Method for separating extracellular microcapsules from cells, tissue culture supernatant or body fluid, specialized separating reagent, kit and application

A technology for separating cells and tissue culture, applied in tissue culture, culture process, animal cells, etc. It can solve the problems of the difference in the concentration of extracellular microcapsules, affect the use, and the sample volume is only 100-200mL, etc. Simple operation and good repeatability

Inactive Publication Date: 2016-07-27
细胞邦(北京)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

Chromatography uses the physical properties of microcapsules to harvest target substances through high-efficiency chromatography columns, but each time the sample volume is only 100-200mL, it takes more than 24 hours
In addition, due to different sources of extracellular microcapsule

Method used

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  • Method for separating extracellular microcapsules from cells, tissue culture supernatant or body fluid, specialized separating reagent, kit and application
  • Method for separating extracellular microcapsules from cells, tissue culture supernatant or body fluid, specialized separating reagent, kit and application
  • Method for separating extracellular microcapsules from cells, tissue culture supernatant or body fluid, specialized separating reagent, kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1. Isolation of extracellular microcapsules from the culture supernatant of human umbilical cord mesenchymal stem cells

[0038] 1. Materials

[0039] Cells: Human umbilical cord mesenchymal stem cells, provided by Cell State (Beijing) Biotechnology Co., Ltd., were isolated and cultured according to conventional methods.

[0040] Main reagents: polyethylene glycol (PEG), purchased from Sigma, USA, product number 81260, with an average molecular weight of 6000. Sodium chloride was purchased from Sigma, USA. Tris and hydrochloric acid were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. Fetal bovine serum was purchased from Invitrogen. alpha-MEM medium was purchased from Invitrogen.

[0041] Preparation of main reagents:

[0042] (1) Prepare 500mM Tris HCl solution with a pH value of 8.0.

[0043] (2) Prepare 0.15M NaCl solution with Tris·HCl solution, Tris·HCl is 100mM.

[0044] (3) A 40% (W / V) polyethylene glycol (PEG) solution...

Embodiment 2

[0060] Example 2. Isolation of extracellular microcapsules from the culture supernatant of human dendritic cells

[0061] 1. Materials

[0062] Cells: Human dendritic cells, provided by Cell State (Beijing) Biotechnology Co., Ltd., were isolated and cultured according to conventional methods.

[0063] Main reagents: polyethylene glycol (PEG), purchased from Sigma, USA, product number 1546605, average molecular weight 8000. Sodium chloride was purchased from Sigma, USA. IL-4, GM-CSF and LPS were purchased from Peprotech. AIM-V serum-free medium is a product of Invitrogen.

[0064] Preparation of main reagents:

[0065] (1) Prepare 2M NaCl solution with deionized water.

[0066] (2) Use 2M NaCl solution to prepare 30% (W / V) polyethylene glycol (PEG) solution.

[0067] 2. Method

[0068] 1) Cultivate human dendritic cells by conventional methods, the medium is AIM-V serum-free medium, and the culture conditions are 37°C, 5% CO 2 , 95% humidity, add IL-4 (final concentrati...

Embodiment 3

[0076] Example 3. Isolation of extracellular microcapsules from human peripheral blood

[0077] 1. Materials

[0078] Cells: Human peripheral blood, derived from healthy donors. Human plasma was harvested according to conventional methods and anticoagulated with heparin.

[0079] Main reagents: polyethylene glycol (PEG), purchased from Sigma, USA, product number 1546569, with an average molecular weight of 4000. Sodium chloride was purchased from Sigma, USA.

[0080] Preparation of main reagents:

[0081] (1) Prepare 2M NaCl solution with deionized water.

[0082] (2) Prepare 48% (W / V) polyethylene glycol (PEG) solution with 2M NaCl solution.

[0083] 2. Method

[0084] 1) Prepare human plasma by conventional methods, the specific method is: centrifuge at 3000 rpm for 10 minutes. Transfer the plasma to a 50 mL centrifuge tube.

[0085] 2) Add 10 mL of 48% (W / V) PEG solution to 30 mL of human plasma, the final concentration of PEG is 12% (W / V), and mix well.

[0086] 3...

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Abstract

The invention discloses a method for separating extracellular microcapsules from cells, tissue culture supernatant or body fluid, a specialized separating reagent, a kit and application. The method comprises the steps of alienating extracellular microcapsules in cells, tissue culture supernatant or body fluid from a water solution by virtue of a polyethylene glycol (PEG) solution, and concentrating and collecting the settle extracellular microcapsules by virtue of a low-speed centrifugation method. By virtue of the method, 1000mL or above specimens can be processed in each time; compared with ultracentrifugation and chromatography methods, the method has the advantages that the operation is simple, the elapsed time is short, large-sized instruments and equipment are not required, and the repeatability is good; and the problem that the extracellular microcapsules are difficult to separate is solved, and the scientific research and the clinical application of the extracellular microcapsules are promoted.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the acquisition technology of extracellular microcapsules, in particular to a method for isolating extracellular microcapsules from cell culture supernatant, tissue culture supernatant or body fluid, and special separation reagents, kits and separation methods thereof. Applications of the obtained extracellular microcapsules. Background technique [0002] Under physiological and pathological conditions, resting and activated cells can release a vesicular structure into the extracellular space and body fluid, which is wrapped by a lipid bilayer structure, with a diameter ranging from tens of nanometers to several microns, called It is an extracellular microcapsule, and its English name is extracellular vesicles (EV). Depending on the source and the size of the vesicles, extracellular microvesicles also have other names, such as oncosomes, prostasomes, apoptotic bodies, etc. At present...

Claims

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Application Information

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IPC IPC(8): C12N5/0784C12N5/078C12N5/0775
CPCC12N5/0665C12N5/0634C12N5/0639C12N2500/50
Inventor 郭子宽
Owner 细胞邦(北京)生物技术有限公司
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