Apparatus and method for isolating extracellular vesicles from a liquid suspension by exposing cells to fluid shear stress

Abstract

An apparatus and method for generating one or more extracellular vesicles from a liquid suspension including one or more cells is disclosed. The apparatus includes a conduit, a pump fluidically coupled to the conduit and a reservoir fluidically coupled to the conduit. The method includes forming a first liquid suspension including one or more cells. The method further includes exposing the first liquid suspension to a fluid shear stress to form a second liquid suspension including one or more fluid shear stressed cells and one or more extracellular vesicles released from the one or more cells in response to the fluid shear stress. The method further includes isolating the one or more extracellular vesicles from the second liquid suspension.

Description

[0001] APPARATUS AND METHOD FOR ISOLATING EXTRACELLULAR VESICLES FROM A LIQUID SUSPENSION BY EXPOSING CELLS TO FLUID SHEAR STRESS

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims priority to United States Provisional Application Number 63 / 622,409 that was filed on January 18, 2024. The entire content of the applications referenced above is hereby incorporated by reference herein.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under CA 263550 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] BACKGROUND

[0007] Extracellular vesicles, such as exosomes, can contain nucleic acids (mRNA, microRNA) and proteins. There is an increasing interest in understanding the role of extracellular vesicles in intercellular communication, the function of extracellular vesicles as a biomarker in various diseases including cancer as well as the potential of extracellular vesicles to deliver therapeutic payloads. For the latter application it is necessary to isolate extracellular vesicles in great quantity and high purity. Current methods for isolating extracellular vesicles rely on spontaneous release of extracellular vesicles from cultured cells followed by one or more methods to purify them. This can be a lengthy (days-weeks long) process and may result in relatively low yield of extracellular vesicles. The current process also relies on a large amount of cells to generate extracellular vesicles in conditioned media which may not optimize the potential of cells to generate extracellular vesicles. For these and other reasons there is a need for the subject matter of the present disclosure.

[0008] SUMMARY

[0009] Consistent with the present disclosure, a method comprises forming a first liquid suspension including one or more cells; exposing the first liquid suspension to a fluid shear stress to form a second liquid suspension including one or more fluid shear stressed cells and one or more extracellular vesicles released from the one or more cells in response to the fluid shear stress; and isolating the one or more extracellular vesicles from the second liquid suspension. In some embodiments, exposing the first liquid suspension to the fluid shear stress to form the second liquid suspension including the one or more fluid shear stressed cells and the one or more extracellular vesicles released from the one or more cells in response to the fluid shear stress comprises flowing the first liquid suspension including the one or more cells through a conduit to form the second liquid suspension including the one or more fluid shear stressed cells and the one or more extracellular vesicles.

[0010] In some embodiments, flowing the first liquid suspension including the one or more cells through a conduit to form the second liquid suspension including the one or more fluid shear stressed cells and the one or more extracellular vesicles comprises flowing the first liquid suspension through the conduit at a flow rate of between about 20 microliters per second and about 250 microliters per second.

[0011] In some embodiments, flowing the first liquid suspension including the one or more cells through the conduit to form the second liquid suspension including the one or more fluid shear stressed cells and the one or more extracellular vesicles comprises flowing the first suspension of cells through the conduit with a force of between 60 dynes / cm2and 6400 dynes / cm2to produce the one or more fluid shear stressed cells.

[0012] In some embodiments, exposing the first liquid suspension to the fluid shear stress to form the second liquid suspension including the one or more fluid shear stressed cells and one or more extracellular vesicles released from the one or more cells in response to the fluid shear stress comprises exposing the first liquid suspension to between one and twenty pulses of fluid shear stress.

[0013] Consistent with the present disclosure, a method comprises introducing the liquid suspension including the one or more cells into a conduit; introducing one or more pressure pulses into the conduit to induce a shear stress in the one or more cells to produce the one or more extracellular vesicles; and collecting the one or more extracellular vesicles.

[0014] In some embodiments, the method further comprises for the liquid suspension including the one or more cells and for the one or more cells having a flow rate through the conduit, controlling the flow rate. In some embodiments, controlling the flow rate comprises setting the flow rate to between about 20 microliters per second and about 250 microliters per second.

[0015] In some embodiments, for the conduit having a conduit length controlling the conduit length. In some embodiments, controlling the conduit length comprises setting the conduit length to between one-half inch and two inches.

