WO2022187319A2 - Acoustofluidic separation of cells and particles via acoustic radiation force dynamics - Google Patents

Abstract

Devices and methods enable in-line separation/filtration of cells or particles during fill- and-finish operations without requiring external processing steps. Cell/particle separation and volume reduction is achieved using cationic microbubbles or microparticles in combination with acoustic radiation force via high pressure ultrasound waves, which deflect the cells or particles toward the wall to concentrate them into one side of the channel. The rate of cell/particle movement is directly dependent on their acoustic contrast, which is primarily a function of size, density, and compressibility. Therefore, cells or particles with different size, density, or compressibility can be separated with this technique.

Description

ACOUSTOFLUIDIC SEPARATION OF CELLS AND PARTICLES VIA ACOUSTIC RADIATION FORCE DYNAMICS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patent application no. 63/155,621, filed March 2, 2021, the complete contents of which are herein incorporated by reference.

FIELD OF THE INVENTION

Embodiments generally relate to cell/particle separation and volume reduction and, more particularly, separation devices and techniques that employ acoustic radiation forces.

BACKGROUND

Cell-based therapies are a rapidly expanding market that have significant potential to improve patient outcomes. Cell/particle separation and volume reduction is a key step in cell therapy manufacturing processes. However, cell therapy manufacturing processes are currently limited by inefficient filtration and separation methods. Current processing methods generally require an additional step using physical filters or other external specialized equipment like centrifuges to separate cells or particles, which adds complexity and increases cost and safety risks.

An existing product from Millipore/FloDesign Sonics (Ekko cell processing system) induces aggregation of cells or particles into pressure nodes for volume reduction but has limited size separation capabilities.

Faridi, et ah, “MicroBubble activated acoustic cell sorting” Biomed Microdevices (2017) 19:23, describes the use of microbubbles for acoustophoresis cell sorting applications. They describe a microfluidic-based microbubble-activated acoustic cell sorting method that utilizes specific antibodies to attach microbubbles onto cells. The method utilized a glass- silicone microfluidic flow chamber with a maximum channel dimension of 535 pm. In addition, the method uses continuous ultrasound waves as the ultrasound source. SUMMARY

Embodiments herein relate to the use of acoustofluidics technology to separate cells and particles via acoustic radiation force in combination with cationic charged microbubbles or particles.

A significant aspect of present embodiments is the use of ultrasound waves in combination with cationic microbubbles or microparticles to enhance separation of cells in solution. Embodiments of the disclosure enable rapid sorting of biological cells using cationic gas-filled microbubbles or solid microparticles (such as metal ion or polymer-based particles). The cationic surface charge of the gas-filled microbubbles or solid microparticles causes them to interact with the anionic surface of biological cells via charge-charge interactions, which enables rapid separation via acoustic radiation forces within the acoustofluidic channel due to significantly higher acoustic responsiveness of microbubbles and microparticles to acoustic pressures compared to individual cells alone. Cells that are associated with the cationic microbubbles or microparticles respond to acoustic radiation forces which push them toward one side of the channel where they can be collected through a separate output port.

Existing acoustic cell separation technologies do not utilize cationic microbubbles or microbeads, and the specific material properties and ultrasound parameters required to enhance separation have not been previously investigated. The unique approach of embodiments of this disclosure bears the advantage of rapidly separating cells and particles with higher efficiency than methods which do not employ cationic microbubbles or microbeads. The technology described herein enables in-line separation/filtration of cells or particles during fill-and-finish operations without requiring external processing steps.

The Examples below demonstrate the utilization of acoustic radiation forces in acoustofluidic channels for cell/particle separation and volume reduction.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A is a perspective view of an exemplary filtration device according to some embodiments. Figure IB is a first end view of the exemplary filtration device, showing an inlet.

Figure 1C is a cross-sectional view C — C taken from Figure ID.

Figure ID is a top plan view of the exemplary filtration device.

Figure IE is a second end view of the exemplary filtration device, showing a plurality of outlets.

