JP2021518750A - Acoustic process for transfection and transduction - Google Patents

Abstract

A method for introducing a foreign nucleic acid into a cell by performing transfection / transduction using an acoustic process is disclosed. Foreign DNA / RNA and cells are co-located in a multidimensional acoustic standing wave or by acoustic streaming.

Description

Cross-reference to related applications This application is filed in US Provisional Patent Application No. 62 / 641,234 filed March 9, 2018 and US Patent Application No. 15 / 947,746 filed April 6, 2018. Claims the priority of the issue, all of which is incorporated herein by reference in its entirety.

Background The present disclosure relates to a method for introducing a foreign nucleic acid into a cell by transfection and transduction using acoustic waves. Also included in the present disclosure are cells and related compositions produced by such methods. Such methods and compositions may be useful in application to cell therapy.

Transfection and transduction are the processes for deliberately introducing nucleic acids (either DNA or RNA) into cells. Transduction is performed using viral vectors such as bacteriophage and other viruses. Viruses such as adenovirus, lentivirus, and paramyxovirus are commonly used. Transfection is the introduction of nucleic acids into cells using a non-viral method.

Brief Description The present disclosure relates to, in various embodiments, a process for introducing a foreign nucleic acid (DNA / RNA) into a cell, such as by transfecting or transducing the cell using acoustic waves. Very commonly, acoustic waves are used to bring cells together with nucleic acids so that they can be transferred to the cells. Acoustic devices can be used for this purpose and such devices are described herein. The cells and related compositions produced by such methods are also included in the description herein.

Disclosed herein in various embodiments are methods for inducing transfection or transduction of cells. The nucleic acid may be bare or in a viral vector. The cells and nucleic acids are placed within the electrophoretic device, for example, by placing them in a bag that is inserted into the electrophoretic device, or by flowing a fluid mixture containing both the cells and nucleic acids through the electrophoretic device. .. The acoustic migration device includes an acoustic chamber in which cells and nucleic acids are placed, an ultrasonic transducer, and a reflector facing the ultrasonic transducer, and the ultrasonic transducer generates a multidimensional acoustic standing wave in the acoustic chamber. Includes driveable piezoelectric material. Driving an ultrasonic transducer produces an acoustic standing wave. As a result, cells and nucleic acids are co-located by acoustic standing waves. In other words, cells and nucleic acids are placed close enough to each other so that they can react with each other. The acoustic standing wave may be a multidimensional acoustic standing wave, a plane standing wave, or a combination of both.

In some embodiments, the nucleic acid is present in a viral vector. Transduction can occur when the virus attaches to the target cell and injects the nucleic acid into the target cell. In other embodiments, pores are opened in the cell membrane of the cell before the cell is co-located with the nucleic acid. Transfection can occur when nucleic acids enter cells, for example through the pores. The pores can be opened by electroporation, sonoporation or exposure to calcium phosphate.

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

In certain embodiments, the cells are 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 delta T cells, Jurkat T cells, CAR-T cells, B cells or NK cells, or peripheral blood mononuclear cells (PBMC), algae, plant cells or bacteria. ..

The ultrasonic transducer may be driven for a period of about 5 to about 15 minutes, which period can be varied as needed. The ultrasonic transducer may be driven at a frequency of about 0.5 MHz to about 20 MHz. In some embodiments, the frequency of the multidimensional acoustic standing wave varies with a sweep pattern to move the cell against the nucleic acid.

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

The document also discloses methods of inducing cell transduction. A viral vector containing cells and nucleic acids can be driven to generate an acoustic chamber and a multidimensional acoustic standing wave, a planar acoustic standing wave, or a combination of a planar and multidimensional acoustic standing wave within the acoustic chamber. It is placed within an acoustic migration device consisting of an ultrasonic transducer containing the material. When the ultrasonic transducer is driven, a multidimensional acoustic standing wave, a planar acoustic standing wave, or a combination of a multidimensional acoustic standing wave and a planar acoustic standing wave is created. Cells and viral vectors are co-located by acoustic standing waves, triggering cell transduction.

Also disclosed herein are methods of inducing transfection of cells. Pore is opened in the cell membrane of the cell. The cells, along with the nucleic acids, are placed in an acoustic migration device consisting of an acoustic chamber and an ultrasonic transducer containing a piezoelectric material that can be driven to generate an acoustic standing wave within the acoustic chamber. The acoustic standing wave can be a multidimensional acoustic standing wave, a planar acoustic standing wave, or a combination of a planar acoustic standing wave and a multidimensional acoustic standing wave. The cells may be opened before being placed in the electrophoretic device or may be opened afterwards. After that, the ultrasonic transducer is driven to generate an acoustic standing wave. Cells and nucleic acids are co-located by acoustic standing waves, triggering cell transfection.

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

The accompanying drawings, which are incorporated herein and form part of this specification, illustrate certain aspects of the subject matter disclosed herein and, along with explanations, explain some of the principles associated with the disclosed embodiments. It is useful for doing.

FIG. 1 is a diagram illustrating the methods / processes of the present disclosure that enhance the efficiency of virus transduction. The cell culture is bound to a viral vector tagged with green fluorescent protein (GFP) and exposed to an acoustic process, where a multidimensional acoustic standing wave brings the cells and virus close to each other, resulting in reaction efficiency. To increase. After washing and overnight incubation, GFP is expressed intracellularly, indicating that transduction has occurred.

