a method and kit for separating exosome. The invention provides a reagent. The reagent is an aqueous solution of a hydrophilic polymer or a salt solution, wherein the hydrophilic polymer is PEG, dextran sulfate, or PVP, or a composition thereof. Experimental results prove that the exosome can be successfully separated by using a plurality of hydrophilic polymers; and the yield of separated exosome can be increased by using a composition of a hydrophilic polymer and an inorganic salt.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for isolating exosomes and a kit thereof. Background technique [0002] Exosomes are small vesicles (usually about 30-100 nanometers) secreted by cells into the extracellular space, and the size of exosomes from different sources varies. All types of cultured cells secrete exosomes and are abundant in body fluids such as blood, urine, saliva, and breast milk (Kosaka et al., Silence 3(2010) 1-7; Mitchell et al., PNAS 105( 2008) 10513-10518), and exosomes can reach other cells or tissues through body fluid circulation and other channels. Exosomes contain nucleic acids, proteins, lipids and other biomolecules, and may be the carriers of these substances in vivo. Relevant studies believe that exosomes may play the role of messengers. They participate in intercellular communication by transmitting various signaling molecules between cells, thereby regulating a variety of physiolo...

Claims

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Application Information

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IPC IPC(8): C12N5/09C12N5/071C12N5/077
Inventor 张必良曹亮刘建世
Owner 广州锐博生物技术有限公司
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