Figure IF is a first side view of the exemplary filtration device.

Figure 1G is a second side view of the exemplary filtration device.

Figure 2 is an exemplary system for cell separation.

Figure 3A is a diagram of the separation section of the filtration device near the inlet end.

Figure 3B is a diagram of the separation section of the filtration device just before the main channel partitions into a plurality of separate outlet conduits.

Figure 4 is a side view of the exemplary filtration device with an enlarged view of a segment of the main channel showing a serpentine flow path applicable to some embodiments.

Figure 5A shows an exemplary serpentine channel path.

Figure 5B shows an exemplary spiral channel path.

Figure 6 is an exemplary method for cell separation.

Figure 7A is an image of polystyrene beads in an acoustofluidic channel before ultrasound exposure.

Figure 7B is an image of the same polystyrene beads in the acoustofluidic channel depicted in Figure 7A, except after ultrasound exposure.

Figure 8 is representative results of acoustofluidic sorting with cationic microbubbles.

DETAILED DESCRIPTION

Figures 1A-1G present different views of an exemplary filtration/separation device 100 for acoustofluidic separation/sorting of cells. The device 100 comprises at least one channel 108 and at least one ultrasound transducer 103. The transducer 103 is positioned or positionable relative to the at least one channel 108 so that ultrasound waves from the transducer propagate into and across the at least one channel 108. A body 110 of the device 100 defines the channel 108 and provides a surface against which the transducer 103 is positioned. The transducer 103 may be, for example, a piezoelectric comprising or made of materials such as but not limited to lead zirconate titanate (PZT) or polyvinylidene fluoride (PVDF). Transducers may have different physical characteristics in different embodiments as well. For instance, transducers may have a round or rectangular face, or a face of some other geometry besides round or rectangular. The transducers may be flat or concave (focused). “Channel” as used in this disclosure may be interchanged with terms including “conduit”, “passage”, “tube”, “tubule”, “pipe”, and “flow chamber”.

The channel 108 has an inlet 101 at one end of the device 100 and multiple outlets 102 at a second end of the device 100. In the illustrated embodiment, the first and second ends are opposite one another. The channel 108 comprises a separation region 109 proximal to the transducer 103 and in which the ultrasound waves propagate. The separation region 109 is the region of the device 100 in which cells are subjectable to acoustic radiation forces. The channel 108 may have a cross-sectional dimension of at least 1 mm. A range of channel dimensions are possible, e.g., the diameter may be between 0.5 - 5 mm. The cross-sectional dimension may be a diameter, for a channel cross-sectional shape that is round/circular, or a width or length, for a cross-sectional shape that is square or rectangular. Other cross-sectional shapes, such as other polygons, may be used in alternative embodiments.

After the separation region 109 the channel 108 divides into a plurality of smaller conduits. In the illustrated example, device 100 has three conduits 112, 113, and 114, leading respectively to separate outlets 102a, 102b, and 102c (collectively identified as outlets 102). The three partitions are of equal size in Figure 1, but these dimensions can be asymmetric in order to achieve additional volume reduction, and additional partitions (or simply a two-way split) may be implemented depending on the embodiment and the particular cells being sorted.

Device 100 and/or fluidic channels of the device may be fabricated via 3D-printing, injection molding, or other processes in polycarbonate plastic or other solid materials like other polymers, metals, or alloys. The relatively large size of the channel 108 compared to many traditional microfluidic channels makes polymer material construction more viable than in such traditional microfluidic systems.