FIG. 2A is an exploded perspective view of an example of an acoustic electrophoretic device according to the present disclosure, which includes a cooling unit for cooling the transducer. FIG. 2B is a perspective view of the assembled device of FIG. 2A.

FIG. 3 is a perspective view of another electrophoretic device that can be used to carry out the methods / processes of the present disclosure. Disposable containers, such as plastic bags, contain a fluid mixture of two particle types that interact with each other in individual acoustic transfer devices, including one or more ultrasonic transducers.

FIG. 4 is a cross-sectional view of a conventional ultrasonic transducer.

FIG. 5 is a cross-sectional view of the ultrasonic transducer of the present disclosure. There is an air gap in this transducer and there is no backing layer or protective plate.

FIG. 6 is a cross-sectional view of the ultrasonic transducer of the present disclosure. There is an air gap in this transducer, a backing layer and a protective plate.

FIG. 7 is a graph showing the electrical impedance amplitude with respect to the frequencies of square transducers driven at different frequencies.

FIG. 8 shows the configuration of capture lines for the seven resonance frequencies (minimum values of the electrical impedance amplitude) in FIG. 7 as viewed from a direction orthogonal to the flow of the fluid.

FIG. 9 is a computer simulation of the acoustic pressure amplitude (Pa on the right scale) and the out-of-plane displacement of the transducer (meters on the left scale). The letters at the top of the scale on the left are read as "^x10-7". The letter with the upward triangle at the top of the scale on the left is read as ^{"1.473 x 10-6".} By the downward triangle at the bottom of the left scale character reads as "1.4612 × ^{10 -10".} The upper part of the character of the right side of the scale is read as "× 10 ^6". Character by the upward triangle at the top of the right-hand side of the scale is read as "1.1129 × 10 ^6". The letter of the downward triangle at the bottom of the scale on the right side is read as "7.357". These triangles show the maximum and minimum values of the scale shown in this figure. The horizontal axis represents the position in the chamber along the X axis in inches, and the vertical axis represents the position in the chamber along the Y axis in inches.

FIG. 10 shows the in-plane displacement and the out-of-plane displacement of the crystal in which the compound wave is present.

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

FIG. 12 is a graph showing the ratio of transduction efficiency (acoustic / no acoustic) to transduction time for three experiments at different execution times (30 minutes, 60 minutes and 90 minutes).

Detailed Description The present disclosure can be more easily understood by reference to the detailed description below for the desired embodiments and examples contained therein. In the following specification and subsequent claims, some terms defined to have the following meanings are referred to.

Although specific terms are used in the following description for clarity, these terms refer only to the particular structure in the embodiments selected for illustration purposes and are within the scope of the present disclosure. Does not define or limit. It should be understood that in the drawings and in the following description, similar numerical codes refer to components of similar function.

The singular "a", "an" and "the" include plural objects unless the context clearly indicates otherwise.

The term "comprising" is used herein as requiring the presence of named components and allowing the presence of other components. The term "comprising" should be construed to include the term "consisting of" which allows the presence of only the named component along with any impurities that may result from the manufacture of the named component. Is.

The numbers are the same when converted to the same number of significant figures, and differ from the numbers described below the experimental error of the types of conventional measurement techniques described in this application to determine the numbers. It should be understood that the numbers are included as an example.

All ranges disclosed herein include endpoints listed and can be combined independently (eg, the range "2 grams to 10 grams" is a boundary point 2 Includes grams and 10 grams and all medians). The range boundaries and arbitrary values disclosed herein are not limited to the exact range or value, but are ambiguous enough to include values close to these range and / or values. To have.

The modifier "about" used in relation to a quantity includes the stated values and has a meaning defined by the context. When used in connection with a range, the modifier "about" also discloses the range defined by the absolute value of the two boundaries. For example, the range "about 2 to about 10" also discloses the range "from 2 to 10". The term "about" can refer to 10% of the plus or minus of the indicated number. For example, "about 10%" may indicate a range of 9% to 11%, and "about 1" may mean a range of 0.9 to 1.1.

Please note that many of the terms used in this document are relative terms. For example, the terms "upper" and "lower" are relative to each other in position, i.e., the upper component is located at a higher altitude than the lower component in a given direction. However, these terms can change when the device is flipped. The terms "inlet" and "exit" are relative to the fluid flowing through them with respect to a given structure, for example, the fluid flows into the structure through the inlet and the structure through the outlet. Outflow from things. The terms "upstream" and "downstream" are relative to the direction in which the fluid flows through the various components, i.e., the fluid passes through the upstream components before flowing through the downstream components. It flows. Note that in the loop, the first component can be explained as both upstream and downstream of the second component.

The terms "horizontal" and "vertical" are used to indicate an absolute reference, that is, the direction relative to the level of the ground. However, these terms should not be construed to require that the structures be absolutely parallel or absolutely vertical. For example, the first vertical structure and the second vertical structure are not necessarily parallel to each other. The terms "top" and "bottom" or "base" are absolute criteria, that is, the plane whose top is always higher than the bottom / bottom with respect to the surface of the earth. Used to point to. The terms "upwards" and "downwards" are also relative to absolute criteria, and upwards always oppose the Earth's gravity.

