CN111565787A

CN111565787A - Acoustic methods for transfection and transduction

Info

Publication number: CN111565787A
Application number: CN201980007887.8A
Authority: CN
Inventors: B·利普肯斯; G·戈沙尔; N·曼兹; T·J·肯尼迪三世; R·托斯通伊斯; B·罗斯-约翰斯鲁德
Original assignee: Flodesign Sonics Inc
Current assignee: Flodesign Sonics Inc
Priority date: 2018-03-09
Filing date: 2019-03-11
Publication date: 2020-08-21
Also published as: AU2019232037A1; KR20200102469A; SG11202004022PA; WO2019173850A9; KR102487074B1; JP2021518750A; AU2019232037B2; CA3088027A1; WO2019173850A1; IL274173A; JP7198286B2; EP3762086A1; BR112020008977A2

Abstract

Disclosed herein are methods for introducing exogenous nucleic acids into cells using an acoustic process, for example, by performing transfection/transduction. The exogenous DNA/RNA and the cell co-localize in the multi-dimensional acoustic standing wave, or co-localize by acoustic streaming.

Description

Acoustic methods for transfection and transduction

Cross Reference to Related Applications

This application claims priority to U.S. provisional patent application serial No. 62/641,234 filed on 3/9/2018 and U.S. patent application serial No. 15/947,746 filed on 6/4/2018, all of which are incorporated herein by reference in their entirety.

Background

The present disclosure relates to methods of introducing exogenous nucleic acids into cells, for example, by transfection and transduction using acoustic waves. Also included are cells produced by such methods and related compositions. Such methods and compositions may be used in cell therapy applications.

Transfection and transduction are methods by which nucleic acids (DNA or RNA) are intentionally introduced into cells. Transduction is performed using viral vectors such as bacteriophage or other viruses. Viruses such as adenovirus, lentivirus or paramyxovirus are commonly used. Transfection refers to the introduction of nucleic acids into cells using non-viral methods.

Disclosure of Invention

In various embodiments, the present disclosure relates to methods for introducing exogenous nucleic acids (DNA/RNA) into cells, e.g., by transfection or transduction of cells using acoustic waves. Very generally, sound waves are used to bring cells and nucleic acids together so that the nucleic acids can be transferred into the cells. Acoustic devices may be used for this purpose, and such devices are described herein. Also included are cells produced by such methods and related compositions.

Disclosed herein, in various embodiments, are methods for causing transfection or transduction of a cell. The nucleic acids may be naked, or they may be in a viral vector. The cells and nucleic acids are placed in the acoustophoresis device, for example, by placing them in a bag inserted into the acoustophoresis device, or by flowing a fluid mixture containing both the cells and nucleic acids through the acoustophoresis device. The acoustophoresis device includes: an acoustic chamber in which cells and nucleic acids are disposed; and an ultrasonic transducer comprising a piezoelectric material and a reflector opposite the ultrasonic transducer, the ultrasonic transducer being drivable to generate a multi-dimensional acoustic standing wave in the acoustic chamber. The ultrasonic transducer is driven to produce an acoustic standing wave. As a result, the cells and nucleic acids co-localize via the acoustic standing wave. In other words, the cell and nucleic acid are placed close enough to each other to allow reaction between each other. The acoustic standing wave may be a multi-dimensional acoustic standing wave, a planar standing wave, or a combination of both.

In some embodiments, the nucleic acid is in a viral vector. Transduction can occur when the virus attaches itself to a target cell and injects nucleic acid into the target cell. In other embodiments, a pore is opened in the cell membrane of the cell prior to co-localizing the cell with the nucleic acid. Transfection may occur when nucleic acid enters a cell, for example, via a pore. The pores may be opened by electroporation, sonoporation, or by exposure to calcium phosphate.

Cells and nucleic acids (naked or in viral vectors) can be suspended in a fluid. Such fluids may include cell culture media, water, saline solutions, and the like.

In particular embodiments, the cell is a Chinese Hamster Ovary (CHO) cell, NS0 hybridoma cell, Baby Hamster Kidney (BHK) cell, human cell, regulatory T cell, helper T cell, cytotoxic T cell, memory T cell, effector T cell, gamma T cell, Jurkat T cell, CAR-T cell, B cell or NK cell, Peripheral Blood Mononuclear Cell (PBMC), algae, plant cell, or bacterium.

The ultrasonic transducer may be driven for a period of time of about 5 minutes to about 15 minutes, although this period of time may vary as desired. The ultrasonic transducer may be driven at a frequency of about 0.5MHz to about 20 MHz. In some embodiments, the frequency of the multi-dimensional acoustic standing wave is varied in a scanning pattern to move the cell relative to the nucleic acid.

The piezoelectric material of the ultrasonic transducer may be lead zirconate titanate (PZT) or lithium niobate. The acoustophoresis device may further include a cooling unit for cooling the ultrasonic transducer.

Also disclosed herein are methods for causing cell transduction. Placing a cell and a viral vector comprising a nucleic acid in an acoustophoresis device, the acoustophoresis device comprising: an acoustic chamber; and an ultrasonic transducer comprising a piezoelectric material, the ultrasonic transducer being drivable to produce a multi-dimensional acoustic standing wave, a planar acoustic standing wave, or a combination of planar and multi-dimensional standing wave sounds in the acoustic chamber. The ultrasonic transducer is driven to produce a multi-dimensional acoustic standing wave, a planar acoustic standing wave, or a combination of multi-dimensional and planar acoustic standing waves. The cell and viral vector are co-localized by the acoustic standing wave to cause transduction of the cell.

Also disclosed are methods for causing transfection of cells. A hole is opened in the cell membrane of the cell. Placing a cell together with a nucleic acid in an acoustophoresis device, the acoustophoresis device comprising: an acoustic chamber; and an ultrasonic transducer comprising a piezoelectric material, the ultrasonic transducer being drivable to generate an acoustic standing wave in the acoustic chamber. The acoustic standing wave may be a multi-dimensional acoustic standing wave, a planar acoustic standing wave, or a combination of planar and multi-dimensional acoustic standing waves. The aperture may be opened before or after the cell is placed in the acoustophoresis device. The ultrasonic transducer is then driven to produce an acoustic standing wave. The cells and nucleic acids are co-localized by acoustic standing waves to cause transfection of the cells.

These and other non-limiting features are described in more detail below.

Drawings

The accompanying drawings incorporated in and forming a part of the specification illustrate certain aspects of the subject matter disclosed herein and, together with the description, help explain some of the principles associated with the disclosed embodiments.

Figure 1 is a diagram illustrating the methods/processes of the present disclosure in which the efficiency of viral transduction is enhanced. Cell cultures were combined with viral vectors tagged with Green Fluorescent Protein (GFP) and exposed to acoustic processing, where a multi-dimensional acoustic standing wave brought the cells and virus into close proximity to each other, enhancing the efficiency of the reaction. After washing and overnight incubation, GFP was expressed in the cells, thus indicating that transduction had occurred.

Fig. 2A is an exploded perspective view of an example acoustophoresis device including a cooling unit for cooling a transducer according to the present disclosure. Fig. 2B is a perspective view of the assembly device of fig. 2A.

Fig. 3 is a perspective view of another acoustophoretic device that can be used to practice the methods/processes of the present disclosure. A disposable container, such as a plastic bag, contains a fluid mixture having two particle types that interact with each other in a separate acoustophoresis device containing one or more ultrasonic transducers.

Fig. 4 is a cross-sectional illustration of a conventional ultrasound transducer.

Fig. 5 is a cross-sectional illustration of an ultrasound transducer of the present disclosure. There is an air gap within the transducer and no backing layer or wear plate is present.

Fig. 6 is a cross-sectional illustration of an ultrasound transducer of the present disclosure. There is an air gap within the transducer and there is a backing layer and wear plate.