Figure 2 shows an exemplary system 200 which includes a device 100. The system 200 comprises a signal generator 201 configured to produce the one or more controls signals for the transducer 103. Arrows in Figure 2 depict the general direction of the acoustic radiation forces across the separation region 109. The signal generator 201 may be part of, or connected to and itself controlled by, one or more processors 202. The system 200 further comprises a source 203 of a population of cells and a source 204 of gas-filled microbubbles or microparticles having cationic charge. Gas-filled microbubbles may have a shell composed of phospholipids or polymers with a positive surface charge, encapsulating a biocompatible gas such as perfluorocarbon (e.g., C3F8 or C4F10). Zeta potential measurements of microbubble or microparticle surface charge may be between +10 mV and +50 mV. Microbubbles or microparticles (e.g., microbeads) may have a diameter between 0.5 - 100 pm (in particular between 1 - 20 pm for some embodiments). The type of charge on a microbubble surface matters, with different effects on cell sorting efficiency depending on whether the charge is cationic (positive), neutral (i.e., no charge), or anionic (negative). The cells typically have a negative charge, so a cationic charge of the microbubbles attracts them to the cell surface, and this coupling significantly increases the ultrasound displacement force.

The source 203 may be an upstream module within a cell therapy manufacturing line, for example. The cells from source 203 and gas-filled microbubbles or microparticles from source 204 may be combined at block 205, which may be a mixing reservoir or mixing line, for example. The resulting mixture then flows into the inlet 101 of device 100 and specifically into the separation region 109. Populations of cells can be passed through an acoustofluidic flow channel at concentrations of lxlO⁴ cells/mL - lxlO⁹ cells/mL with microbubble or microparticle concentrations at lxl0⁵/mL - lxlO¹¹ /mL for rapid sorting (typically microbubble or microparticle concentrations are between 10-fold to 100-fold higher than the cell concentration in the solution).

Figures 3A and 3B diagrammatically depict exemplary effects of acoustic radiation forces within the separation region 109 of the device 100. The net flow of the population of cells is depicted by a vertical arrow, according to the figures’ orientation on the page. Horizontal arrows depict the primary direction of the acoustic radiation forces, according to the figures’ orientation on the page. A nonhomogeneous population of cells is represented by a plurality of circles which, for ease of illustration, include three different sizes to represent three different cell types (not to scale). The dashed lines are merely a visual guide that visually separate respective thirds of the channel 108 which, according to the longitudinal direction of the device 100, align with the openings to conduits 112, 113, and 114. Figure 3 A shows the cell population at the start of the separation region 109 nearest the inlet 101. Figure 3B shows the cell population at the end of the separation region 109 nearest the outlets 102.

Figures 3A and 3B respectively show a “before” separation and “after” separation juxtaposition. The cationic surface charge of the gas-filled microbubbles or solid microparticles causes them to interact with the anionic surface of biological cells via charge-charge interactions, which enables rapid separation via the acoustic radiation forces within the separation region 109 due to significantly higher acoustic responsiveness of microbubbles and microparticles to acoustic pressures compared to individual cells alone. Cells that are associated with the cationic microbubbles or microparticles respond to acoustic radiation forces which push them toward one side of the channel 108. Generally, based on the acoustic radiation force, the associated microbubbles/cells would only move in one direction (away from the transducer). Focusing the microbubbles/cells along one axis, such as depicted by Figure 3A, may be done in some embodiments to help separate them during acoustic radiation force exposure. This can be achieved using, for example, a sheath fluid at the input to focus the microbubbles/cells toward the center or near side of the channel.

Returning to Figure 2, the multiple conduits and outlet ports allow siphoning the concentrated cells or particles within specific size, density, and compressibility characteristics. When the channel 108 divides into conduits 112, 113, and 114, the three different cell types now separated within chamber 108 (as depicted in Figure 3B) flow respectively into different conduits and, from there, they can be collected from the separate outlets 102 into downstream destinations 212, 213, and 214 such as reservoirs or downstream lines, as depicted in Figure 2.