In this application, "the size of the same order" is referred to. If the quotient of the larger number divided by the smaller number is at least 1 and is less than 10, the two numbers will be of the same digit size.

The acoustic devices described herein may operate in multimode, planar mode, or a combination thereof. Multi-mode means that an acoustic wave is generated by an acoustic transducer that generates acoustic force three-dimensionally. A multimode acoustic wave, which may be an ultrasonic wave, can be generated by a single acoustic transducer and is sometimes referred to herein as a multidimensional acoustic standing wave or a three-dimensional acoustic standing wave. Planar mode means that an acoustic wave is generated by an acoustic transducer that generates an acoustic force that is substantially one-dimensional, for example, along the propagation direction. Such acoustic waves, which may be ultrasonic waves, generated in planar mode are sometimes referred to herein as one-dimensional acoustic standing waves.

The acoustic transducer may be made of a piezoelectric material such as lead zirconate titanate (PZT) or lithium niobate. Such an acoustic transducer can be electrically excited to generate a planar acoustic wave or a multimode acoustic wave. The three-dimensional acoustic force generated by the multimode acoustic wave includes a radial or lateral force that does not coincide with the propagation direction of the acoustic wave. Lateral forces may act in two dimensions. The lateral force is present in addition to the axial force of the multimode acoustic wave that substantially coincides with the propagation direction of the acoustic wave. The lateral force may be on the same order as the magnitude of the axial force in such a multimode acoustic wave. The acoustic transducer excited by the multimode operation may generate a standing wave on its surface, thereby generating a multimode acoustic wave. The standing wave on the surface of the acoustic transducer may be related to the operating mode of the multimode acoustic wave. Further, when the acoustic transducer is electrically excited to generate a planar acoustic wave, a one-dimensional acoustic standing wave may be generated by the surface of the transducer exhibiting a piston-like action. Compared to planar acoustic waves, multimode acoustic waves continuously exhibit very high particle capture activity at the same input power. One or more acoustic transducers may be used to generate a combination of planar acoustic standing waves and multidimensional acoustic standing waves. For example, two acoustic transducers may be arranged to face each other in order to generate a standing wave. In such an example, one of the acoustic transducers is passive and may act as a reflector of the incident wave. Alternatively or additionally, each transducer may be active to generate acoustic waves, including acoustic waves as described elsewhere in this document.

Acoustic electrophoresis is the manipulation of matter using acoustic waves. In some exemplary implementations, acoustic electrophoresis is used for the separation of substances and is a solid state with low power consumption, no pressure loss and no clogging to separate the particles from the fluid dispersion. Represents the approach of. As a result of the scattering of the acoustic field from the particles, a three-dimensional acoustic radiation force is generated, which acts as a three-dimensional capture field. When the particles are small relative to the wavelength, the acoustic radiation force is proportional to the volume of the particles (eg, the cube of the radius). Acoustic radiation is proportional to frequency and acoustic contrast factors. Acoustic radiation is proportional to sound energy (eg, the square of acoustic pressure amplitude). In the case of harmonic excitation, the spatial variation of the force sine wave drives the particle to a stable position within the standing wave. If the acoustic radiation exerted on the particle is stronger than the combined effect of fluid drag and buoyancy / gravity, the particle is trapped in the field of an acoustic standing wave. As a result of lateral and axial acoustic forces acting on the captured particles, particles heavier than the host fluid will continuously settle under the action of gravity when the critical size is reached, or more than the host fluid. Light particles rise by the action of buoyancy, and through the concentration, clustering, agglomeration, agglomeration and / or coalescence of such particles, tightly packed clusters are formed. In addition, secondary intermolecular forces such as Bjerkness force help the particles to agglomerate.

Acoustic standing waves create local areas of high and low pressure. The particles are pushed into the nodes or antinodes of the standing wave, depending on the compressibility and density of the surrounding fluid. Particles with high density and compression ratio move to the node of the standing wave, and the secondary phase with low density moves to the antinode. The force applied to the particles also depends on the size of the particles, and the larger the particles, the greater the force. The magnitude of the force depends on the density and compression ratio of the particles with respect to the fluid medium and increases with the volume of the particles.

For the purposes of the present disclosure, biological cells can be considered particles. The acoustic contrast factor between the cell and the medium has a positive value because most biological cell types are denser and less compressible than the medium in which they are suspended. As a result, axial acoustic radiation (ARF) drives the cell towards the pressure node of the standing wave. The axial component of acoustic radiation drives cells with a positive contrast factor into the pressure node, while cells with a negative contrast factor or other particles are driven into a pressure antinode. The radial or lateral component of acoustic radiation is the force that captures cells. The radial or lateral component of the ARF is greater than the combined effect of fluid drag and gravity.

Additional theoretical and numerical models have been developed to calculate the acoustic radiation force of particles without being constrained by particle size with respect to wavelength. These models also include the effects of fluid and particle viscosities, allowing for more accurate acoustic radiative calculations. The implemented model is based on the theoretical study of Yurii Ilinskii and Evgenia Zabolotskaya described in "AIP Conference Proceedings" (Vol. 1474-1, pp. 255-258 (2012)). ing. In addition, an additional in-house model has been developed to calculate the acoustic capture force for cylindrical objects such as "hockey packs" of particles captured during standing waves.