FIG. 7 is a graph of electrical impedance magnitude versus frequency for a square transducer driven at different frequencies.

FIG. 8 shows the capture line configuration of FIG. 7 for seven resonance frequencies (minimum of electrical impedance magnitude) from a direction orthogonal to the fluid flow.

Fig. 9 is a computer simulation of sound pressure amplitude (right scale in Pa) and transducer out-of-plane displacement (left scale in meters). The text at the top of the left scale is shown as "x 10^-7". The text at the top of the left scale by the upward triangle is shown as "1.473 x10^-6". The text at the bottom of the left scale by the downward triangle is shown as "1.4612 x10^-10". The text at the top of the right scale is shown as "x 10^-6". The text at the top of the right scale by the upward triangle is shown as "1.1129 x10⁶". Through the direction ofThe text of the lower triangle at the bottom of the right scale is shown as "7.357". The triangles show the maxima and minima depicted in this figure for a given scale. The horizontal axis is the position in inches within the chamber along the X-axis, and the vertical axis is the position in inches within the chamber along the Y-axis.

Fig. 10 shows in-plane and out-of-plane displacements of the crystal in which the complex wave is present.

Fig. 11 is a photograph of a plastic bag in which T cells and viruses interact with each other.

Fig. 12 is a graph showing the ratio of transduction efficiency (acoustic/non-acoustic) versus transduction time for three experiments with different run times (30, 60 and 90 minutes).

Detailed description of the preferred embodiments

The present disclosure may be understood more readily by reference to the following detailed description of the desired embodiments and the examples included therein. In the following specification and the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings.

Although specific terms are used in the following description for the sake of clarity, these terms are intended to refer only to the particular structure selected for the embodiments shown in the drawings, and are not intended to define or limit the scope of the present disclosure. In the drawings and the following description below, it is to be understood that like numeric designations refer to components of like function.

The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

The term "comprising" is used herein when it is desired to specify the presence of a component and to allow the presence of other components. The term "comprising" should be interpreted as including the term "consisting of … …" which only allows the presence of the specified component, along with any impurities that may result from the manufacture of the specified component.

Numerical values should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement techniques of the type used to determine the value in question in this application.

All ranges disclosed herein are inclusive of the recited endpoints and independently combinable (e.g., a range of "2 grams to 10 grams" is inclusive of the endpoints 2 grams and 10 grams and all intermediate values). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values close to these ranges and/or values.

The modifier "about" used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context. The modifier "about" when used in the context of a range should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, "about 2 to about 10" also discloses the range "2 to 10". The term "about" may refer to plus or minus 10% of the indicated number. For example, "about 10%" may indicate a range of 9% to 11%, and "about 1" may mean 0.9-1.1.

It should be noted that many of the terms used herein are relative terms. For example, the terms "upper" and "lower" are positionally relative to one another, i.e., the upper component is positioned at a higher elevation than the lower component in a given orientation, but these terms may change if the device is flipped. The terms "inlet" and "outlet" relate to a fluid flowing therethrough with respect to a given structure, e.g., a fluid flows into a structure through an inlet and exits the structure through an outlet. The terms "upstream" and "downstream" are relative to the direction in which fluid flows through various components, i.e., fluid flows through an upstream component before flowing through a downstream component. It should be noted that in a ring, a first component may be described as being upstream and downstream of a second component.

The terms "horizontal" and "vertical" are used to indicate directions relative to an absolute reference, i.e., the ground plane. However, these terms should not be construed as requiring structures to be absolutely parallel or absolutely perpendicular to each other. For example, the first and second vertical structures need not be parallel to each other. The terms "top" and "bottom" or "base" are used to refer to surfaces in which the top is always higher than the bottom/base relative to an absolute reference, i.e., the earth's surface. The terms "upward" and "downward" are also relative to absolute reference; the upward flow is always against the earth's gravity.

The present application relates to "same order of magnitude". If the quotient of the larger number divided by the smaller number is a value of at least 1 and less than 10, the two numbers are of the same order of magnitude.

The acoustophoresis devices discussed herein may operate in either a multi-mode or a planar mode, or a combination of both. Multiple modes refer to sound waves generated by an acoustic transducer that produces acoustic forces in three dimensions. Multiple modes of acoustic waves, which may be ultrasonic waves, may be generated by a single acoustic transducer and are sometimes referred to herein as multi-dimensional or three-dimensional acoustic standing waves. A planar mode refers to an acoustic wave generated by an acoustic transducer that generates an acoustic force substantially in one dimension, e.g., along a propagation direction. Such acoustic waves, which may be ultrasonic waves, generated in planar modes are sometimes referred to herein as one-dimensional acoustic standing waves.

The acoustic transducer may comprise a piezoelectric material, such as lead zirconate titanate (PZT) or lithium niobate. Such acoustic transducers may be electrically excited to generate planar or multi-mode acoustic waves. The three-dimensional acoustic forces generated by the multiple modes of acoustic waves include radial or lateral forces that are not aligned with the direction of acoustic wave propagation. The lateral force may act in two dimensions. The transverse force is a complement to the axial force in the multi-mode acoustic wave, which is substantially aligned with the direction of acoustic wave propagation. The transverse force may be of the same order of magnitude as the axial force of such a multi-mode acoustic wave. An acoustic transducer excited in multi-mode operation may exhibit a standing wave on its surface, thereby generating multi-mode acoustic waves. Standing waves at the surface of the transducer may be related to the operating mode of the multi-mode acoustic wave. When an acoustic transducer is electrically excited to generate a planar acoustic wave, the surface of the transducer may exhibit a piston-like motion, thereby generating a one-dimensional acoustic standing wave. Multimode acoustic waves exhibit significantly greater particle capture activity on a continuous basis for the same input power compared to planar acoustic waves. One or more acoustic transducers may be used to generate a combination of planar and multi-dimensional acoustic standing waves. For example, two acoustic transducers may be arranged opposite each other to generate a standing wave. In such instances, one of the acoustic transducers may be passive and act as a reflector of the incident wave. Alternatively or additionally, each transducer may be active to generate acoustic waves, including such acoustic waves as described elsewhere herein.

Acoustophoresis is the manipulation of materials using acoustic waves. In some example embodiments, acoustophoresis is used for separation of materials, and may represent a low-power, non-pressure-drop, non-clogging, solid-state approach to particle separation from fluid dispersions. Scattering of the acoustic field from the particles results in three-dimensional acoustic radiation forces, which act as three-dimensional trapping fields. When the particles are small relative to the wavelength, the acoustic radiation force is proportional to the particle volume (e.g., the cube of the radius). The acoustic radiation force is proportional to the frequency and the acoustic contrast factor. Acoustic radiation force is proportional to acoustic energy (e.g., the square of the acoustic pressure amplitude). For harmonic excitation, the sinusoidal spatial variation of force drives the particles to stable positions within the standing wave. Particles are trapped within the acoustic standing wave field when the acoustic radiation force applied to the particles is stronger than the combined effect of the fluid drag and buoyancy/gravity forces. The effect of the transverse and axial acoustic forces on the captured particles results in the formation of tightly packed clusters by concentration, clustering, agglomeration, coagulation and/or coalescence of the particles, which clusters, when reaching a critical size, continuously settle by enhanced gravity for particles heavier than the bulk fluid or rise by enhanced buoyancy for particles lighter than the bulk fluid. In addition, secondary interparticle forces, such as the Bjerkness force, contribute to particle agglomeration.

The acoustic standing wave creates localized regions of high and low pressure. Depending on its compressibility and density relative to the surrounding fluid, the particles are pushed to a standing wave node or antinode. The higher density and compressible particles move to nodes in the standing wave, while the lower density secondary phase moves to the antinodes. The force applied to the particles also depends on their size, with larger particles being subjected to larger forces. The magnitude of the force depends on the particle density and compressibility with respect to the fluid medium and increases with particle volume.

