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Methods of detecting therapeutic exosomes

An exosome, therapeutic technology, applied in the field of cell biology, molecular biology and genetics, medicine, can solve the problem that the molecular or biochemical basis has not been elucidated

Inactive Publication Date: 2014-03-12
AGENCY FOR SCI TECH & RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To date, most bioefficacy studies have been limited to the immune response of immune cells to exosomes, especially dendritic cells 2 , but the molecular or biochemical basis of these biological responses has not been elucidated

Method used

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  • Methods of detecting therapeutic exosomes
  • Methods of detecting therapeutic exosomes
  • Methods of detecting therapeutic exosomes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0332] Example 1. Materials and Methods - Preparation of Exosomes Exosomes

[0333] Exosomes were purified from huES9.E1-derived MSC conditioned cultures (CM) using HPLC as previously described. Briefly, CM collected from MSC cultures was concentrated 50-fold by tangential flow filtration (TFF) using a membrane with a 100 kDa MWCO (Sartorius, Goettingen, Germany).

[0334] Then, CM passed through a chromatography column (TSK Guard column SWXL, 6x40mm and TSK gel G4000SWXL, 7.8x300mm, Tosoh Corp., Tokyo, Japan).

[0335] Exosomes were collected from the first peak of elution and concentrated using 100 kDa MWCO filters (Sartorius). Filter exosomes with a 0.22 µm filter before storage or use.

Embodiment 2

[0336] Example 2. Materials and methods - LC MS / MS

[0337] Proteins in 2 ml dialyzed exosomes were reduced, alkylated and trypsinized as described (20).

[0338] Then, the samples were desalted by passing the digestion mixture through a conditioned Sep-Pak C-18SPE kit (Waters, Milford, MA, USA) with 3% acetonitrile (ACN) (New JT Baker, Phillipsburg, NJ) and 0.1% Wash twice with formic acid (FA) buffer and elute with 70% ACN and 0.1% FA buffer.

[0339] Eluted samples were then dried to approximately 10% of their original volume by removing the organic solvent in a vacuum centrifuge.

[0340] To reduce sample complexity, offline peptide fractionation was performed with an HPLC system (Shimadzu, Japan) through a Polysulfoethyl SCX column (200 mm × 4.6 mm) (PolyLC, USA).

[0341] 1ml / min, mobile phase A (5mM KH 4 PO 4 +30% acetonitrile) and mobile phase B (5mM KH 4 PO 4 +30% acetonitrile +350mM KCl).

[0342] Collect and dry into 8 fractions in a vacuum centrifuge.

[03...

Embodiment 3

[0352] Example 3. Materials and Methods - Antibody Chips

[0353] According to the manufacturer's instructions, use a Biotinylated Human Antibody Chip I (RayBio, Norcross, GA) detected the presence of cytokines and other proteins from 500 l of unconditioned cultures and exosomes from three independent preparations.

[0354] Cytokines and other proteins were considered present in exosomes if the signal intensity was 2-fold higher (p<0.05) than in non-conditioned cultures.

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Abstract

We describe a method of detecting a therapeutic exosome, the method comprising detecting an activity of an exosome. The activity may be selected from the group consisting of: (a) immunodulatory activity; (b) complement inhibition activity; (c) proteasome activity; (d) glycolytic enzyme activity; (e) anti-oxidative activity; (f) extracellular matrix (ECM) modifying activity; (g) NT5E (CD73) ecto-5'-ectonucleotidase activity; (h) ion homeostasis activity; and (i) chaperone activity. If the exosome is detected as having one or more such activities, the exosome is likely to comprise a therapeutic exosome having therapeutic activity.

Description

technical field [0001] The present invention relates to the fields of medicine, cell biology, molecular biology and genetics. The present invention relates to the field of medicine. Background technique [0002] Exosomes were once thought of as cells' "garbage bags" for discarding unwanted proteins 1 . However, exosomes are increasingly recognized to have important physiological functions, especially in cellular communication. [0003] Exosomes are 50-100ηm double lipid membrane vesicles secreted by many cell types 2 . They belong to a class of secreted cellular products called microsomes, which broadly encompasses all secreted membrane vesicles. In addition to exosomes, microparticles include microvesicles (100-1000ηm), ectosomes (50-200ηm), membrane granules (50-80ηm), exosome-like vesicles (20-50ηm) and apoptotic vesicles (50 -500ηm). The main distinguishing parameter of these different classes of particles is their size, with the most well-defined class being exos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/48
CPCG01N33/5005G01N2570/00G01N33/6848G01N33/68A61P11/00A61P11/02A61P17/00A61P17/02A61P17/06A61P25/16A61P25/28A61P29/00A61P35/00A61P37/02A61P37/08A61P9/00A61P9/10A61P3/10
Inventor 林赛娟
Owner AGENCY FOR SCI TECH & RES
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