[0016] In some embodiments, for the conduit having an conduit inside diameter, controlling the conduit inside diameter. In some embodiments, controlling the conduit inside diameter comprises setting the conduit inside diameter to between about 100 millimeters and 250 millimeters.

[0017] In some embodiments, introducing the one or more pressure pulses into the conduit to apply the shear stress to the one or more cells and to generate the one or more extracellular vesicles comprises introducing between about one and about twenty pressure pulses into the conduit.

[0018] Consistent with the present disclosure, an apparatus for generating one or more extracellular vesicles from a liquid suspension including one or more cells, the apparatus comprises a conduit; a pump fluidically coupled to the conduit, the pump to provide the liquid suspension including the one or more cells to the conduit; and a reservoir fluidically coupled to the conduit, the reservoir to receive one or more extracellular vesicles released from the one or more cells in response to a fluid shear stress applied to the one or more cells in the conduit.

[0019] In some embodiments, the pump provides a force at the conduit of between about 60 dynes / cm2and about 6400 dynes / cm2.

[0020] In some embodiments, the conduit has an conduit inside diameter of between about 100 millimeters and 250 millimeters. In some embodiments, the pump comprises a syringe pump. In some embodiments, the syringe pump includes the conduit, the conduit having a length of between about one-half inch and about two inches.

[0021] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosed or claimed subject matter.

[0022] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosed subject matter and together with the description, serve to explain the principles of the disclosed subject matter.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Fig. 1 A shows an illustration of an apparatus for generating one or more extracellular vesicles from a liquid suspension including one or more cells in accordance with some embodiments of the present disclosure;

[0025] Fig. IB shows an illustration of a conduit in accordance with some embodiments of the present disclosure;

[0026] Fig. 2 shows a flow diagram of a method for processing a liquid suspension including one or more cells in accordance with some embodiments of the present disclosure; and Fig. 3 shows a flow diagram of a method for generating one or more extracellular vesicles from a liquid suspension including one or more cells in accordance with some embodiments of the present disclosure.

[0027] Description

[0028] Reference will now be made in detail to the exemplary embodiments of the present disclosure described below and illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout to refer to same or like parts.

[0029] While the present invention is described herein with reference to illustrative embodiments for particular applications, it should be understood that the invention is not limited thereto. Those having ordinary skill in the art and access to the teachings provided herein will recognize additional modifications, applications, embodiments, and substitution of equivalents, that all fall within the scope of the disclosure. Accordingly, the disclosure is not to be considered as limited by the foregoing or following descriptions.

[0030] Fig. 1 A shows an illustration of an apparatus 100 for generating one or more extracellular vesicles 102 from a liquid suspension 104 including one or more cells 106 in accordance with some embodiments of the present disclosure. The apparatus 100 includes a conduit 108, a pump 110, and a reservoir 112. The pump 110 is fluidically coupled to the conduit 108. The conduit 108 is fluidically coupled to the reservoir 112. The pump 110 is not limited to a particular type of pump or a pump providing a particular force. In some embodiments, the pump 110 provides a force at the conduit 108 of between about 60 dynes / cm2and about 6400 dynes / cm2. In some embodiments, the pump 110 provides a force at the conduit 108 of between about 60 and 100, 100 and 500, 500 and 1000, 1000 and 1500, 1500 and 2000, 2000 and 2500, 2500 and 3000, 3000 and 3500, 3500 and 4000, 4000 and 4500, 4500 and 5000, 5000 and 5500, 5500 and 6000, 6000, or 6400 dynes / cm2. The term “about” as used herein indicates that the quatity being modified can be plus or minus 10% of the value of the quantity. In some embodiments, the pump 110 is a syringe pump. The pump 110 is not limited to a particular type of syringe pump. In some embodiments, the syringe pump 110 is a motor- driven precision pump that uses one or more syringes to deliver precise and accurate amounts of fluid. In some embodiments, the pump 110, for example a syringe pump, includes the conduit 108.