For the purposes of this disclosure, a biological cell may be considered a particle. Most biological cell types exhibit higher density and lower compressibility than the medium in which they are suspended, so that the acoustic contrast factor between the cells and the medium has a positive value. As a result, the axial Acoustic Radiation Force (ARF) drives the cell towards the standing wave pressure node. The axial component of the acoustic radiation force drives cells with positive contrast factors to the pressure node, while cells or other particles with negative contrast factors are driven to the pressure antinode. The radial or lateral component of the acoustic radiation force is the force that captures the cell. The radial or lateral component of the ARF is greater than the combined effect of fluid resistance and gravity.

Additional theoretical and numerical models have been developed for calculating the acoustic radiation force of particles without any limitation regarding the particle size with respect to wavelength. These models also include the effects of fluid and particle viscosity and are therefore more accurate calculations of acoustic radiation force. The model implemented works on the basis of the theories of Yurii Iiinskii and Evgenia Zabolotskaya, as described in AlPConreference Proceedings, Vol.1474-1, p.255-. Additional internal models have also been developed to calculate the acoustic trapping force of cylindrical objects, such as the "hockey" trapping particles in standing waves, which closely resemble a cylinder.

Desirably, the ultrasonic transducer generates a multi-dimensional standing wave in the fluid that exerts a lateral force on the suspended particles to accompany the axial force. Common results published in the literature state that transverse forces are two orders of magnitude smaller than axial forces. In contrast, the technology disclosed in the present application provides a lateral force that is of the same order of magnitude as the axial force. However, in certain embodiments further described herein, the device uses both a transducer that produces a multi-dimensional acoustic standing wave and a transducer that produces a planar acoustic standing wave. The lateral force component of the total Acoustic Radiation Force (ARF) generated by the ultrasound transducer of the present disclosure is significant and sufficient to overcome fluid resistance at linear velocities up to 1cm/s and produces close-packed clusters and is of the same order of magnitude as the axial force component of the total acoustic radiation force.

The present disclosure relates to methods of transfection or transduction using such an acoustophoretic device containing an ultrasonic transducer. Briefly, an acoustophoresis device is used to aggregate cells with nucleic acids (DNA or RNA or both) in a localized volume. This allows the transfer of nucleic acids into the cell. Current transduction methods can have relatively high cost, low efficiency and poor ability to scale up for commercialization. The approaches described herein may reduce cost, increase efficiency, and have an extensible platform for commercialization.

Very generally, cells and carriers (containing RNA or DNA) are placed in the acoustic chamber of an acoustophoresis device. Generally, they are suspended in a fluid to form a fluid mixture. In some example embodiments, an acoustophoresis device includes an acoustic chamber having an ultrasonic transducer and a reflector opposite the ultrasonic transducer (e.g., on an opposite wall of the chamber). The ultrasonic transducer includes a piezoelectric material, which can be driven to produce an acoustic standing wave, such as a multi-dimensional acoustic standing wave and/or a planar acoustic standing wave, within the acoustic chamber. The acoustic standing wave has regions of higher pressure and regions of lower pressure. In some examples, the lower pressure region captures and retains the cells. The nucleic acid flows into the acoustic chamber along with the retained cells. This action results in co-localization of the cell and the nucleic acid, so that the nucleic acid can enter the cell. In the case of transfection, a hole is opened in the cell membrane of the cell to allow the entry of naked nucleic acid into the cell. In the case of transduction, the nucleic acid is part of a viral vector. Viral vectors enter cells or insert nucleic acids into cells. Specific viral vectors that may be used for transduction in the present disclosure include adenovirus, lentivirus, or paramyxovirus.

In some example embodiments, the acoustophoretic force generated by the acoustic standing wave on cells and/or nucleic acids (naked or in viral vectors) may be sufficient to overcome the fluidic resistance exerted on these particles by the moving fluid. In other words, acoustophoretic forces may act as a mechanism to capture cells and/or nucleic acids in an acoustic field. The acoustophoretic force can drive the cells and/or nucleic acids to a stable position of reduced or minimal acoustophoretic force amplitude. These locations of reduced or minimal acoustophoretic force amplitude may be nodes of standing acoustic waves. Over a period of time, the collection of cells and/or nucleic acids at the nodes steadily increases. The collection of cells and/or nucleic acids may take the shape of a beam-like collection of disks over a certain period of time, which may be several minutes or less, depending on their concentration. Each disc may be spaced apart by half a wavelength of the acoustic field.

In some embodiments, the acoustic standing wave captures cells and/or nucleic acids and co-localizes them, improving the efficiency of the transfection/transduction reaction. Many different mechanisms may be implemented for these embodiments. In one such mechanism, cells and nucleic acids may have similar acoustic contrast factors, such that both types of particles may be driven to nodes or antinodes of the standing wave. This mechanism is more effective than relying on brownian motion (as with conventional agitation) to bring cells and nucleic acids into close spatial proximity to one another. In other words, cells and nucleic acids are trapped in a small three-dimensional space created by the multi-dimensional acoustic standing wave relative to the size of the acoustic chamber. In certain exemplary embodiments, both the cell and the nucleic acid have a positive acoustic contrast factor, or both have a negative acoustic contrast factor. In other words, their acoustic contrast factors have the same sign. Again, the nucleic acid may be naked or in a viral vector.

In another mechanism, one of the two types of particles (cells or nucleic acids) can be driven to a node while the other type of particle is driven to an anti-node. However, at higher frequencies, the nodes and antinodes are close enough to each other so that the cell and nucleic acid can react with each other. In such embodiments, the cell or nucleic acid has a positive acoustic contrast factor, while the other group (i.e., nucleic acid or cell) has a negative acoustic contrast factor. In other words, their acoustic contrast factors have opposite signs. Particles with a positive contrast factor are driven to the node, while particles with a negative contrast factor are driven to the antinode. Some relevant factors for this reaction mechanism include the size of the cell and the support, and the frequency at which the ultrasound transducer operates.

Finally, as the cells and/or nucleic acids continue to be captured and concentrated, they can reach a size and weight such that gravity settling occurs, wherein clusters of particles will fall from the acoustic standing wave to the bottom of the acoustic chamber. A new set of particles may then be captured and reacted within the acoustic field generated by the acoustic standing wave.

In some example embodiments, an acoustophoresis device is provided with an acoustic chamber including an ultrasonic transducer comprising a piezoelectric material, the ultrasonic transducer being drivable to produce an acoustic standing wave, such as a multi-dimensional acoustic standing wave acoustic wave and/or a planar acoustic standing wave, within the acoustic chamber. The acoustic standing wave has regions of higher pressure and regions of lower pressure. A recirculation loop is provided to the acoustic chamber that allows the output fluid and particles to be reintroduced into the acoustic chamber. In some examples, the lower pressure region captures and retains cells, but does not capture or retain viral vectors or nucleic acids, which can flow out of the acoustic chamber and be reintroduced via the recirculation loop. In the example of viral vectors, cells are retained in the acoustic field generated by the acoustic standing wave, and viral vectors flow into the field to allow the cells and viral vectors to interact. By recirculating viral vectors that did not interact on previous passes to the acoustic chamber, an increased number of interactions between viral vectors and cells can be achieved. In the example of nucleic acids, the cells remaining in the acoustic field may be subjected to additional processing to allow for the formation of pores in the cell membrane, as discussed in more detail elsewhere herein. The nucleic acid flows into the acoustic chamber with the retained cells to allow the nucleic acid to interact with the cells and to enter the cells via the pores. The nucleic acid is not retained by the acoustic field, but is recirculated to the acoustic chamber to allow transfection to occur in multiple passes.

