Systems and methods for controlled membrane disruption

Patent metadata

Field	Value
Publication number	US-12465912-B2
Application number	US-202017602728-A
Country	US
Kind code	B2
Filing date	Apr 10, 2020
Priority date	Apr 11, 2019
Publication date	Nov 11, 2025
Grant date	Nov 11, 2025

Abstract

A module of microfluidic device used for applying shear stress to cells ( 501 ) in order to deliver payloads ( 502 ) to the cells ( 501 ). The module includes cross junction modules ( 105 ), serpentine modules and squeezing-relaxing modules ( 1101 ). The devices and related methods result in increased payload ( 502 ) delivery and reduced loss in cell ( 501 ) viability.

First claim

The invention claimed is:

1 . A microfluidic device for delivering a payload into a cell, the device comprising:
a microfluidic chip of a generally planar shape and having a plurality of channels and defined therein and which includes:
a first input port configured to receive cells suspended in a first fluid solution;
a first channel of the plurality, the first channel extending along a first axis;
a second input port configured to receive a payload suspended in a second fluid solution;
a second channel of the plurality of channels, the second channel extending along a second axis transverse to the first axis;
a third channel of the plurality of channels, the third channel extending along the first axis;
a fourth channel of the plurality, the fourth channel extending along the second axis;
a cross-junction, at which the first channel, the second channel, the third channel and the fourth channel intersect at a central intersection along a center of the cross-junction,
wherein the first input port is fluidly coupled to the first channel and the first channel extends between the first input port and the intersection such that a flow of the first fluid solution from the first input port flows through the first channel into the central intersection;
wherein the second input port is fluidly coupled to the third channel and the third channel extends between the second input port and the intersection such that a flow of the second fluid solution from the second input port flows through the third channel into the central intersection;
wherein the first and third channels extend from opposite directions toward the intersection such that, when the first and second fluid solutions are introduced into the first and second input ports, the flows of the first and second fluid solutions combine in the cross-junction and form a stagnation point in the central intersection which causes shear to be applied onto cellular membranes of the cells in the first fluid solution so that the cellular membranes stretch and form pores that receive and associate with the payload from the second fluid solution, thereby loading cells with the payload; and
an output port that is fluidly coupled to both the second and fourth channels, wherein the second and fourth channels extend from the intersection in opposite directions along the second axis such that, when the first and second fluid solutions are introduced into the first and second input ports and combine at the central intersection, the loaded cells in the combined first and second solution flow from the central intersection and out the output port.

2 . The microfluidic device of claim 1 , wherein a width of an opening of each of the first and third channels at the central intersection are the same, and wherein a width of each of the openings of the second and fourth channels at the central intersection are same, so that the stagnation point is at a center of the central intersection.

3 . The microfluidic device of claim 1 , wherein a width of an opening of each of the first and third channels at the central intersection are greater than a width of each of the openings of the second and fourth channels at the central intersection.

4 . The microfluidic device of claim 1 , wherein a width of an opening of each of the first and third channels at the central intersection are between 15 and 30 micrometers, and wherein a width of an opening of each of the second and fourth channels at the central intersection are between 20 and 50 micrometers.

5 . The microfluidic device of claim 1 , wherein the first and third channels taper toward the central intersection so that flows of the first and second fluid solutions are focused toward a center of the central intersection.

6 . The microfluidic device of claim 1 , wherein the first channel tapers toward the central intersection and wherein the third channel is wider than the first channel at the central intersection.

7 . The microfluidic device of claim 1 , further comprising a constriction zone in one or both of the second and fourth channels between the central intersection and the output port,
wherein the constriction zone comprises a narrowing of the respective channel that, when the fluid flows through the plurality of channels, stretches the loaded cells to cause the payload associated with the cell membranes to be encapsulated within the cells.

8 . The microfluidic device of claim 7 , wherein the constriction zone is a serpentine pathway comprising a plurality of S-shaped bends that, when the fluid flows through the plurality of channels, induce rotation of the loaded cells, thereby increasing interaction between the payload and cells.

9 . The microfluidic device of claim 8 , wherein an internal width of the serpentine pathway is less than an internal width of a post-mix channel between the cross-junction and the serpentine pathway.

10 . The microfluidic device of claim 7 , wherein the constriction zone has a squeezing-relaxing portion in which the respective channel includes a series of squeezing sections alternating with a series of relaxing sections, wherein the squeezing sections have an internal width that is narrower than an internal width of the relaxing sections that cause the payload to be further encapsulated within the loaded cells when the fluid flows through the plurality of channels.

11 . The microfluidic device of claim 7 , further comprising a relaxing zone disposed in the second and/or fourth channel between the intersection and the constriction zone,
wherein the respective channel in the relaxing zone has an enlarged width that, when fluid flows through the plurality of channels, reduces stress in the cell membranes of the loaded cells.

12 . The microfluidic device of claim 1 , wherein the device facilitates loading of both nucleated and enucleated cells.

13 . A method for delivering a payload into a cell, the method comprising: providing the microfluidic device of claim 1 ;
introducing a plurality of cells into the first channel, via the first port, the first channel extending to the cross-junction module of the microfluidic device introducing a payload into the third channel via the second port; and causing a flow of fluid from the third channel, through the central intersection, and into the second and fourth channels forming the stagnation point in the central intersection, wherein the flow causes shear to be applied onto cellular membranes of the plurality of cells at the stagnation point so that the cellular membrane stretches and forms pores that receive and associate the payload with the cellular membrane, and wherein after receiving the payload the cell flows from the central intersection through one of the second or fourth channels.

14 . The method of claim 13 , wherein widths of openings of the first and third channels at the central intersection are the same so that a flow of fluid from the first channel and the flow of fluid from the third channel are equal so that the stagnation point is at a center of the central intersection.

15 . The method of claim 13 , wherein the first and third channels taper toward the central intersection so that a flow of fluid from the first channel including the cell is focused toward a center of the central intersection.

16 . The method of claim 13 , wherein after receiving the payload and flowing through the second channel or the fourth channel the cell flow through a constriction zone of the microfluidic device that causes the payload associated with the cell membranes to be encapsulated within the cell.

17

Assignees

• Univ Hong Kong Chinese

Inventors

• Ho Yi-Ping
• Chan Michael Kenneth
• Lee Ming Ming Marianne
• Wong Chung Hong Nathaniel
• Rahman Md Habibur

Classifications

• B01L2400/0478

  pistons · CPC title

• B01L2300/0883

  Serpentine channels · CPC title

• B01L2300/0867

  Multiple inlets and one sample wells, e.g. mixing, dilution · CPC title

• B01L2300/0663

  Whole sensors · CPC title

• B01L2200/16

  Reagents, handling or storing thereof · CPC title