Methods for isolating vesicles from biological fluids

Patent metadata

Field	Value
Publication number	US-9719129-B2
Application number	US-201114122920-A
Country	US
Kind code	B2
Filing date	Jun 10, 2011
Priority date	Jun 10, 2011
Publication date	Aug 1, 2017
Grant date	Aug 1, 2017

Abstract

Provided is a device that collects vesicles and vesicle-like materials from biological fluids. Such devices comprise at least one sample loading region; at least one corresponding vesicle-capture material, wherein said vesicle-capture material comprises glass-like materials; and at least one corresponding sample receiving region, wherein passage of the biological fluid from the sample loading region through the vesicle capture material and into the sample receiving region results in the capture of vesicles. Additional methods provide for a method of isolating vesicles and vesicle-like materials from biological fluids are also provided.

First claim

What is claimed is:

1. A method of isolating vesicles from biological fluid, comprising:
(a) obtaining a biological fluid sample comprising vesicles having a diameter of less than 0.5 micrometers;
(b) loading at least a portion of said biological fluid sample into a sample loading region of a vesicle capture device, said portion of said biological fluid sample comprising blood plasma and vesicles having a diameter of less than 0.5 micrometers;
(c) passing said portion of said biological fluid sample from said sample loading region through a vesicle-capture material in said vesicle capture device, said vesicle-capture material comprising at least a first layer and a second layer, both of glassfiber, at least a portion of said vesicles having a diameter of less than 0.5 micrometers adsorbing to and being retained by the vesicle-capture material, thereby producing a supernatant,
wherein said first layer is closer to said sample loading region than said second layer,
wherein said first layer has a porosity configured to capture material that is 1.6 micrometers or greater in diameter, wherein said second layer has a porosity configured to capture material that is 0.6 micrometers to 0.8 micrometers or greater in diameter; and
(d) passing said supernatant to a sample receiving region of said vesicle capture device, thereby isolating vesicles from the biological fluid sample.

2. The method of claim 1 , wherein said vesicle-capture material further comprises a non glass-like material.

3. The method of claim 2 , wherein said non glass-like material comprises nitrocellulose.

4. The method of claim 1 , wherein said passings are accomplished through the application of vacuum pressure to the device.

5. The method of claim 1 , wherein said passings are accomplished through the application of positive pressure to the device.

6. The method of claim 1 , wherein said passings are accomplished through low-speed centrifugation of the device.

7. The method of claim 1 , wherein said vesicle capture device is configured in a multi-well plate format.

8. The method of claim 1 , further comprising eluting said vesicles from said vesicle-capture material.

9. A method of isolating vesicle-associated RNA from a biological fluid comprising vesicles, the method comprising:
(a) obtaining a biological fluid sample comprising blood plasma and vesicles having a diameter of less than 0.5 micrometers;
(b) loading said biological fluid sample into a sample loading region of a vesicle capture device;
(c) passing said biological fluid sample from said sample loading region through a vesicle-capture material in said vesicle capture device, said vesicle-capture material comprising at least a first layer and a second layer, both of glassfiber, thereby producing a supernatant,
wherein said first layer has a porosity configured to capture particles that are 1.6 micrometers or greater in diameter, and
wherein said second layer has a porosity configured to capture particles that are 0.6 micrometers to 0.8 micrometers or greater in diameter;

(d) passing said supernatant to a sample receiving region of said vesicle capture device and discarding the supernatant,
wherein said passings result in capture of at least a portion of said vesicles having a diameter of less than 0.5 micrometers from said biological fluid sample on or in said vesicle-capture material;

(e) lysing said vesicle on or in said vesicle-capture material to create a lysate; and
(f) collecting the lysate from both of said first and second layers of said vesicle-capture material into a single receiving region, thereby isolating vesicle-associated RNA from the biological fluid sample comprising vesicles.

Assignees

• Mitsuhashi Masato
• Hitachi Chemical Co Ltd
• Hitachi Chemical Company America Ltd

Inventors

• Mitsuhashi Masato

Classifications

• B01D39/2017

  the material being filamentary or fibrous · CPC title

• C12N15/1003 Primary

  Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title