Desirably, the ultrasonic transducer generates a multidimensional standing wave in the fluid, which accompanies an axial force to exert a lateral force on the suspended particles. Typical results disclosed in the literature are that the magnitude of the lateral force is two orders of magnitude smaller than the axial force. In contrast, the techniques disclosed herein provide those in which the magnitude of the lateral force is on the same order as the axial force. However, in certain embodiments further described herein, the device uses both a transducer that produces a multidimensional acoustic standing wave and a transducer that produces a planar acoustic standing wave. The lateral force components of all acoustic radiation (ARF) produced by the ultrasonic transducers of the present disclosure are important, overcoming fluid drag and producing tightly packed clusters at linear velocities up to 1 cm / s. It is sufficient to do so and has the same magnitude as the axial force component of the total acoustic radiation.

The present disclosure relates to methods of using an electrophoretic device, such as an ultrasonic transducer, to perform transfection or transduction. Briefly, electrophoretic devices are used to combine cells with nucleic acids (DNA and / or RNA) within a local volume. This allows the nucleic acid to be transferred into the cell. Current transduction processes can be relatively costly, inefficient, and incapable of scaling up for commercialization. The methods described in this document can reduce costs, increase efficiency, and have a scalable platform for commercialization.

Very commonly, cells and carriers (including RNA or DNA) are placed within the acoustic chamber of the electrophoretic device. Generally, they are suspended in a fluid to form a fluid mixture. In some exemplary embodiments, the acoustic electrophoresis device comprises an acoustic chamber having an ultrasonic transducer and a reflector facing the ultrasonic transducer (eg, on the opposite wall of the chamber). The ultrasonic transducer contains a piezoelectric material that can be driven to generate an acoustic standing wave, such as a multidimensional acoustic standing wave and / or a planar acoustic standing wave, in the acoustic chamber. The acoustic standing wave has a place where the pressure is high and a place where the pressure is low. In some examples, low pressure areas capture and retain cells. Nucleic acid flows into the acoustic chamber along with the retained cells. This movement allows the cell and nucleic acid to co-locate and allow the nucleic acid to enter the cell. In the case of transfection, pores are opened in the cell membrane of the cell, allowing bare nucleic acid to enter the cell. In the case of transduction, the nucleic acid is part of the viral vector. The viral vector enters the cell or inserts the nucleic acid into the cell. Specific viral vectors that can be used to perform transduction in the present disclosure include adenovirus, lentivirus, or paramyxovirus.

In some exemplary embodiments, the acoustic drag generated by acoustic standing waves on cells and / or nucleic acids (bare or in a viral vector) is the fluid drag exerted by the moving fluid on these particles. Can be enough to overcome. In other words, electrophoretic power can act as a mechanism for capturing cells and / or nucleic acids in the acoustic field. The acoustic migration force can drive cells and / or nucleic acids to a stable position where the amplitude of the acoustic migration force is reduced or minimized. These positions where the amplitude of the acoustic electrophoretic force is reduced or minimized can be a node of a stationary acoustic wave. Over time, cell and / or nucleic acid aggregates in the nodes grow steadily. Within a period that can be within minutes depending on their concentration, the cell and / or nucleic acid aggregates can take the form of a beam-like disc aggregate. Each disc can be spaced half a wavelength apart from the acoustic field.

In some embodiments, acoustic standing waves capture cells and / or nucleic acids and co-locate them to improve the efficiency of transfection / transduction reactions. In these embodiments, several different mechanisms may be implemented. In one such mechanism, cells and nucleic acids may have similar acoustic contrast factors so as to drive both types of particles to the nodes or antinodes of the standing wave. This mechanism brings cells and nucleic acids spatially close to each other more efficiently than relying on Brownian motion (similar to conventional agitation). In other words, cells and nucleic acids are captured in a three-dimensional volume produced by a multidimensional acoustic standing wave, which is small compared to the size of the acoustic chamber. In certain exemplary embodiments, the cell and nucleic acid both have a positive acoustic contrast factor, or both have a negative acoustic contrast factor. In other words, those acoustic contrast factors have the same sign. Again, the nucleic acid may be bare or in a viral vector.

In another mechanism, one of the two types of particles (cells or nucleic acids) may be driven to the node and the other type of particles to the belly. However, at higher frequencies, the nodes and belly are close enough that the cells and nucleic acids can react with each other. In such an embodiment, the cell or nucleic acid has a positive acoustic contrast factor and the other set (ie, nucleic acid or cell) has a negative acoustic contrast factor. In other words, those acoustic contrast factors have opposite signs. Particles with a positive contrast factor are driven to the nodes and particles with a negative contrast factor are driven to the belly. Several factors related to this reaction mechanism include cell and carrier size and the frequency at which the ultrasonic transducer operates.

Eventually, as cells and / or nucleic acids continue to be captured and concentrated, they can reach a size and weight that causes gravitational sedimentation, where particle clusters from acoustic standing waves to acoustic chambers. Will fall to the bottom of the. A new aggregate of particles can then be captured and reacted in the acoustic field generated by the acoustic standing wave.