Additionally or alternatively, in some instances, the ultrasonic transducer is driven to induce an acoustic flow within the acoustic chamber. In short, acoustic flow refers to the fluid flow generated within an acoustic chamber when the fluid absorbs acoustic energy transmitted by an ultrasonic transducer (vibrations from the ultrasonic transducer). The velocity of the fluid is induced by the oscillating acoustic waves generated by the ultrasonic transducer. Typically, when an acoustic flow is generated, it causes a circulatory motion or vortex, which can cause agitation in the fluid mixture. This phenomenon is non-linear and can cause cells and nucleic acids to interact with each other.

The cells and nucleic acids are brought into proximity so that they can react with each other. In the present disclosure, the terms "interaction" and "reaction" are used to indicate that a physical change occurs in a cell. For example, in transduction, a virus containing a nucleic acid may penetrate into a cell to cause transduction to occur. The virus may be, for example, a retrovirus, such as a lentivirus.

For the transfection example, a hole is opened in the cell membrane of the cell to allow the nucleic acid to enter the cell. For example, cells may be subjected to electroporation, wherein the cells are exposed to an electric field to increase the permeability of the cell membrane. Sonoporation may also be used when cells are exposed to ultrasound to induce pore formation in the cell membrane. Calcium phosphate may also be used to cause transfection. The location at which the opening of the aperture occurs is not critical and may occur in the acoustophoresis device upstream of the acoustic chamber where the cells and nucleic acids mix or may occur externally of the acoustophoresis device.

For reference, it is noted that productive transfection and gene transfer not only requires DNA into the cell and subsequent transcription from an appropriate promoter, but also many intracellular events that allow DNA to move from the outer surface of the cell into and through the cytoplasm, and eventually across the nuclear envelope and into the nucleus, before any transcription can begin. Immediately after entry into the cytoplasm, naked DNA (delivered by physical techniques or after disintegration of the DNA-carrier complex) binds to a number of cellular proteins that mediate subsequent interactions with the microtubule network for movement towards the microtubule tissue center and the nuclear envelope. The plasmid then enters the nucleus upon mitotic disassembly of the nuclear envelope, or through the nuclear pore complex in the absence of cell division, using a different set of proteins.

Specific viral vectors that may be used for transduction in the present disclosure include adenovirus, lentivirus, or paramyxovirus. Retroviruses are characterized by their ability to reverse transcribe their RNA genome into cDNA copies that are then stably integrated into the genome of the host cell. Thus, the virus carries the nucleic acid into the cell.

Retroviruses can be classified as simple viruses or complex viruses (e.g., lentiviruses). Both types of viral particles contain two copies of positive-stranded RNA, and the associated viral Reverse Transcriptase (RT) located in the inner core. Structural and enzymatic proteins are also located within this compartment, including the Nucleocapsid (NC), Capsid (CA), Integrase (IN) and Protease (PR). The inner core is surrounded by an outer protein layer consisting of Matrix (MA) proteins, which in turn is surrounded by a host cell membrane-derived envelope that is enchased by envelope glycoproteins (ENV).

Lentiviral and retroviral gene delivery systems (or vectors) take advantage of aspects of retroviral replication to provide stable integration of a desired nucleic acid sequence. Although transfection of exogenous nucleic acids only results in transient transgene expression, viral integrase activity in retrovirus and lentivirus-based systems allows stable integration of exogenous transgenes that are subsequently inherited and expressed sequentially through repeated cell divisions. A key feature of both lentiviral and retroviral vectors is that they produce replication-defective or self-inactivating particles. This allows delivery of the desired sequence without sustained viral replication in the target cell.

A common method of verifying the successful introduction of exogenous nucleic acid into a cell is to measure protein expression. This is usually done by western blotting or immunostaining.

Examples of cells that can be transfected/transduced by the methods of the present disclosure include Chinese Hamster Ovary (CHO) cells, NS0 hybridoma cells, Baby Hamster Kidney (BHK) cells, human cells, regulatory T cells, helper T cells, cytotoxic T cells, memory T cells, effector T cells, gamma T cells, Jurkat T cells, CAR-T cells, B cells or NK cells, Peripheral Blood Mononuclear Cells (PBMCs), algae, plant cells, or bacteria. The cells themselves may be attached to other materials, such as beads. Examples of beads include polymeric beads, magnetic beads, superparamagnetic beads, and microspheres. These can be used for biochemical reactions or for labeling purposes.

In some embodiments, the additive may be included in a mixture of cells and nucleic acids. Such additives may include Polybrene^TMAnd retroNectin^TM。Polybrene^TMIs a cationic polymer (polyhexamethylenediammonium bromide) used to increase the efficiency of transduction of certain cells with retroviruses (and lentiviruses) in cell culture, and is typically used in amounts of 10 μ g/mL or less. RetroNectin^TMIs a polypeptide consisting of three functional domains (the C domain, the H domain and the CS-1 site) derived from human fibronectin. RetroNectin^TMEnhancing the efficiency of retrovirus-and lentivirus-mediated transduction in hematopoietic cells, including hematopoietic stem cells and terminally differentiated cells, such as primary T cells and macrophages. Other additives may include lipofectin, lipofectamine, and cationic peptides, such as protamine. These additives may be used to enhance transduction, as appropriate.

Without being limited by theory, it is believed that the frequency of the multi-dimensional acoustic standing wave determines the diameter of the particle that can be captured by the acoustic standing wave. For example, for a 2MHz wave, the particle size is about 1 to about 100 microns.

Fig. 1 is a diagram illustrating an example method of the present disclosure as applied to viral transduction. In this example, cells were labeled with Green Fluorescent Protein (GFP). Starting from the left side of the figure, first, cell culture 100 is combined with viral vector 110. The fluid mixture containing the cells and viruses is then placed in an acoustic chamber 120, the acoustic chamber 120 being located between an ultrasonic transducer 122 and a reflector 124. An acoustic standing wave was generated for 10 minutes at room temperature. As shown here, cells and viruses are trapped in the acoustic standing wave. Cells are captured at nodes, while viruses are captured at antinodes. However, due to their relative sizes, the cells and virus are co-localized, and the virus is able to infect the cells (identified by reference numeral 128). After washing to remove unreacted material, the cells were incubated overnight at 37 ℃ and GFP was expressed in the labeled cells. Similar methods can be used to make Chimeric Antigen Receptor (CAR) expressing T cells or CAR T cells.

The methods of the present disclosure may be performed in a continuous process in which a fluid mixture containing cells and nucleic acids suspended in a host fluid is flowed through an acoustophoresis device. The nucleic acids may be naked or they may be contained in a viral vector such as a bacteriophage or other virus.

Fig. 2A is an exploded view of an acoustophoretic device 200 that can be used in a continuous process. Fig. 2B is a view of device 200 in a fully assembled condition.

Referring to fig. 2A, the acoustophoresis device may be constructed such that each component is modular and can be changed or switched separately from each other. Thus, when a new revision or modification is made to a given component, that component may be replaced while remaining the same in the remainder of the device.

The apparatus includes an ultrasonic transducer 220 and a reflector 250 on the opposite wall of the acoustic chamber 210. Note that the reflector 250 may be made of a transparent material so that the inside of the flow chamber 210 can be seen. The ultrasonic transducer is proximate to the first wall of the acoustic chamber. The reflector is adjacent to, or may constitute, the second wall of the acoustic chamber.

The cooling unit 260 may be located between the ultrasonic transducer 220 and the flow chamber 210. As shown here, the cooling unit 260 includes a separate flow path that is separate from the flow path through the acoustic chamber. The coolant inlet 262 allows cooling fluid to enter the cooling unit. The coolant and waste heat exit the cooling unit through coolant outlet 264. The coolant flowing through the cooling unit may be any suitable fluid. For example, the coolant may be water, air, alcohol, ethanol, ammonia, or some combination thereof. The coolant may be a liquid, gas or gel. The coolant may be a non-conductive fluid to prevent electrical shorting.