Figure 4 depicts a side view of the device 100 and channel 108, as well as an enlarged view of a section of the channel 108. The enlarged view shows how the channel’s interior may, in at least some embodiments, follow a non-linear path, in this case a serpentine path. Figures 5A and 5B juxtapose two alternative flow path geometries, namely a serpentine path and a spiral path. In both of these non-limiting examples, the main direction of the acoustic radiation forces would generally be into or out of the page. A snaking/serpentine channel geometry or other non linear path increases residence time within the separation region, and thus the ultrasound beam, and enable larger displacement of cells or particles. Other channel geometries besides those depicted may be utilized in some embodiments (e.g., concentric spiral, rectilinear, etc.). Figure 6 presents a flowchart 600 of an exemplary method 600 for cell separation and/or volume reduction. At block 601, a cell population is combined with microbubbles or microparticles having cationic surface charge. At block 602, the combination is flowed through a fluidic channel, as described above. The combination of block 601 may occur before or within the flow channel. At block 603, the flow is subjected to acoustic radiation forces by the transmission of ultrasound waves across the channel to separate target cells associated with the cationic microbubbles or microparticles from other cells or particles in the sample. The flow stream is physically divided into separate subchannels/conduits at block 604. At block 604 the target cells associated with the cationic microbubbles or microparticles and/or other cells from other conduits are collected. After the bubbles/particles associate with the cells, the resulting combination may have a net charge of positive or negative, or simply neutral.

In this approach, cell/particle separation and volume reduction is achieved using acoustic radiation force via high pressure ultrasound waves, which deflect the cells or particles toward the wall to concentrate them into one side of the channel. Pulsed ultrasound waves to induce acoustic radiation force dynamics may be used without standing waves. The rate of cell/particle movement is directly dependent on their acoustic contrast, which is primarily a function of size, density, and compressibility. Therefore, cells or particles with different size, density, or compressibility can be separated with this technique. This technology enables in-line filtration and volume reduction during aseptic fill-and-finish processes for cell therapy manufacturing, which simplifies processing and reduces safety risks.

The specific parameters of the ultrasound source signal (e.g., center frequency, ultrasound pressure, pulse duration, pulse interval, etc.), cationic microbubbles or microbeads (e.g., size, concentration, material properties, etc.), and acoustofluidic chamber design (e.g., diameter, angle, flow rate, etc.) are factors that may be selected and/or adjusted to support and maximize efficiency of cell sorting within the acoustofluidic device. Continuous wave has a duty cycle of 100%. By definition a pulse would be any signal with a duty cycle less than 100%, but in practice many exemplary embodiments use a duty cycle of 50% or lower for pulses.

Previously acoustic cell separation technologies utilized acoustic standing waves to entrap and separate particles without any microbubbles or microbeads. However, some exemplary methods herein utilize short ultrasound pulses instead of continuous waves to induce acoustic radiation forces on cell/microbubbles or cell/microbead complexes that enable rapid separation within the flow chamber. These optimal parameters are highly dependent on the acoustofluidic chamber design, microbubble or microbead properties, and ultrasound parameters. Therefore, our method is unique by utilizing cationic microbubbles or microbeads and ultrasound parameters with specific properties and parameters that enable efficient sorting of cells. Advantageously, the microbubbles and microparticles do not need to be, and are not in some embodiments, conjugated with an antibody.

The ultrasound frequency may be selected between 0.5-100 MHz, e.g. 1-5 MHz, to optimize separation of desired size ranges. With microbubbles or microparticles utilized for sorting, the peak negative pressure for inducing acoustic radiation force effects may be between 0.1-10.0 MPa (e.g., between 0.5-2.0 MPa in some embodiments). The duty cycle may be between 10-100% (typically between 50-100%). Duty cycles approaching 100% will typically form standing acoustic waves, but cell/particle separation is achievable without standing waves by using lower duty cycles in the range of 10-50%. Ultrasound parameters used in other applications, for instance gene delivery applications, are not suited for cell sorting via acoustic radiation force dynamics, especially in combination with cationic microbubbles or microbeads.

In gene delivery the typical desire is to cause microbubble destruction, whereas in cell sorting lower energies are desirable which cause acoustic radiation forces without rapid microbubble destruction.