In some exemplary embodiments, the acoustic transducer has a driveable piezoelectric material in the acoustic chamber to generate an acoustic standing wave, such as a multidimensional acoustic standing wave and / or a planar acoustic standing wave. It has an acoustic chamber with an ultrasonic transducer including. This acoustic standing wave has a high pressure location and a low pressure location. The acoustic chamber is provided with a recirculation loop that allows the output fluids and particles to be reintroduced into the acoustic chamber. In some examples, low pressure areas capture and retain cells, but not viral vectors or nucleic acids that are shed from the acoustic chamber or reintroduced through a recirculation loop. .. In the example of a viral vector, the cells are held in an acoustic field generated by an acoustic standing wave, which flows into the acoustic field to allow the cells to interact with the viral vector. By recirculating the viral vector that did not interact in the previous pass into the acoustic chamber, the number of interactions between the viral vector and the cell can be increased. In the nucleic acid example, additional treatment of cells held in the acoustic field to allow the formation of pores in the cell membrane, as discussed in more detail elsewhere in this book. It can be performed. The nucleic acid is allowed to flow into the acoustic chamber along with the retained cells to allow the nucleic acid to interact with the cell, allowing the nucleic acid to enter the cell through the pores. Nucleic acid is not retained by the acoustic field and is recirculated into the acoustic chamber to allow multiple passes to occur during transfection.

Additional or alternative, in some embodiments, the ultrasonic transducer is driven to cause acoustic streaming within the acoustic chamber. Simply put, acoustic streaming refers to the flow of fluid that occurs in an acoustic chamber as the fluid absorbs the sound energy transmitted by the ultrasonic transducer (from the vibration of the ultrasonic transducer). The velocity of the fluid is induced by the vibration of the acoustic wave generated by the ultrasonic transducer. Typically, when acoustic streaming occurs, circulatory motion or vortices can occur, causing agitation of the fluid mixture. This phenomenon is non-linear and can cause cell-nucleic acid interactions.

The cells and nucleic acids are brought close together 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 nucleic acid may invade a cell to induce transduction. The virus can be a retrovirus, such as a lentivirus.

For the transfection example, pores are opened in the cell membrane of the cell to allow the nucleic acid to enter the cell. For example, electroporation can be performed by exposing cells to an electric field to increase the permeability of cell membranes. Also, sonoporation, in which cells are exposed to ultrasonic waves to induce pore formation in the cell membrane, can be used. Calcium phosphate can also be used to trigger transfection. The location of the pore opening is not important and can occur within the electrophoretic device upstream of the acoustic chamber where the cells and nucleic acids are mixed, or outside the electrophoretic device. ..

For reference, productive transfection and gene transfer are not only the entry of DNA into the cell and subsequent transcription from the appropriate promoter, but also the DNA from the cell surface to the cytoplasm and cytoplasm. It should be noted that it also requires a number of intracellular events that allow it to migrate through and eventually into the nucleus before transcription is initiated across the nuclear membrane. As soon as they enter the cytoplasm, the bare DNA (delivered using physical techniques or after degradation of the DNA-carrier complex) is a microtubule network for migration towards the microtubule formation center and the nuclear envelope. Binds to numerous cytoplasms that mediate subsequent interactions with. The plasmid then enters the nucleus using different sets of proteins during nuclear envelope division and degradation, or via the nuclear pore complex in the absence of cell division.

Specific viral vectors that can be used to perform transduction in the present disclosure include adenovirus, lentivirus, or paramyxovirus. Retroviruses are characterized by the ability to reverse transcrib their RNA genome into a cDNA copy, which is then stably integrated into the host cell genome. In this way, the virus carries the nucleic acid into the cell.

Retroviruses can be classified as simple or complex (eg, lentivirus). Both types of viral particles contain two copies of positive-strand RNA associated with viral reverse transcriptase (RT) located within the inner core. Structural and enzymatic proteins such as nucleocapsid (NC), capsid (CA), integrase (IN), and protease (PR) are also located in this compartment. The inner core is surrounded by an outer protein layer composed of matrix (MA) proteins, which is surrounded by envelopes from the host cell membrane interspersed with envelope glycoproteins (ENVs). ..

Lentivirus and retrovirus gene delivery systems (or carriers) utilize aspects of retrovirus replication to provide stable integration of the desired nucleic acid sequence. Transfection of foreign nucleic acids results only in transient transgene expression, whereas viral integrase activity in retrovirus and lentivirus-based systems allows for stable integration of foreign transgenes. It is inherited after that and is continuously expressed while repeating cell division. An important feature of both lentiviral and retroviral vectors is that they produce replication-deficient or self-inactivating particles. This allows delivery of the desired sequence without continued viral replication in the target cell.

A common method for verifying that a foreign nucleic acid has been successfully introduced into a cell is to measure protein expression. This is typically done by Western blot or immunostaining.

Examples of cells that can be transfected / transduced by the processes 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, Contains cytotoxic T cells, memory T cells, effector T cells, gamma delta T cells, jarcut T cells, CAR-T cells, B cells or NK cells, or peripheral blood mononuclear cells (PBMC), algae , Plant cells or bacteria. The cells themselves may be attached to other materials such as beads. Examples of beads include polymer beads, magnetic beads, superparamagnetic beads, microspheres and the like. These can be used for biochemical reactions or for labeling purposes.

