Isolation of Different Extracellular Vesicle (EV) Subpopulations

Patent metadata

Field	Value
Publication number	US-2022118452-A1
Application number	US-202117504947-A
Country	US
Kind code	A1
Filing date	Oct 19, 2021
Priority date	Oct 20, 2020
Publication date	Apr 21, 2022
Grant date	—

Abstract

An extracellular vesicle-containing sample can be processed using a device for isolating one or more subpopulations of the extracellular vesicles. The extracellular vesicle-containing sample is flowed through a flow chamber of the device under an applied fluid pressure, in which the device has one or more inlets and two or more outlets in fluid communication with one another via the flow chamber. The device has one or more filters in the flow chamber between the inlet(s) and at least one of the outlet(s). The extracellular vesicle-containing sample is flowed through the filter(s) in the flow chamber to sort the extracellular vesicles of extracellular vesicle-containing sample by size into two or more subpopulations of the extracellular vesicles. At least one of the subpopulations that has been sorted flows out of a corresponding one of the outlets. Surface marker-based exosome sorting using magnetic beads may be used after the size-based exosome isolation.

First claim

What is claimed is:

1 . A method of processing an extracellular vesicle-containing sample using a device for extracellular vesicle isolation and isolating one or more subpopulations of the extracellular vesicles, the method comprising:
flowing the extracellular vesicle-containing sample through a flow chamber of the device for extracellular vesicle isolation under an applied fluid pressure, the device having one or more inlets and at least two outlets which are placed in fluid communication with one another via the flow chamber, the device further having one or more filters in the flow chamber between at least one of the inlets and at least one of the at least two outlets; during the step of flowing, flowing the extracellular vesicle-containing sample through the one or more filters in the flow chamber to sort the extracellular vesicles of extracellular vesicle-containing sample by size into two or more subpopulations of the extracellular vesicles; and flowing at least one of the subpopulations of the extracellular vesicles that have been sorted by size after passing through the one or more filters out of a corresponding one of the outlets.

2 . The method of claim 1 , further comprising:
after the step of flowing the extracellular vesicle-containing sample through the one or more filters in the flow chamber to sort the extracellular vesicles of extracellular vesicle-containing sample by size into two or more subpopulations of the extracellular vesicles, introducing labeled magnetically-responsive particles to at least one of the subpopulations of the extracellular vesicles that are labeled to bind to at least some members of the subpopulations of the extracellular vesicles; and flowing the subpopulations of the extracellular vesicles through a channel having a magnetic field applied thereto to further separate members of the subpopulation that are bound to the labeled magnetically-responsive particles from members of the subpopulation that are not bound to the labeled magnetically-responsive particles and flowing those further separated members from different outlets of the channel.

3 . The method of claim 2 , further comprising, during introducing labeled magnetically-responsive particles to at least one of the subpopulations of the extracellular vesicles that are labeled to bind to at least some members of the subpopulations of the extracellular vesicles, acoustic mixing the magnetically-responsive particles with the at least one of the subpopulations of the extracellular vesicles.

4 . The method of claim 2 , wherein a combination of separation by mechanical filtration and binding to labeled magnetically-responsive particles of the at least one of the subpopulations of the extracellular vesicles fractionalizes extracellular vesicles by both size and surface markers.

5 . The method of claim 1 , wherein the one or more inlets include different inlets including (i) at least one inlet for introducing the extracellular vesicle-containing sample to the flow chamber for flow through the one or more filters and (ii) at least one inlet for providing an evacuating flow to flow least one of the subpopulations of the extracellular vesicles from a portion of the flow chamber.

6 . The method of claim 5 , wherein at least one inlet for providing the evacuating flow is positioned between two filters of the one or more filters in the flow chamber such that the evacuating flow isolates the extracellular vesicles in the extracellular vesicle-containing sample having sizes between the two filter filtering sizes associated with the two filters and causes the extracellular vesicles that have been isolated to flow out a corresponding outlet of the at least two outlets.

7 . The method of claim 1 , wherein the at least two outlets include (i) at least one waste outlet for a portion of the extracellular vesicle-containing sample that has passed through all of the one or more filters and (ii) at least one filtered extracellular vesicle outlet out of which flows at least one of the subpopulations of the extracellular vesicles that have been sorted by size after passing through some, but not all, of the one or more filters.

8 . The method of claim 1 ,
wherein the one or more inlets include (i) at least one inlet for introducing the extracellular vesicle-containing sample to the flow chamber for flow through the one or more filters and (ii) at least one inlet for providing an evacuating flow to flow at least one of the subpopulations of the extracellular vesicles from a portion of the flow chamber; and wherein the at least two outlets includes: (i) at least one waste outlet for a portion of the extracellular vesicle-containing sample that has passed through all of the one or more filters and (ii) at least one filtered extracellular vesicle outlet out of which flows at least one of the subpopulations of the extracellular vesicles that have been sorted by size after passing through some, but not all, of the one or more filters.

9 . The method of claim 8 , wherein the at least two outlets include three or more outlets including at least one waste outlet for a portion of the extracellular vesicle-containing sample that has passed through all of the one or more filters and at least two filtered extracellular vesicle outlets out of each of which flow a corresponding one of the subpopulations of the extracellular vesicles that have been sorted by size after passing through some, but not all, of the one or more filters.

10 . The method of claim 9 , where the at least two filtered extracellular vesicle outlets sort the extracellular vesicle-containing sample into subpopulations of the extracellular vesicles having different and non-overlapping size ranges.

11 . The method of claim 8 , wherein in series, a filtration size of the one or more filters in the flow chamber progressively decreases between the at least one inlet for introducing the extracellular vesicle-containing sample and the at least one waste outlet for a portion of the extracellular vesicle-containing sample that has passed through all of the one or more filters.

12 . The method of claim 11 , wherein the filtration size of the one or more filters in the flow chamber are filters between 200 nanometers and 30 nanometers in size.

13 . The method of claim 1 , wherein the extracellular vesicle-containing sample includes exosomes released from pancreatic tumor tissue.

14 . The method of claim 1 , wherein the extracellular vesicles in the extracellular vesicle-containing sample are between 50-200 nanometers in size.

15 . The method of claim 1 , wherein, prior to the step of flowing the extracellular vesicle-containing sample through a flow chamber of the device for extracellular vesicle isolation under an applied fluid pressure, the extracellular vesicle-containing sample is pre-filtered to remove cells, bacterial contaminants, and other large cellular fragments.

16 . The method of claim 1 , wherein the extracellular vesicle-containing sample is or is derived from one of blood, plasma, urine, cerebral spinal fluid, semen, and breast milk.

17 . A system for processing an extracellular vesicle-containing sample to isolate one or more subpopulations of the extracellular vesicles, the system comprising:
a flow chamber defining a fluid pathway through the flow chamber extending from at least one inlet at which the extracellular vesicle-containing sample is introduced into the flow chamber, through a plurality of filters in the flow chamber in which the filters are of progressively decreasing filtration size along the fluid pathway so as t

Assignees

• Univ Leland Stanford Junior

Inventors

• Demirci Utkan
• Ozen Mehmet Ozgun
• Durmus Naside Gozde
• Gupta Rakhi
• Pitteri Sharon
• Garcia Marques Fernando Jose

Classifications

• B01L2300/0681

  Filter · CPC title

• B01L2200/0652

  Sorting or classification of particles or molecules · CPC title

• B01L3/502761

  specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads or physically stretching molecules · CPC title

• B01L2400/043

  magnetic forces · CPC title

• B01L2300/0877

  Flow chambers · CPC title