One non-limiting application for embodiments of this disclosure is automated cell processing systems, for example, T cell therapy manufacturing. An exemplary embodiment may be a module of such systems. An exemplary module is connectable to automated cell therapy systems, including those which are commercially available as of the filing date of this disclosure. The device 100 of Figure 1, with or without the transducer, may be configured as a single use module that connects directly to tubing on existing automated cell processing systems for in-line processing. The plastic flow chamber maintains the closed system for cell processing, and it is producible as a sterile cartridge for GMP processes used in clinical cell therapy manufacturing. Cell therapy products do not come into contact with any components of the acoustofluidic module except the sterile channel within the single-use module which is consumable.

Some embodiments of the present invention may be a system, a device, a method, and/or a computer program product. A system, device, or computer program product may include a computer readable storage medium (or media) having computer readable program instructions thereon for causing a processor to carry out aspects of the present invention, e.g., processes or parts of processes or a combination of processes described herein.

The computer readable storage medium can be a tangible device that can retain and store instructions for use by an instruction execution device. The computer readable storage medium may be, for example, but is not limited to, an electronic storage device, a magnetic storage device, an optical storage device, an electromagnetic storage device, a semiconductor storage device, or any suitable combination of the foregoing. A non-exhaustive list of more specific examples of the computer readable storage medium includes the following: a portable computer diskette, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or Flash memory), a static random access memory (SRAM), a portable compact disc read-only memory (CD-ROM), a digital versatile disk (DVD), a memory stick, a floppy disk, a mechanically encoded device such as punch-cards or raised structures in a groove having instructions recorded thereon, and any suitable combination of the foregoing. A computer readable storage medium, as used herein, is not to be construed as being transitory signals per se, such as radio waves or other freely propagating electromagnetic waves, electromagnetic waves propagating through a waveguide or other transmission media (e.g., light pulses passing through a fiber-optic cable), or electrical signals transmitted through a wire.

Processes described herein, or steps thereof, may be embodied in computer readable program instructions which may be paired with or downloaded to respective computing/processing devices from a computer readable storage medium or to an external computer or external storage device via a network, for example, the Internet, a local area network, a wide area network and/or a wireless network. The network may comprise copper transmission cables, optical transmission fibers, wireless transmission, routers, firewalls, switches, gateway computers and/or edge servers. A network adapter card or network interface in each computing/processing device receives computer readable program instructions from the network and forwards the computer readable program instructions for storage in a computer readable storage medium within the respective computing/processing device.

Computer readable program instructions for carrying out operations of the present invention may be assembler instructions, instmction-set-architecture (ISA) instructions, machine instructions, machine dependent instructions, microcode, firmware instructions, state-setting data, or either source code or object code written in any combination of one or more programming languages, including an object oriented programming language such as Java, Smalltalk, C++ or the like, and conventional procedural programming languages, such as the "C" programming language or similar programming languages. The computer readable program instructions may execute entirely on the user's computer, partly on the user's computer, as a stand-alone software package, partly on the user's computer and partly on a remote computer or entirely on the remote computer or server. In the latter scenario, the remote computer may be connected to the user's computer through any type of network, including a local area network (LAN) or a wide area network (WAN), or the connection may be made to an external computer (for example, through the Internet using an Internet Service Provider). In some embodiments, electronic circuitry including, for example, programmable logic circuitry, field-programmable gate arrays (FPGA), or programmable logic arrays (PLA) may execute the computer readable program instructions by utilizing state information of the computer readable program instructions to personalize the electronic circuitry, in order to perform aspects of the present invention.

Aspects of the present invention are described herein with reference to flowchart illustrations and/or block diagrams of methods, apparatus (systems), and computer program products according to embodiments of the invention. It will be understood that each block of the flowchart illustrations and/or block diagrams, and combinations of blocks in the flowchart illustrations and/or block diagrams, can be implemented by computer readable program instructions and in various combinations.