In some embodiments, additives can be included in the mixture of cells and nucleic acids. Such additives may include Polybrene (registered trademark) and RetroNectin (registered trademark) as examples. Polybrene (registered trademark) is a cationic polymer (polyhexadimethrin bromide) used to increase the efficiency of transduction of predetermined cells using retroviruses (and lentiviruses) in cell culture. , Usually used in an amount of 10 μg / mL or less. RetroNectin (registered trademark) is a polypeptide consisting of three functional domains (C domain, H domain, CS-1 site) derived from human fibronectin protein. RetroNectin (registered trademark) enhances the efficiency of retrovirus and lentivirus-mediated transmission in hematopoietic cells, including hematopoietic stem cells, and terminally differentiated cells such as primary T cells and macrophages. Other additives may include cationic peptides such as lipofectin, lipofectamine, and protamine as examples. These additives can be used to enhance transduction, if desired.

Without being limited by theory, the frequency of a multidimensional acoustic standing wave is thought to determine the diameter of the particles that can be captured by the acoustic standing wave. For example, in the case of a 2 MHz wave, the particle size is about 1 to about 100 microns.

FIG. 1 is a diagram illustrating an exemplary method of the present disclosure applied to viral transduction. In this example, the cells are labeled with green fluorescent protein (GFP). Starting from the left side of the figure, cell culture 100 is first combined with viral vector 110. The fluid mixture containing the cells and the virus is then placed in an acoustic chamber 120 located between the ultrasonic transducer 122 and the reflector 124. The acoustic standing wave is generated at room temperature for 10 minutes. As illustrated here, cells and viruses are captured by acoustic standing waves. The cells are trapped in the nodes and the virus is trapped in the abdomen. However, due to their relative size, the cell and the virus are co-located and the virus can infect the cell (indicated by reference numeral 128). After washing to remove unreacted material, cells are incubated overnight at 37 ° C. to express GFP in labeled cells. Similar methods can be used to generate T cells or CAR-T cells that express the chimeric antigen receptor (CAR).

The methods of the present disclosure can be carried out in a continuous process, where a fluid mixture containing nucleic acids suspended in cells and host fluids flows through an electrophoretic device. The nucleic acid may be exposed or may be contained in a viral vector such as a bacteriophage or other virus.

FIG. 2A is an exploded view of the acoustic electrophoresis device 200 that can be used for continuous processing. FIG. 2B is a diagram of the fully assembled device 200.

As shown in FIG. 2A, the electrophoretic device is constructed with each component modularized and can be modified or interchanged separately from each other. Thus, when new modifications or changes are made to a given component, the rest of the device remains the same and that component can be replaced.

The device has an ultrasonic transducer 220 and a reflector 250 provided on the opposing walls 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 in close proximity to the first wall of the acoustic chamber. The reflector may be in close proximity to the second wall of the acoustic chamber or may form a second wall of the acoustic chamber.

The cooling unit 260 can be arranged between the ultrasonic transducer 220 and the flow chamber 210. As illustrated herein, the cooling unit 260 has an independent flow path separate from the flow path through the acoustic chamber. The coolant inlet 262 allows the cooling fluid to enter the cooling unit. The coolant and waste heat exit the cooling unit through the coolant outlet 264. The cooling fluid flowing through the cooling unit can be any suitable fluid. For example, the coolant can be water, air, alcohol, ethanol, ammonia, or some combination thereof. The coolant can be a liquid, gas or gel. The coolant can be an electrically non-conductive fluid to prevent electrical short circuits.

Alternatively, the cooling unit can also be in the form of a thermoelectric generator that utilizes the Seebeck effect to convert heat flux (ie, temperature difference) into electrical energy, thereby removing heat from the flow chamber. In other words, electricity can be generated from unwanted waste heat while the electrophoretic device is operating.

The cooling unit can be used to cool the ultrasonic transducer, which is especially advantageous when the device is operated continuously over a long period of time (eg during perfusion) with repeated processing and recirculation. Can be. Alternatively, the cooling unit can also be used to cool the fluid passing through the acoustic chamber 210. For desired applications, cells should be maintained near room temperature (~ 20 ° C.), at most around 28 ° C. This is because when a cell experiences a high temperature, its metabolic rate increases. However, without a cooling unit, the temperature of cells flowing through the acoustic chamber can rise to 34 ° C.

Note that the acoustic chamber 210 is exemplified here to have at least an inlet 212 and an outlet 214. This provides access to the internal volume 216 of the acoustic chamber. Additional inlets and outlets (eg, fluid inlets, concentrate outlets, permeate outlets, recirculation outlets, bleed / harvest outlets) may be included as appropriate. The internal volume 216 can be considered to be surrounded by the ultrasonic transducer 220, the cooling unit 260, the acoustic chamber 210 and the reflector 250.

The flow direction of the acoustic electrophoresis device 200 can be directed to a direction other than horizontal. For example, the flow of fluid may be either vertical, either upward or downward, or at some angle to vertical or horizontal. One or more transducers can be included in the system.

FIG. 3 illustrates another electrophoretic device 300 that can be used to carry out the methods and processes of the present disclosure. Very generally, the system includes an acoustic electrophoresis device 300 and a container 310 that is substantially permeable to acoustics. These two components are separable from each other.