Alternatively, the cooling unit may be in the form of a thermoelectric generator which uses the seebeck effect to convert the heat flux (i.e. temperature difference) into electrical energy, thus removing heat from the flow chamber. In other words, electricity can be generated from the waste heat that is not needed when operating the acoustophoresis device.

The cooling unit may be used to cool the ultrasound transducer, which may be particularly advantageous when the device is to be operated continuously for extended periods of time (e.g. perfusion) with repeated processing and recirculation. Alternatively, the cooling unit may also be used to cool the fluid running through the acoustic chamber 210. For the desired application, the cells should be maintained at about room temperature (. about.20 ℃) and at most about 28 ℃. This is because as cells experience higher temperatures, their metabolic rate increases. However, without a cooling unit, the temperature of the cells flowing through the acoustic chamber may rise up to 34 ℃.

Note that the acoustic chamber 210 is shown here as including at least an inlet 212 and an outlet 214. This provides access to the interior volume 216 of the acoustic chamber. Additional inlets and outlets (e.g., fluid inlet, concentrate outlet, permeate outlet, recycle outlet, bleed/harvest outlet) may be included, as desired. The interior volume 216 may be considered to be bounded by the ultrasonic transducer 220, the cooling unit 260, the acoustic chamber 210, and the reflector 250.

The flow direction of the acoustophoresis device 200 may be oriented in a direction other than horizontal. For example, the fluid flow may be vertically upward or downward or at an angle relative to vertical or horizontal. More than one transducer may be included in the system.

Fig. 3 illustrates another acoustophoresis device 300 that may be used to practice the methods and processes of the present disclosure. Very generally, the system includes an acoustophoresis device 300 and a substantially acoustically transparent container 310. The two components may be separate from each other.

The container 310 of the acoustophoretic device is generally formed of a substantially acoustically transparent material, such as plastic, glass, polycarbonate, low density polyethylene, and high density polyethylene (all in a suitable thickness). However, the container may be formed of any material suitable for allowing the acoustic standing wave of the present disclosure to pass therethrough. The container may be in the form of a bottle or a bag. The difference between these forms is in their composition and structure. The bottle is stronger than the bag. When empty, the bag is generally unable to support itself and the bottle is able to stand upright. For example, the container 310 shown here is a high density polyethylene bag. The vessel 310 generally has an upper end 312 and a lower end 314, and an interior space in which the fluid mixture (containing the first particles and the second particles in the bulk fluid) is located.

The acoustophoretic device 300 is defined by at least one wall 332 and typically a plurality of walls forming sides thereof. For example, the acoustophoresis device may be in the shape of a cylinder, or rectangular (as depicted). The wall is solid. There is an opening 326 in the upper end of the acoustophoresis device for receiving container 310 therethrough. Again, the acoustophoretic device 300 is detachable from the container 310 such that the container can be disposable or reusable depending on the desired application of the acoustophoretic device. As shown here, the base of the acoustophoresis device 300 is solid.

The acoustophoresis device 300 includes at least one ultrasonic transducer 330 on a wall 334. The ultrasonic transducer 330 has a piezoelectric material that is driven by a voltage signal to produce an acoustic standing wave. A cable 332 is shown for conveying power and control information to the ultrasonic transducer 330. A reflector 340 may be present and located on the wall 336 opposite the ultrasound transducer 330. A standing wave is thus generated by the initial wave radiated from the transducer and the reflected wave from the reflector. In some embodiments, a separate reflector is not required. For example, the chamber walls or release boundaries, such as may be provided by ambient air, may be used to reflect incident waves and create standing waves. It should be understood that various transducer and reflector combinations may be used. Planar and/or multi-dimensional acoustic standing waves are generated within the vessel and are used to cause particle interactions within the vessel 310. It should be noted that there is no contact between the ultrasonic transducer and the fluid mixture within the container 310.

In certain embodiments, the acoustophoresis device includes a plurality of ultrasonic transducers 330 positioned on a common wall 334, the common wall 334 being opposite a wall 336 on which a reflector 340 is located. Alternatively, the ultrasonic transducers may be positioned relative to each other without reflectors. Additionally, the acoustophoretic device 300 can include a viewing window 324 in another wall 338. As shown here, when a viewing window is provided, it may be in a wall adjacent to the wall on which the ultrasound transducer and reflector are located, such that the lower end 314 of the container 310 may be viewed through the viewing window 324 in the separate chamber 320. In other embodiments, a viewing window may be substituted for the reflector.

In certain embodiments, a fluid, such as water, may be placed in interstitial space 305 between container 310 and acoustophoresis device 300 such that the acoustic standing wave passes through both the fluid in the interstitial space and the fluid mixture in the container. The interstitial fluid may be any fluid, although it should have an acoustic impedance value that allows good transmission of the acoustic standing wave, and preferably has low acoustic attenuation.

In certain exemplary embodiments, the ultrasonic transducer is driven at a frequency of about 0.5MHz to about 20MHz (MHz). Higher frequency standing wave fields result in steeper pressure gradients, which in turn are more suitable for trapping smaller particles such as viruses. The ultrasonic transducer may be driven for a period of time of about 5 minutes to about 15 minutes. This is a much shorter period of time than, for example, conventional viral transduction processes in which the cell culture and viral vector are incubated together for about 30 minutes to about 120 minutes. Such a long incubation period is due to the fact that the reaction between the cells and the virus only occurs when brownian motion brings them into proximity with each other. The use of the acoustophoresis device of the present disclosure greatly increases the likelihood that the cell and virus are close enough to react with each other. This results in higher reaction efficiency using fewer particles. However, if desired, the ultrasonic transducer may be driven for a desired period of time, for example up to 120 minutes or more.

It may be helpful to now describe the ultrasonic transducer used in the acoustic filtering device in more detail. Fig. 4 is a cross-sectional illustration of a conventional ultrasound transducer. This transducer has a wear plate 50 at the bottom end, an epoxy layer 52, a ceramic piezoelectric element 54 (made of, for example, lead zirconate titanate (PZT) or lithium niobate), an epoxy layer 56, and a backing layer 58. On either side of the ceramic piezoelectric element there is one electrode: a positive electrode 61 and a negative electrode 63. An epoxy layer 56 attaches a backing layer 58 to the piezoelectric element 54. The entire assembly is contained in a housing 60, which housing 60 may be made of, for example, aluminum. The shell serves as a ground electrode. An electrical adapter 62 provides a connection for wires passing through the housing and connected to leads (not shown) attached to the piezoelectric element 54. Typically, the backing layer is designed to increase damping and produce a broadband transducer with uniform displacement across a wide range of frequencies, and to suppress excitation of specific vibrational eigenmodes of the piezoelectric element. Wear plates are typically designed as impedance transformers to better match the characteristic impedance of the medium into which the transducer radiates.

Fig. 5 is a cross-sectional view of an ultrasonic transducer 81 according to an example of the present disclosure, for use in an acoustic filtering device of the present disclosure. The transducer 81 is shaped as a square and has an aluminum housing 82. The aluminum shell has a topEnd and bottom ends. The transducer housing may also be composed of plastic such as medical grade HDPE or other metals. The piezoelectric elements are perovskite ceramic masses, each made of a mixture of a relatively large divalent metal ion (usually lead or barium) and O^2-The smaller tetravalent metal ion (usually titanium or zirconium) in the lattice of the ion. For example, a PZT (lead zirconate titanate) piezoelectric element 86 defines the bottom end of the transducer and is exposed from the outside of the bottom end of the case. The piezoelectric element is supported on its periphery by a small resilient layer 98, such as epoxy, silicone or similar material, said resilient layer 98 being located between the piezoelectric element and the housing. In other words, there is no wear plate or backing material present. However, in some embodiments, there is a layer of plastic or other material separating the piezoelectric element from the fluid in which the acoustic standing wave is generated. The piezoelectric material/element/crystal has an outer surface (which is exposed) and an inner surface.