These computer readable program instructions may be provided to one or more processors of one or more general purpose computers, special purpose computers, or other programmable data processing apparatuses to produce a machine or system, such that the instructions, which execute via the processor(s) of the computer or other programmable data processing apparatus, create means for implementing the functions/acts specified in the flowchart and/or block diagram block or blocks. These computer readable program instructions may also be stored in a computer readable storage medium that can direct a computer, a programmable data processing apparatus, and/or other devices to function in a particular manner, such that the computer readable storage medium having instructions stored therein comprises an article of manufacture including instructions which implement aspects of the function/act specified in the flowchart and/or block diagram block or blocks. The computer readable program instructions may also be loaded onto a computer, other programmable data processing apparatus, or other device to cause a series of operational steps to be performed on the computer, other programmable apparatus or other device to produce a computer implemented process, such that the instructions which execute on the computer, other programmable apparatus, or other device implement the functions/acts specified in the flowchart and/or block diagram block or blocks.

The flowchart and block diagrams in the Figures illustrate the architecture, functionality, and operation of possible implementations of systems, methods, and computer program products according to various embodiments of the present invention. In this regard, each block in the flowchart or block diagrams may represent a module, segment, or portion of instructions, which comprises one or more executable instructions for implementing the specified logical function(s). In some alternative implementations, the functions noted in the block may occur out of the order noted in the figures. For example, two blocks shown in succession may, in fact, be executed substantially concurrently, or the blocks may sometimes be executed in the reverse order, depending upon the functionality involved. It will also be noted that each block of the block diagrams and/or flowchart illustration, and combinations of blocks in the block diagrams and/or flowchart illustration, can be implemented by special purpose hardware-based systems that perform the specified functions or acts or carry out combinations of special purpose hardware and computer instructions.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are described.

It is noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

While exemplary embodiments of the present invention have been disclosed herein, one skilled in the art will recognize that various changes and modifications may be made without departing from the scope of the invention as defined by the appended claims.

EXAMPLES

Example 1

This example demonstrates acoustic radiation force displacement of erythrocytes and lOO-pm polystyrene microspheres. As shown in the figures, ultrasound exposure forced erythrocytes and polystyrene microspheres toward the walls of a cuvette.

Figure 7A is a microscopy image of polystyrene beads (visible as dark spots) in an acoustofluidic channel before ultrasound exposure. Figure 7B is a microscopy image of the same polystyrene beads in the acoustofluidic channel depicted in Figure 7A, except after ultrasound exposure. The before and after side-by-side comparison of these images visually shows that the ultrasound waves in the acoustofluidic channel forced the polystyrene beads toward the wall of the channel opposite the transducer. Note that the 100-micron microspheres can be displaced via acoustic radiation force without microbubbles. Cells, by contrast, are much less responsive to acoustic radiation force and require extended sonication, therefore microbeads or microbubbles can increase the acoustic radiation forces and reduce the sonication time. For the erythrocytes in this Example, there were no microbubbles, but the acoustic radiation force displacement required ultrasound exposure of at least 30 seconds or longer, which generally is not optimal for cell sorting applications. Example 2

This Example concerns separation of human T cells using cationic microbubbles within an acoustofluidic chamber. As illustrated by Figure 8, the ultrasound pressure within a narrow range provides an advantage in separating cells efficiently. Acoustic attenuation measurements indicate a narrow range of ultrasound pressures for efficient separation (n=5 -6/condition). Higher attenuation coefficients indicate the presence of microbubbles after passing through the flow chamber. At lower ultrasound pressures there was very little displacement of microbubbles toward the far channel, so the attenuation was similar for both the near channel and the far channel. At high ultrasound pressures there was a lot of microbubble destruction, so attenuation was much lower for both the near channel and the far channel. In the middle range of ultrasound pressures, however, the microbubbles were mostly not destroyed, but there was more acoustic radiation force so the attenuation increased in the far channel (the direction microbubbles were pushed toward) and decreased in the near channel (the direction microbubbles were pushed away from). At 5 MPa there is a slight increase in attenuation for both channels which may be due to coalescence of microbubbles caused by ultrasound disruption (which is consistent with higher ultrasound pressures causing complete microbubble destruction).