The container 310 of the acoustic migration device is generally made of a substantially acoustically permeable material (all at suitable thickness) such as plastic, glass, polycarbonate, low density polyethylene, high density polyethylene. However, the vessel may be formed from any material suitable to allow the passage of the acoustic standing waves of the present disclosure. The container may be in the form of a bottle or bag. The difference between these forms lies in their composition and structure. Bottles are more rigid than bags. In the empty state, the bag generally cannot support itself, but the bottle can stand on its own. For example, the container 310 shown here is a high density polyethylene bag. The container 310 generally has an upper end 312 and a lower end 314 and has an internal volume in which the fluid mixture (including primary and secondary particles in the host fluid) is located.

The shape of the electrophoretic device 300 is defined by at least one wall 332, usually a plurality of walls, forming its side surface. For example, the electrophoretic device may be cylindrical or rectangular (as shown). The walls are solid. An opening 326 for receiving the container 310 is provided at the upper end of the electrophoretic device. Again, the electrophoretic device 300 is separable from the container 310, and the container can be disposable or reusable, depending on the desired application of the electrophoretic device. As illustrated here, the base of the electrophoretic device 300 is solid.

The acoustic electrophoresis device 300 includes at least one ultrasonic transducer 330 on the wall 334. The ultrasonic transducer 330 has a piezoelectric material driven by a voltage signal to generate an acoustic standing wave. Cable 332 is shown for transmitting power and control information to the ultrasonic transducer 330. The reflector 340 may be present and the reflector is placed on the wall 336 facing the ultrasonic transducer 330. As a result, the standing wave is generated via the initial wave radiated from the transducer and the reflected wave from the reflector. Some embodiments do not require a separate reflector. For example, a chamber wall, or an open boundary that can be provided by ambient air, may be used to reflect incident waves to generate standing waves. It should be understood that various transducer and reflector combinations may be used. Planar and / or multidimensional acoustic standing waves are generated within the vessel and are used to trigger the interaction of particles within the vessel 310. Note that there is no contact between the ultrasonic transducer and the fluid mixture in the vessel 310.

In certain embodiments, the electrophoretic device comprises a plurality of ultrasonic transducers 330 located on a common wall 334 facing the wall 336 where the reflector 340 is located. Alternatively, the ultrasonic transducers may be arranged to face each other in the absence of reflectors. Further, the acoustic electrophoresis device 300 may include an observation window 324 in another wall 338. As illustrated herein, if an observation window is provided, it may be on the wall adjacent to the wall on which the ultrasonic transducers and reflectors are located, in which case the lower end 314 of the container 310. Can be seen through the observation window 324 of the separation chamber 320. In other embodiments, the observation window can replace the reflector.

In certain embodiments, a fluid such as water is placed in the clearance space 305 between the vessel 310 and the sound absorbing device 300, and an acoustic standing wave passes through both the fluid in the clearance space and the fluid mixture in the vessel. You may try to do it. The crevice fluid should have an acoustic impedance value that allows good transmission of acoustic standing waves, preferably low acoustic attenuation, but can be any fluid.

In certain exemplary embodiments, the ultrasonic transducer is driven at a frequency of about 0.5 MHz to about 20 MHz (megahertz). Higher frequency standing wave fields result in steeper pressure gradients, which are suitable for capturing small particles such as viruses. The ultrasonic transducer can be driven for a period of about 5 to about 15 minutes. This is significantly shorter than, for example, a conventional viral transduction process in which the cell culture and the viral vector are incubated together for about 30 to about 120 minutes. This long incubation time is due to the reaction between Brownian motion only when the cells and viruses are brought close to each other. The use of the acoustic electrophoresis devices of the present disclosure greatly increases the probability that cells and viruses are close enough to react with each other. This results in higher reaction efficiency using fewer particles. However, if desired, the ultrasonic transducer can be driven for a desired time, eg, a time of up to 120 minutes or longer.

Here, it may be useful to describe the ultrasonic transducers used in acoustic filtering devices in more detail. FIG. 4 is a cross-sectional view of a conventional ultrasonic transducer. This transducer is composed of a protective plate 50 at the lower 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. And have. Positive electrodes 61 and negative electrodes 63 are provided on both sides of the ceramic piezoelectric element. The epoxy layer 56 attaches the backing layer 58 to the piezoelectric element 54. The entire assembly is housed in a housing 60 that can be made from, for example, aluminum. The housing is used as a ground electrode. The electrical adapter 62 provides a connection for a wire that passes through the housing and connects to a lead wire (not shown) attached to the piezoelectric element 54. Generally, the backing layer is designed to add damping to form a wideband transducer with uniform displacement over a wide frequency range and to suppress the excitation of a particular vibration-specific mode of the piezoelectric element. The protective plate is usually designed as an impedance transducer to better match the characteristic impedance of the medium from which the transducer radiates.