The screw 88 attaches the aluminum top plate 82a of the housing to the main body 82b of the housing via threads. The top plate includes a connector 84 for powering the transducer. The top surface of the piezoelectric element 86 is connected to a positive pole 90 and a negative pole 92 separated by an insulating material 94. The electrodes may be made of any conductive material, such as silver or nickel. Power is supplied to the piezoelectric element 86 through electrodes on the piezoelectric element. Note that the piezoelectric element 86 does not have a backing layer or epoxy layer. In other words, in the transducer, there is an internal volume or air gap 87 between the aluminum top plate 82a and the piezoelectric element 86 (i.e., the air gap is completely empty). In some embodiments, a minimal backing 58 and/or wear plate 50 may be provided, as seen in fig. 6.

The design of the transducer can affect the performance of the system. A typical transducer is a layered structure in which a ceramic piezoelectric element is bonded to a backing layer and a wear plate. Conventional design guidelines for wear plates (e.g., half-wavelength thickness for standing wave applications or quarter-wavelength thickness for radiation applications) and manufacturing methods may be inadequate due to the high mechanical impedance presented by the standing wave loaded transducer. In contrast, in one embodiment of the present disclosure, the transducer has no wear plate or backing, allowing the piezoelectric element to vibrate in a combination of one or several of its high-Q factor eigenmodes. The vibrating ceramic piezoelectric element/disc is directly exposed to the fluid flowing through the fluid cell.

Removing the backing (e.g., air backing the piezoelectric element) also allows the ceramic piezoelectric element to vibrate in higher order vibration modes (e.g., higher order mode displacements) with little damping. In a transducer having a piezoelectric element with a backing, the piezoelectric element vibrates like a piston with a more uniform displacement. Removing the backing allows the piezoelectric element to vibrate in a non-uniform displacement mode. The higher the mode shape of the piezoelectric element, the more nodal lines the piezoelectric element has. Higher order mode displacements of the piezoelectric element produce more trapping lines, although the correlation between trapping lines and nodes is not necessarily one-to-one, and driving the piezoelectric element at higher frequencies does not necessarily produce more trapping lines.

In some embodiments of the acoustic filtering devices of the present disclosure, the piezoelectric element can have a backing that minimally affects the Q factor of the piezoelectric element (e.g., less than 5%). The backing may be made of a substantially acoustically transparent material, such as balsa wood, foam, or cork, which allows the piezoelectric element to vibrate in a high order mode shape and maintain a high Q factor while still providing some mechanical support for the piezoelectric element. The backing layer may be a solid or may be a lattice with holes through the layer such that the lattice follows the nodes of the vibrating piezoelectric element in certain higher order vibration modes, providing support at the node locations while allowing the remaining piezoelectric element to vibrate freely. The purpose of the lattice or acoustically transparent material is to provide support without reducing the Q factor of the piezoelectric element or interfering with the excitation of specific mode shapes.

Placing the piezoelectric element in direct contact with the fluid also contributes to a high Q factor by avoiding the damping and energy absorbing effects of the epoxy and wear plates. Other embodiments of the transducer may have a wear plate or wear surface to prevent PZT containing lead from contacting the bulk fluid. This may be desirable, for example, in biological applications such as separation of blood, biopharmaceutical perfusion, or fed-batch filtration of mammalian cells. Such applications may use wear resistant layers such as chromium, electrolytic nickel or electroless nickel. Chemical vapor deposition may also be used to apply a layer of poly (p-xylene) (e.g., Parylene) or other polymer. Organic and biocompatible coatings (e.g., silicone or polyurethane) may also be used as wear resistant surfaces. Thin films such as Polyetheretherketone (PEEK) films can also be used as a covering for the transducer surface exposed to the fluid, with the advantage of biocompatible materials. In one embodiment, the PEEK film is adhered to the face of the piezoelectric material using a Pressure Sensitive Adhesive (PSA). Other membranes may also be used.

In some embodiments, the ultrasound transducer has a nominal 2MHz resonant frequency. Each transducer may consume approximately 28W of power for droplet capture at a flow rate of 3GPM (gallons per minute). This translates into 0.25kW hr/m³The energy cost of (a). This is an indication that the energy cost of this technique is extremely low. Desirably, each transducer is powered and controlled by its own amplifier. In other embodiments, the ultrasound transducer uses square piezoelectric elements, for example having a 1 "x 1" size. Alternatively, the ultrasonic transducer may use rectangular piezoelectric elements, for example having dimensions of 1 "x 2.5". The power consumption of each transducer was 10W per 1 "x 1" transducer cross-sectional area and per inch of acoustic standing wave span in order to obtain sufficient acoustic capture force. For a 4 "span for a medium scale system, each 1" x1 "square transducer consumes 40W. The larger 1 "x 2.5" rectangular transducer uses 100W in a medium scale system. An array of three 1 "x 1" square transducers consumes a total of 120W, while an array of two 1 "x 2.5" transducers consumes about 200W. Closely spaced transducer arrays represent an alternative potential implementation of this technique. The size, shape, number and location of the transducers may be varied as desired to generate a desired multi-dimensional acoustic standing wave pattern.

The size, shape and thickness of the transducer determine the transducer displacement at different excitation frequencies, which in turn affects the separation efficiency. Typically, the transducer operates at a frequency near the thickness resonance frequency (half wavelength). The gradient of transducer displacement generally results in more capture locations for cells/biomolecules. High order mode displacements generate three dimensional acoustic standing waves with strong gradients in the acoustic field in all directions, resulting in equally strong acoustic radiation forces in all directions, resulting in a number of trapping lines, where the number of trapping lines is associated with a particular mode shape of the transducer.

To investigate the effect of transducer displacement distribution on acoustic capture force and separation efficiency, ten experiments were repeated using a 1 "x 1" square transducer, where all conditions except excitation frequency were identical. Ten consecutive acoustic resonance frequencies indicated by the circled numbers 1-9 and the letter a on fig. 7 are used as excitation frequencies. The conditions were 30 minutes duration of the experiment, a 1000ppm oil concentration of approximately 5 microns SAE-30 oil droplets, a flow rate of 500 ml/minute and 20W applied power. Oil droplets are used because oil is less dense than water and can be separated from water by acoustophoresis.

FIG. 7 shows the measured electrical impedance magnitude of a square transducer as a function of frequency in the vicinity of the 2.2MHz transducer resonance. The minimum in the transducer electrical impedance corresponds to the acoustic resonance of the water column and represents a potential frequency for operation. There are additional resonances at other frequencies where a multi-dimensional standing wave is excited. Digital modeling has indicated that the transducer displacement profile varies significantly at these acoustic resonance frequencies and thereby directly affects the acoustic standing wave and the resulting trapping force. Since the transducer operates near its thickness resonance, the displacements of the electrode surfaces are substantially out of phase. The typical displacement of the transducer electrodes is not uniform and varies depending on the excitation frequency. For example, at one excitation frequency where a single line captures an oil droplet, the displacement has a single maximum in the middle of the electrode and a minimum near the transducer edge. At another excitation frequency, the transducer spectrum has multiple maxima, resulting in multiple capture lines of oil droplets. The higher order transducer displacement modes result in higher trapping forces and multiple stable trapping lines for the trapped oil droplets.

The capture line of the oil droplets was observed and characterized as the oil-water emulsion passed through the transducer. This characterization involves observation and pattern of the number of capture lines across the fluidic channel for seven of the ten resonance frequencies identified in fig. 7, as shown in fig. 8. Different displacement profiles of the transducer may produce different (more) capture lines in the standing wave, with a larger gradient in the displacement profile generally producing a higher capture force and more capture lines.