[0031] Fig. IB shows an illustration of the conduit 108 in accordance with some embodiments of the present disclosure. The conduit 108 is not limited to a particular shape. In some embodiments the conduit 108 is a cylinder having an inside diameter 114 of between about 100 millimeters and 250 millimeters. In some embodiments, the conduit 108 has an inside diameter of between about 100 and 150, 150 and 200, or 200 and 250 millimeters. In some embodiments, the conduit 108 has a length 116 of between about one-half inch and about two inches. In some embodiments, the conduit 108 has a length 116 of between about one-half and one, one and one and one-half, or one and one-half and two inches. The reservoir 112 provides a pool, or collection vehicle, for the one or more extracellular vesicles 102 released from the one or more cells 106.

[0032] In operation, the pump 110 provides the liquid suspension 104 including the one or more cells 106 to the conduit 108. The reservoir 112 receives one or more extracellular vesicles 102 released from the one or more cells 106 in response to a fluid shear stress applied to the one or more cells 106 in the conduit 108.

[0033] Fig. 2 shows a flow diagram 200 of a method for processing a liquid suspension including one or more cells in accordance with some embodiments of the present disclosure. The method includes forming a first liquid suspension including one or more cells (block 202), exposing the first liquid suspension to a fluid shear stress to form a second liquid suspension including one or more fluid shear stressed cells and one or more extracellular vesicles released from the one or more cells in response to the fluid shear stress (block 204), and isolating the one or more extracellular vesicles from the second liquid suspension (block 206).

[0034] In some embodiments, exposing the first liquid suspension to the fluid shear stress to form the second liquid suspension including the one or more fluid shear stressed cells and the one or more extracellular vesicles released from the one or more cells in response to the fluid shear stress includes flowing the first liquid suspension including the one or more cells through a conduit to form the second liquid suspension including the one or more fluid shear stressed cells and the one or more extracellular vesicles.

[0035] In some embodiments, flowing the first liquid suspension including the one or more cells through a conduit to form the second liquid suspension including the one or more fluid shear stressed cells and the one or more extracellular vesicles comprises flowing the first liquid suspension through the conduit at a flow rate of between about 20 microliters per second and about 250 microliters per second.

[0036] In some embodiments, flowing the first liquid suspension including the one or more cells through the conduit to form the second liquid suspension including the one or more fluid shear stressed cells and the one or more extracellular vesicles comprises flowing the first suspension of cells through the conduit with a force of between about 60 dynes / cm2and 6400 dynes / cm2to produce the one or more fluid shear stressed cells.

[0037] In some embodiments, exposing the first liquid suspension to the fluid shear stress to form the second liquid suspension including the one or more fluid shear stressed cells and one or more extracellular vesicles released from the one or more cells in response to the fluid shear stress comprises exposing the first liquid suspension to between about one and twenty pulses of fluid shear stress.

[0038] Fig. 3 shows a flow diagram 300 of a method for generating one or more extracellular vesicles 102 from a liquid suspension 104 including one or more cells 106 in accordance with some embodiments of the present disclosure. The method includes introducing the liquid suspension including the one or more cells into a conduit (block 302), introducing one or more pressure pulses into the conduit to induce a shear stress in the one or more cells to produce the one or more extracellular vesicles (block 304), and collecting the one or more extracellular vesicles (block 306).

[0039] In some embodiments the method includes for the liquid suspension including the one or more cells and for the one or more cells having a flow rate through the conduit, controlling the flow rate. In some embodiments, controlling the flow rate comprises setting the flow rate to between about 20 microliters per second and about 250 microliters per second. In some embodiments, the flow rate is between about 20 and 50, 50 and 100, 100 and 150, and 150 and 200, or 200 and 250 microliters per second.

[0040] In some embodiments, for the conduit having a conduit length, controlling the conduit length. In some embodiments, controlling the conduit length comprises setting the conduit length to between about one-half inch and two inches. In some embodiments, for the conduit having an conduit inside diameter, controlling the conduit inside diameter. In some embodiments, controlling the conduit inside diameter comprises setting the conduit inside diameter of between about 100 millimeters and 250 millimeters. In some embodiments, introducing the one or more pressure pulses into the conduit to apply the shear stress to the one or more cells and to generate the one or more extracellular vesicles comprises introducing between about one and about twenty pressure pulses into the conduit.

[0041] Reference throughout this specification to “an embodiment,” “some embodiments,” or “one embodiment.” means that a particular feature, structure, material, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases such as “in some embodiments,” “in one embodiment,” or “in an embodiment,” in various places throughout this specification are not necessarily referring to the same embodiment of the present disclosure. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments. As used herein, “about” includes values that are plus or minus ten percent of the recited value.