Claims

CLAIMS What is claimed is:

1. A cell separation device, comprising at least one channel configured to accommodate the flow of a nonhomogeneous population of cells together with gas-filled microbubbles or microparticles having cationic surface charges; and at least one ultrasound transducer configured to produce ultrasound waves in response to one or more control signals and positioned or positionable relative to the at least one channel so that the ultrasound waves propagate into and across the at least one channel, wherein the one or more control signals are such that the resulting ultrasound waves produce acoustic radiation forces on cells associated with the gas-filled microbubbles or microparticles.

2. The cell separation device of claim 1, further comprising a signal generator configured to produce the one or more controls signals.

3. The cell separation device of claim 1, wherein the one or more control signals do not induce standing waves.

4. The cell separation device of claim 1, wherein the at least one channel has a cross-sectional dimension of at least 1 mm.

5. The cell separation device of claim 1, wherein the cross-sectional dimension is a diameter.

6. The cell separation device of claim 1, wherein the device is configured as an in-line device within a cell therapy manufacturing line.

7. The cell separation device of claim 1, wherein the cells comprise T cells.

8. The cell separation device of claim 1, wherein the microbubbles have a phospholipid or polymer shell and are gas-filled.

9. The cell separation device of claim 8, wherein the gas is perfluorocarbon.

10. The cell separation device of claim 1, wherein the microparticles are metal ion particles.

11. The cell separation device of claim 1, wherein the microparticles are polymer-based particles.

12. The cell separation device of claim 1, wherein the microbubbles or microparticles have a surface charge of +10 mV to +70 mV.

13. The cell separation device of claim 1, wherein the microbubbles or microparticles have a diameter of 0.5-100 pm.

14. The cell separation device of claim 1, wherein the microbubbles or microparticles have a diameter of 1-20 pm.

15. The cell separation device of claim 1, wherein the concentration of cells is lxlO⁴ cells/mL to lxlO⁹ cells/mL.

16. The cell separation device of claim 1, wherein the concentration of microbubbles or microparticles is 10-fold to 100-fold higher than the concentration of cells in the sample.

17. The cell separation device of claim 1, wherein the microbubbles and microparticles are not conjugated with an antibody.

18. The cell separation device of claim 1, wherein a frequency of the ultrasound waves is from 0.5-100 MHz.

19. The cell separation device of claim 1, wherein the frequency of the ultrasound waves is from 1-5 MHz.

20. The cell separation device of claim 1, wherein the channel has a serpentine or spiral geometry.

21. A method for cell separation, comprising: flowing cationic microbubbles or microparticles and a sample comprising a population of cells through a fluidic channel; transmitting ultrasound waves through the microfluidic channel to separate target cells associated with the cationic microbubbles or microparticles from other cells or particles in the sample; and collecting the target cells associated with the cationic microbubbles or microparticles.

22. The method of claim 21, wherein the target cells are T cells.

23. The method of claim 21, wherein the microbubbles have a phospholipid or polymer shell and are gas-filled.

24. The method of claim 23, wherein the gas is perfluorocarbon.

25. The method of claim 21, wherein the microparticles are metal ion particles.

26. The method of claim 21, wherein the microparticles are polymer-based particles.

27. The method of claim 21, wherein the microbubbles or microparticles have a surface charge of +10 mV to +70 mV.

28. The method of claim 21, wherein the microbubbles or microparticles have a diameter of 0.5- 100 pm.

29. The method of claim 21, wherein the microbubbles or microparticles have a diameter of 1-20 pm.

30. The method of claim 21, wherein the concentration of cells in the sample is lxlO⁴ cells/mL to lxlO⁹ cells/mL.

31. The method of claim 21, wherein the concentration of microbubbles or microparticles is 10- fold to 100-fold higher than the concentration of cells in the sample.

32. The method of claim 21, wherein the microbubbles and microparticles are not conjugated with an antibody.

33. The method of claim 21, wherein the frequency of the ultrasound waves is from 0.5-100 MHz.

34. The method of claim 21, wherein the frequency of the ultrasound waves is from 1-5 MHz.

35. The method of claim 21, wherein the fluidic channel has a serpentine or spiral geometry.