FIG. 9 is a digital model showing the pressure field matched to 9 trapping line patterns. The digital model is a two-dimensional model; and therefore, only three capture lines are observed. In a third dimension perpendicular to the plane of the page, there are two other sets of three capture lines.

In contrast to the vibration mode where the crystal effectively moves as a piston with uniform displacement, the lateral force of the acoustic radiation force generated by the transducer can be increased by driving the transducer in a higher order mode shape. In some example embodiments, the higher order mode shape is a bezier function. The sound pressure is proportional to the drive voltage of the transducer. Power is proportional to the square of the voltage. The transducer is typically a thin piezoelectric plate with an electric field in the z-axis and a dominant displacement in the z-axis. The transducers are typically coupled on one side by air (i.e., an air gap within the transducer) and on the other side by a fluid mixture containing particles that interact with each other. The type of wave generated in the plate is called a complex wave. The subset of the composite waves in the piezoelectric plate resemble leaky symmetric (also called compression or expansion) lamb waves. The piezoelectric properties of the plate typically result in the excitation of a symmetric lamb wave. These waves are leaky because they radiate into the water layer, which results in the generation of acoustic standing waves in the water layer. Lamb waves exist in an infinite range of thin sheets with stress-free conditions on their surfaces. Because the transducer of this embodiment is limited in nature, the actual mode displacement is more complex.

Fig. 10 shows the general variation of in-plane displacement (x-displacement) across the thickness of the plate and out-of-plane displacement (y-displacement), which is an even function across the thickness of the plate and out-of-plane displacement is an odd function. Due to the limited size of the plates, the displacement component varies across the width and length of the plates. In general, the (m, n) mode is a displacement mode of the transducer in which there are m fluctuations in the transducer displacement in the width direction and n fluctuations in the length direction, and the thickness variation as described in fig. 10. The maximum number of m and n is a function of the size of the piezoelectric material (e.g., piezoelectric crystal) and the excitation frequency. There are additional three-dimensional modes that do not have the form (m, n).

The transducer is driven such that the piezoelectric element vibrates in a higher order mode of the general formula (m, n), where m and n are independently 1 or greater. Generally, the transducer vibrates in a higher order mode than (2, 2). Higher-order modes generate more nodes and antinodes, resulting in a three-dimensional standing wave in the water layer, characterized by strong gradients in the acoustic field in all directions, not only in the direction of the standing wave, but also in the lateral direction. As a result, the acoustic gradient results in a stronger trapping force in the lateral direction.

Generally, an ultrasonic transducer may be driven by an electrical signal that may be controlled based on voltage, current, phase angle, power, frequency, or any other electrical signal characteristic. In particular, the drive signal for the transducer may be based on a voltage, current, magnetic, electromagnetic, capacitive, or any other type of signal to which the transducer is responsive. In embodiments, the voltage signal driving the transducer may have a sinusoidal, square, sawtooth, pulsed, or triangular waveform; and has a frequency of 500kHz to 10 MHz. The voltage signal may be driven with pulse width modulation, which produces any desired waveform. The voltage signal may also have amplitude or frequency modulated start/stop capability to eliminate flow. In particular embodiments, the voltage signal may have a frequency of about 0.5MHz to about 30MHz, such that such frequencies are generated by the ultrasound transducer.

The transducer is used to generate a pressure field that generates an acoustic radiation force orthogonal to and of the same order of magnitude as the direction of the standing wave. Particles of size 0.1 to 300 microns will move more effectively towards the "capture line" when the forces are of approximately the same order of magnitude, so that the cells and nucleic acids (whether naked or in a viral vector) co-localize against each other, allowing them to react with each other.

In biological applications, all parts of the system (e.g., the bioreactor, the acoustic filtering device, the tubing fluidly connecting them, etc.) may be separate from one another and disposable. Avoiding a centrifuge and filter allows for better separation of biological cells from the fluid without reducing the viability of the cells, the acoustophoretic separator can provide improved performance over centrifuges and filters. The transducer may also be driven to produce rapid pressure changes to prevent or clear blockages due to coagulation of biological cells. The frequency of the transducer may also be varied to obtain optimum effectiveness for a given power.

The techniques and embodiments described herein may be used for integrated, continuous automated bioprocessing. Control may be distributed to some or all of the units involved in bioprocessing. Feedback from the unit may be provided to allow for a summary of the biological process, which may be in the form of screen displays, control feedback, reports, status reports, and other information transfer. Distributed processing allows a high degree of flexibility in achieving the desired process control, for example by coordinating the steps in a unit and providing batch processing.

Acoustophoresis devices utilizing acoustic wave systems may be implemented with biocompatible materials, and may include gamma sterilized and disposable components. The processing system also allows for ultrasonic flow measurements, which are non-invasive and capable of operating with high viscosity fluids. The system may be implemented with a single-use sterile connector and a simple Graphical User Interface (GUI) for control. The acoustophoretic device is expandable. For example, relatively small units can be operated on a scale of 2L to 50L.

The methods, systems, and apparatus discussed above are examples. Various configurations may omit, substitute, or add various programs or components as appropriate. For example, in alternative configurations, the methods may be performed in a different order than described, and various steps may be added, omitted, or combined. In addition, features described with respect to certain configurations may be combined in various other configurations. Different aspects and elements of the configurations may be combined in a similar manner. In addition, technology is evolving and, thus, many elements are examples and do not limit the scope of the disclosure or claims.

Specific details are given in the description to provide a thorough understanding of example configurations, including embodiments. However, configurations may be practiced without these specific details. For example, well-known methods, structures and techniques have been shown without unnecessary detail in order to avoid obscuring the arrangement. This description provides example configurations only, and does not limit the scope, applicability, or configuration of the claims. Rather, the previous description of the configurations provides a description for implementing the techniques. Various changes may be made in the function and arrangement of elements without departing from the spirit or scope of the disclosure.

A statement that a value exceeds (or exceeds) a first threshold is equivalent to a statement that the value meets or exceeds a second threshold that is slightly greater than the first threshold, e.g., the second threshold is a value that is higher than the first threshold in the resolution of the associated system. A statement that a value is less than (or within) a first threshold is equivalent to a statement that the value is less than or equal to a second threshold that is slightly lower than the first threshold, e.g., the second threshold is a value that is lower than the first threshold in the resolution of the associated system.

Additionally, a configuration may be described as a method depicted as a flowchart or a block diagram. Although each may describe the operations as a sequential process, many of the operations can be performed in parallel or concurrently. In addition, the order of the operations may be rearranged. The method may have further stages or functions not included in the figures.

The following examples are provided to illustrate the apparatus and methods of the present disclosure. These examples are illustrative only and are not intended to limit the disclosure to the materials, conditions, or process parameters set forth therein.

Examples

Example 1:

fig. 11 is a photograph of a plastic bag containing a fluid mixture with T cells and viruses. The plastic bag is placed in an acoustophoresis device filled with water. A multi-dimensional acoustic standing wave is generated that encourages T cells to interact with the virus. This is visible within the plastic bag as a series of disc bundles.

Example 2:

the system (ThermoFisher Scientific) used baculovirus for transduction and for green fluorescencePhotoprotein (GFP) was transduced into Jurkat T cells. Such systems are used for various experiments. Five results are shown below. They are labeled control, method control 1, method control 2, acoustic 3MHz, and acoustic 10 MHz.

Control, method control 1, and method control 2 experiments were not exposed to the acoustic standing wave.

For acoustic 3MHz experiments, the use of an acoustic standing wave at a nominal frequency of 3Hz enhanced the interaction between T cells and viruses.

For acoustic 10MHz experiments, the use of an acoustic standing wave at a nominal frequency of 10Hz enhanced the interaction between T cells and viruses.

The results are set forth in Table A below. The MOI is the multiplicity of infection, or number of viral vector particles per cell. GFP + is% of cells expressing GFP.

