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Methods of measuring exosomes using intrinsic fluorescence

a technology of exosomes and fluorescence, applied in the direction of fluorescence/phosphorescence, material analysis through optical means, instruments, etc., can solve the problems of inability to accurately and rapidly measure the absolute number of extracellular vesicles, laborious and time-consuming current methods, and current ultra-centrifugation protocols are commercially unreproducibl

Inactive Publication Date: 2020-01-23
LONZA SALES AG
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AI Technical Summary

Benefits of technology

The patent describes methods for detecting extracellular vesicles without using additional dyes or fluorescences. The methods involve measuring the intrinsic fluorescence of the vesicles using a specific excitation wavelength and emission wavelength range. The sample can be separated into fractions, such as by chromatography, prior to the detection step. The amount of extracellular vesicles can be determined by comparing the fluorescence emission signal to a standard. The extracellular vesicles can also contain therapeutic payloads.

Problems solved by technology

Although extracellular vesicles are beginning to be used in commercial processes and produced in industrial quantities, methods of accurately and rapidly measuring extracellular vesicle concentration and absolute number remain scarce.
Current approaches for the detection, isolation and purification of extracellular vesicles derived from cell culture or other biological samples requires laborious and time consuming methods.
For example, current ultra-centrifugation protocols are commercially unreproducible, as they produce a heterogeneous mix of extracellular vesicles, other cellular vesicles and macromolecular complexes and can lead to vesicle aggregation.

Method used

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  • Methods of measuring exosomes using intrinsic fluorescence
  • Methods of measuring exosomes using intrinsic fluorescence
  • Methods of measuring exosomes using intrinsic fluorescence

Examples

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example 1

ion Exchange Chromatography Profiles of Purified Exosomes and Cell Culture Harvest at Different Excitation and Emmission Spectra

[0083]To understand fundamental properties of exosomes and develop analytical tools for studying exosomes, Optiprep™ purified exosome preparations and cell culture harvest were analyzed by strong anion exchange chromatography (AEX). Exosomes from HEK293SF cells grown in serum-free conditions were processed as described in the methods above. For cell culture harvest, HEK293SF cells were grown in serum-free medium, and 600 ml of supernatant were collected. Cell culture harvest was then centrifuged at 1600 g for 10 minutes and filtered through a 0.8 μm filter to remove cellular debris before chromatographic analysis. AEX was monitored at 210, 254, and 280 nm and was performed using a CIMac monolithic QA-1 mL column from BIA Separations. The Optiprep™ purified exosome stock concentration was measured to be 4×1012 particles / mL by nanoparticle tracking assay (NTA...

example 2

ng the Optimal Fluorescence Spectrum for Analyzing AEX-Purified Exosomes

[0086]To optimize the excitation and emission wavelengths for the detection of exosomes, wavelength scanning on Optiprep™ purified exosomes was carried out. To determine the optimal excitation wavelength, Optiprep™ purified exosomes were analyzed by AEX, while the emission wavelength was held constant at 573 nm, and the excitation wavelength was scanned from 280 nm to 560 nm (FIG. 4A). To determine the optimal emission wavelength, Optiprep™ purified exosomes were analyzed by AEX, while the excitation wavelength was held constant at 556 nm, and the emission wavelength was scanned from 570 nm to 770 nm (FIG. 4B). These results indicate that the optimal wavelengths for detecting intrinsic fluorescence of exosomes is an excitation wavelength of 556 nm and emission wavelength of 573 nm.

example 3

Liposomes and Cell-Derived Exosomes Do Not Have the Same Fluorescence Spectra

[0087]To determine if the fluorescence profile determined in Example 1 was specific to cell-derived exosomes, synthetic liposomes were also analyzed by AEX at ex556 / em573. Synthetic clodronate liposome controls formulated in PBS were purchased. The liposomes were composed of a mixture of phosphatidylcholine and cholesterol and formulated to be between 0.15-3 μm in size. Before analysis, the liposomes were filtered through a 0.45 μm cellulose acetate filter to remove the larger sized liposomes. The resulting filtered liposomes were analyzed by NTA (FIG. 5A) and transmission electron microscopy (FIG. 5B) confirming that they are similar in size and shape to cell-derived exosomes (see FIG. 1B).

[0088]To confirm that the synthetic liposomes had similar size-dependent characteristics of cell-derived exosomes, both populations were analyzed by size-exclusion chromatography (SEC) and monitored by UV absorbance. As ...

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Abstract

Described herein are novel rapid and reliable methods of detection of extracellular vesicles and quantifying extracellular vesicle concentrations and absolute number from various sources, including raw cell harvest. The methods described herein comprise detection of intrinsic fluorescence of extracellular vesicles in biological samples. Extracellular vesicles analyzed by the methods of this application have a stereotypical elution profile distinct from known contaminants. The methods described herein are a significant improvement over the state of the art and fulfills an unmet need in the field of extracellular vesicle manufacturing and quality control.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application claims the priority benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62 / 434,985 filed on 15 Dec. 2016, and U.S. Provisional Application No. 62 / 542,697 filed on Aug. 8, 2017, both of which are incorporated by reference herein in their entirety.BACKGROUND OF THE INVENTIONDescription of the Related Art[0002]Exosomes and other small extracellular vesicles are increasingly well-recognized biological particles. Although extracellular vesicles are beginning to be used in commercial processes and produced in industrial quantities, methods of accurately and rapidly measuring extracellular vesicle concentration and absolute number remain scarce. Current approaches for the detection, isolation and purification of extracellular vesicles derived from cell culture or other biological samples requires laborious and time consuming methods. For example, current ultra-centrifugation protocols are commercially unreproduc...

Claims

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Application Information

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IPC IPC(8): G01N21/64G01N21/76
CPCG01N21/76G01N21/6486
Inventor HOUDE, DAMIAN J.CHOI, YOUNG JUNKULMAN, JOHN D.WILLIAMS, DOUGLAS E.
Owner LONZA SALES AG
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