[0042] Although explanatory embodiments have been shown and described, it would be appreciated by those skilled in the art that the above embodiments cannot be construed to limit the present disclosure, and changes, alternatives, and modifications can be made in the embodiments without departing from spirit, principles and scope of the present disclosure.

Claims

What is claimed:

1. A method comprising: forming a first liquid suspension including one or more cells; exposing the first liquid suspension to a fluid shear stress to form a second liquid suspension including one or more fluid shear stressed cells and one or more extracellular vesicles released from the one or more cells in response to the fluid shear stress; and isolating the one or more extracellular vesicles from the second liquid suspension.

2. The method of claim 1, wherein exposing the first liquid suspension to the fluid shear stress to form the second liquid suspension including the one or more fluid shear stressed cells and the one or more extracellular vesicles released from the one or more cells in response to the fluid shear stress comprises flowing the first liquid suspension including the one or more cells through a conduit to form the second liquid suspension including the one or more fluid shear stressed cells and the one or more extracellular vesicles.

3. The method of claim 2, wherein flowing the first liquid suspension including the one or more cells through a conduit to form the second liquid suspension including the one or more fluid shear stressed cells and the one or more extracellular vesicles comprises flowing the first liquid suspension through the conduit at a flow rate of between about 20 micro liters per second and about 250 micro liters per second.

4. The method of claim 2, wherein flowing the first liquid suspension including the one or more cells through the conduit to form the second liquid suspension including the one or more fluid shear stressed cells and the one or more extracellular vesicles comprises flowing the first suspension of cells through the conduit with a force of between about 60 dynes / cm2and about 6400 dynes / cm2to produce the one or more fluid shear stressed cells.

5. The method of claim 1, wherein exposing the first liquid suspension to the fluid shear stress to form the second liquid suspension including the one or more fluid shear stressed cells and one or more extracellular vesicles released from the one or more cells in response to the fluid shear stress comprises exposing the first liquid suspension to between one and twenty pulses of fluid shear stress.

6. A method for generating one or more extracellular vesicles from a liquid suspension including one or more cells, the method comprising: introducing the liquid suspension including the one or more cells into a conduit; introducing one or more pressure pulses into the conduit to induce a shear stress in the one or more cells to produce the one or more extracellular vesicles; and collecting the one or more extracellular vesicles.

7. The method of claim 6, further comprising for the liquid suspension including the one or more cells and for the one or more cells having a flow rate through the conduit, controlling the flow rate.

8. The method of claim 7, wherein controlling the flow rate comprises setting the flow rate to between about 20 micro liters per second and about 250 micro liters per second.

9. The method of claim 6, for the conduit having a conduit length controlling the conduit length.

10. The method of claim 9, wherein controlling the conduit length comprises setting the conduit length to between about one-half inch and about two inches.

11. The method of claim 10, for the conduit having a conduit inside diameter, controlling the conduit inside diameter.

12. The method of claim 11, wherein controlling the conduit inside diameter comprises setting the conduit inside diameter to between about 100 millimeters and about 250 millimeters.

13. The method of claim 6, wherein introducing the one or more pressure pulses into the conduit to apply the shear stress to the one or more cells and to generate the one or more extracellular vesicles comprises introducing between about one and about twenty pressure pulses into the conduit.

14. An apparatus for generating one or more extracellular vesicles from a liquid suspension including one or more cells, the apparatus comprising: a conduit;a pump fluidically coupled to the conduit, the pump to provide the liquid suspension including the one or more cells to the conduit; and a reservoir fluidically coupled to the conduit, the reservoir to receive one or more extracellular vesicles released from the one or more cells in response to a fluid shear stress applied to the one or more cells in the conduit.

15. The apparatus of claim 14, wherein the pump provides a force at the conduit of between about 60 dynes / cm2and about 6400 dynes / cm2.

16. The apparatus of claim 14, wherein the conduit has an conduit inside diameter of between about 100 millimeters and about 250 millimeters.

17. The apparatus of claim 14, wherein the pump comprises a syringe pump.

18. The apparatus of claim 17, wherein the syringe pump includes the conduit, the conduit having a length of between about one -half inch and about two inches.

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