TABLE A

Experiment of	MOI	GFP+(％)
			Control	-	-
Method control 1	50	28.4
			Method control 2	50	48.8
Acoustic 3MHz	10	21.8
			Acoustic 10MHz	10	48.4

The use of acoustics results in equivalent transduction efficiency with 80% fewer viral particles per cell.

Example 3:

the acoustic chamber includes an ultrasonic transducer and a reflector. The recirculation loop draws fluid from one end of the acoustic chamber and then circulates the fluid back into the acoustic chamber through the other end. The acoustic chamber has a volume of about 1mL, while the recirculation loop has a volume of about 3 mL. Transduction testing was performed using an acoustic chamber.

Four acoustic tests were performed using an acoustic chamber with recirculation. For both acoustic tests, the acoustics were turned on at a power level of 1W to determine their effect on conversion efficiency. For the other two tests, the acoustics were turned off (only recirculation).

Each acoustic test was also performed with a control run and a static run. For the control run, the virus was added to the cells in the culture dish and then immediately washed away. For static operation, the virus was added to the cells in the culture dish for 90 minutes and then washed away. The MOI (number of viral vector particles per cell) used for these tests was 25. For all runs, cell viability was measured by nucleocounter. Cell viability was also measured using a Vi-Cell viability analyzer for each acoustic run.

Table B indicates the parameters for each test. Table C indicates the results of each test run. Table D provides the Vi-Cell analysis. Table E provides the Nucleocounter analysis. In table C, the transduction gain is the acoustic efficiency divided by the static efficiency.

Table B.

Table C.

Table D.

Table E.

As can be seen in table C, the use of recirculation improves transduction efficiency. The use of acoustics plus recirculation improves transduction efficiency even more. Referring to table D, the two runs with acoustics and recycling (runs 1 and 2) showed about 72% more total viable cells transduced compared to the two runs with recycling but without acoustics (runs 3 and 4). Referring to table E, the two runs with acoustics and recycling (runs 1 and 2) showed about 68% more total viable cells transduced compared to the two runs with recycling but without acoustics (runs 3 and 4).

Example 4:

three acoustic tests were performed using an acoustic chamber with recirculation, as described in example 3, but run times of 30 minutes, 60 minutes and 90 minutes. The acoustics operate at a frequency of 2MHz and a power of 3.5W. Control and static runs were also performed as described in example 3, but for 30, 60 or 90 minutes. Table F indicates the parameters for each test. The results are shown in fig. 12. As can be seen therein, as the run time increases, the transduction efficiency improves compared to no acoustics.

Table F.

Having described several example configurations, various modifications, alternative constructions, and equivalents may be used without departing from the spirit of the disclosure. For example, the above elements may be components of a larger system in which other structures or processes may take precedence over or otherwise modify the application of the invention. In addition, many actions may be taken before, during, or after the above elements are considered. Accordingly, the above description does not limit the scope of the claims.

Claims

1. A method for introducing an exogenous nucleic acid into a cell, comprising:

placing the cells and nucleic acids in an acoustophoresis device, the device comprising:

an acoustic chamber in which the cell and nucleic acid are disposed; and

an ultrasonic transducer comprising a piezoelectric material, the ultrasonic transducer being drivable to produce an acoustic standing wave in the acoustic chamber; and

driving the ultrasonic transducer to produce a multi-dimensional acoustic standing wave;

wherein at least the cell is retained by the acoustic standing wave, the nucleic acid being co-localized with the cell to allow introduction of the exogenous nucleic acid into the cell.

2. The method of claim 1, wherein the nucleic acid is in a viral vector.

3. The method of claim 1, further comprising opening a hole in a cell membrane of the cell prior to co-localizing the cell with the nucleic acid.

4. The method of claim 3, wherein the pores are opened by electroporation, sonoporation, or by exposure to calcium phosphate.

5. The method of claim 1, wherein the acoustophoresis device further comprises a recirculation loop coupled with the acoustic chamber; and one or more of the cells or nucleic acids is recirculated through the acoustic chamber.

6. The method of claim 1, wherein the cell is a Chinese Hamster Ovary (CHO) cell, NS0 hybridoma cell, Baby Hamster Kidney (BHK) cell, human cell, regulatory T cell, helper T cell, cytotoxic T cell, memory T cell, effector T cell, gamma T cell, Jurkat T cell, CAR-T cell, B cell or NK cell, Peripheral Blood Mononuclear Cell (PBMC), algae, plant cell, or bacterium.

7. The method of claim 1, wherein the acoustic standing wave is a multi-dimensional acoustic standing wave, a planar standing wave, or a combination of a multi-dimensional acoustic standing wave and a planar standing wave.

8. The method of claim 1, wherein the ultrasound transducer is driven at a frequency of about 0.5MHz to about 20 MHz.

9. The method of claim 1, wherein the frequency of the acoustic standing wave is varied in a scanning pattern to move the cell relative to the nucleic acid.

10. A method for causing cell transduction comprising:

placing the cells and a viral vector comprising a nucleic acid in an acoustophoresis device, the device comprising:

an acoustic chamber in which the cell and nucleic acid are disposed; and

wherein the cell and viral vector are co-localized by an acoustic standing wave to allow transduction of the cell.

11. The method of claim 10, wherein the cells and viral vector are suspended in a fluid.

12. The method of claim 10, wherein the cell is a Chinese Hamster Ovary (CHO) cell, NS0 hybridoma cell, Baby Hamster Kidney (BHK) cell, human cell, regulatory T cell, helper T cell, cytotoxic T cell, memory T cell, effector T cell, gamma T cell, Jurkat T cell, CAR-T cell, B cell or NK cell, Peripheral Blood Mononuclear Cell (PBMC), algae, plant cell, or bacterium.

13. The method of claim 10, wherein the acoustophoresis device further comprises a recirculation loop coupled with the acoustic chamber; and one or more of the cells or nucleic acids is recirculated through the acoustic chamber.

14. The method of claim 10, wherein the ultrasonic transducer is driven at a frequency of about 0.5MHz to about 20 MHz.

15. The method of claim 10, wherein the frequency of the acoustic standing wave is varied in a scanning pattern to move the cells relative to the viral vector.

16. A method of causing transfection of a cell, comprising:

opening a pore in a cell membrane of the cell;

an acoustic chamber in which the cell and nucleic acid are disposed; and

driving the ultrasonic transducer to produce an acoustic standing wave;

wherein the cell and nucleic acid are co-localized by the acoustic standing wave to cause transfection of the cell.

17. The method of claim 16, wherein the pores are opened by electroporation, sonoporation, or by exposure to calcium phosphate.

18. The method of claim 16, wherein the well is opened before or after placing the cell in the acoustophoresis device.

19. The method of claim 16, wherein the acoustophoresis device further comprises a recirculation loop coupled with the acoustic chamber; and one or more of the cells or nucleic acids is recirculated through the acoustic chamber.

20. The method of claim 16, wherein the cell is a Chinese Hamster Ovary (CHO) cell, NS0 hybridoma cell, Baby Hamster Kidney (BHK) cell, human cell, regulatory T cell, helper T cell, cytotoxic T cell, memory T cell, effector T cell, gamma T cell, Jurkat T cell, CAR-T cell, B cell or NK cell, Peripheral Blood Mononuclear Cell (PBMC), algae, plant cell, or bacterium.

21. The method of claim 16, wherein the acoustic standing wave is a multi-dimensional acoustic standing wave, a planar standing wave, or a combination of a multi-dimensional acoustic standing wave and a planar standing wave.

22. The method of claim 16, wherein the ultrasonic transducer is driven at a frequency of about 0.5MHz to about 20 MHz.

23. The method of claim 16, wherein the frequency of the acoustic standing wave is varied in a scanning pattern to move the cell relative to the nucleic acid.

24. A cell produced by the method of claim 1.

25. A cell produced by the method of claim 10.

26. A cell produced by the method of claim 16.