High-quality exosome and preparation method thereof

An exosome and bold technology, applied in the fields of biology and medicine, can solve the problems of large volume, difficult to meet clinical needs, low yield, etc.

Active Publication Date: 2015-09-09
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the large volume of cell culture supernatant and the limitation of centrifuge specifications, this method has low yield and low efficiency, and it is difficult to meet clinical needs.

Method used

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  • High-quality exosome and preparation method thereof
  • High-quality exosome and preparation method thereof
  • High-quality exosome and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] Preparation of exosomes

[0116] Human colon cancer cells sw480 (expressing carcinoembryonic antigen CEA) were cultured, and the cells were cultured to a confluence of 60%, and the medium containing 10% exosome-free serum was replaced to continue culturing for 24 hours, and then the cell culture supernatant was collected. The culture supernatant was passed through a 3+0.8 μm filter using a 50 rpm / min peristaltic pump to remove cells, cell debris, organelles, and impurities in the supernatant. The filtered supernatant was concentrated at 500KD using an ultrafilter, further washed and filtered with PBS, and then centrifuged at 100,000g for 2 hours with a 26%-42% (w / v) discontinuous sucrose density gradient, and the middle layer was washed and filtered with PBS for 5 times , 0.22μm filter bacteria. The exosomes prepared in this way can be used as tumor vaccines for clinical research.

Embodiment 2

[0118] Identification of exosomes prepared by the present invention

[0119] Exosome precipitation was fixed with 4% paraformaldehyde at 4°C for 1 hour, washed twice with PBS, made into suspension drops, fixed with 1% osmic acid, dehydrated with gradient alcohol, stained with lead-uranium in ultra-thin sections, and then examined for transmission electron microscopy View the photo below. The result is as figure 1 As shown, exosomes are uniform 30-90nM vesicles.

Embodiment 3

[0121] The invention prepares the exosomes characteristically expressing TfR, LAMP, HSP70, HSC70, CD9 and the tumor antigen CEA specifically expressed by the source cells.

[0122] Using western-blot ( figure 2 ) and flow cytometry analysis ( image 3 ) Analysis of exosome protein composition, tumors expressing marker proteins lysosome-associated membrane protein (lysosome-associated membrane protein, LAMP), HSP70, HSC70, transferrin receptor (Transferin receptor, TfR), CD9 and sw480 Antigen CEA.

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Abstract

The present invention discloses a high-quality exosome and a preparation method thereof, and particularly relates to an exosome, which is vesicles produced by cells and extracellular secreted, wherein the vesicles have the following characteristics that: a) the diameter is 30-90 nm; b) the density is 1.11-1.19 g/ml; c) and the vesicles contain one or a plurality of feature molecules such as TfR, LAMP, HSP70, HSC70 and CD9. The exosome of the present invention has characteristics of good uniformity and high safety, and can be subjected to mass production through the method of the present invention so as to meet clinical requirements.

Description

technical field [0001] The invention relates to the fields of biology and medicine, in particular to an exosome with good uniformity and high safety and a method for preparing the exosome on a large scale. Background technique [0002] Malignant tumors are major diseases that seriously threaten human life and quality of life. Tumor immunotherapy is a necessary supplement to the three major treatment methods of surgery, chemotherapy and radiotherapy, and it is also a hot spot in tumor treatment and immunology research. With the successive discovery and identification of many tumor-specific antigens and tumor-associated antigens, as well as the deepening of people's research on the mechanism of antigen presentation and T cell activation, the application of various forms of tumor vaccines to induce the body's T cell-mediated The application research of specific anti-tumor immune function has been developed rapidly. However, due to the weak immunogenicity of most tumors, in th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09C12N5/0784C12N5/0783A61K39/00A61P35/00A61P37/02A61P25/00A61P1/00
Inventor 王建莉杨菲蔡志坚
Owner ZHEJIANG UNIV
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