FIG. 5 is a cross-sectional view of the ultrasonic transducer 81 of the present disclosure used in the acoustic filtering device of the present disclosure. The transducer 81 has a square shape and has an aluminum housing 82. The aluminum housing has an upper end and a lower end. The transducer housing may also be made of plastic or other metal such as medical grade HDPE. A piezoelectric element is a mass of perovskite-structured ceramics, each of which is a small tetravalent metal, usually titanium or zirconium, within a lattice of large divalent metal ions, usually lead or barium. It consists of an ion and an O ^{2-ion lattice.} In one example, a PZT (lead zirconate titanate) piezoelectric element 86 defines the lower end of the transducer and is exposed from the outside of the lower end of the housing. The piezoelectric element is supported around it by a small elastic layer 98 arranged between the piezoelectric element and the housing, such as epoxy, silicone or similar material. In other words, there is no protective plate or backing material. However, in some embodiments, there is a layer of plastic or other material that separates the piezoelectric element from the fluid in which the acoustic standing wave is generated. Piezoelectric materials / elements / crystals have an outer surface (exposed) and an inner surface as well.

The screw 88 attaches the aluminum upper plate 82a of the housing to the main body 82b of the housing via a screw thread. The top plate includes a connector 84 for powering the transducer. The upper surface of the piezoelectric element 86 is connected to the positive electrode 90 and the negative electrode 92 separated by the insulating material 94. The electrodes can be made from any conductive material such as silver or nickel. Electric power is supplied to the piezoelectric element 86 via an electrode on the piezoelectric element. The piezoelectric element 86 does not have a backing layer or an epoxy layer. In other words, there is an internal volume or air gap 87 within the transducer between the aluminum top plate 82a and the piezoelectric element 86 (ie, the air gap is completely empty). As seen in FIG. 6, in some embodiments, a minimal backing 58 and / or protective plate 50 may be provided.

Transducer design can affect the performance of the system. A typical transducer has a layered structure in which a ceramic piezoelectric element is bonded to a backing layer and a protective plate. Because the transducer is loaded with the high mechanical impedance exhibited by standing waves, conventional protective plates such as, for example, half-wave thickness for standing wave applications or 1/4 wavelength thickness for radiation applications. Design guidelines and manufacturing methods may not be appropriate. Rather, in the transducer of one embodiment of the present disclosure, there is no protective plate or backing, and the piezoelectric element can oscillate in one or a combination of multiple unique modes having its high Q value. The vibrating ceramic piezoelectric element / disk is directly exposed to the fluid flowing through the fluid cell.

Removing the backing (eg, the back of the piezo is in contact with air) also allows the ceramic piezo to vibrate in higher vibration modes with slight damping (eg, higher mode displacement). To. In a transducer having a piezoelectric element with a backing, the piezoelectric element vibrates with a more uniform displacement, like a piston. When the backing is removed, the piezoelectric element vibrates in a non-uniform displacement mode. The higher the mode shape of the piezoelectric element, the more nodes the piezoelectric element has. Higher-order modal displacements of the piezo give rise to more capture lines, but the correlation between the capture lines and the nodes is not always one-to-one, and driving the piezo at higher frequencies does not necessarily result in more. Not many capture lines occur.

In some embodiments of the acoustic filtering devices of the present disclosure, the piezoelectric element may have a backing that has minimal (eg, less than 5%) effect on the Q value of the piezoelectric element. The backing allows the piezo to vibrate in a higher mode shape and provides some mechanical support to the piezo while maintaining a high Q value, acoustically substantial, such as balsa, foam or cork. It may be made of a transparent material. The backing layer may be solid or may be a grid with holes through the layer, such a grid following the nodes of the piezoelectric element vibrating in a particular higher order vibration mode. The rest of the piezoelectric element is supported at the node position, allowing it to vibrate freely. The action of the lattice or the purpose of the acoustically transparent material is to support the piezoelectric element without lowering the Q value or interfering with the excitation of a particular mode shape.

Placing the piezoelectric element in direct contact with the fluid also contributes to a high Q value by avoiding the damping and energy absorption effects of the epoxy layer and protective plate. In other embodiments of the transducer, it may have a protective plate or surface to prevent the lead-containing PZT from coming into contact with the host fluid. This may be desirable in biological applications such as, for example, blood separation, biopharmaceutical perfusion, or feed-batch filtration of mammalian cells. In such applications, a protective layer such as chromium, electrolytic nickel or electroless nickel may be used. Chemical vapor deposition can also be used to coat layers of poly (p-xylylene) (eg parylene) or other polymers. Organic and biocompatible coatings such as silicone or polyurethane can also be used as protective surfaces. Thin films such as polyetheretherketone (PEEK) films also have the advantage of being biocompatible and can be used as a cover for transducer surfaces exposed to fluids. In one embodiment, the PEEK film is adhered to the surface of the piezoelectric material using a pressure sensitive adhesive (PSA). Other films can be used as well.

In some embodiments, the ultrasonic transducer has a nominal resonance frequency of 2 MHz. Each transducer can consume about 28 W of power to capture droplets at a flow rate of 3 GPM (gallons / minute). This corresponds to energy costs 0.25kWhr / ^{m 3.} This shows that the energy cost of this technology is very low. Desirably, each transducer is powered and controlled by its own amplifier. In another embodiment, the ultrasonic transducer uses, for example, a square piezoelectric element having dimensions of 1 inch x 1 inch. Alternatively, the ultrasonic transducer can use, for example, a rectangular piezoelectric element having a dimension of 1 inch × 2.5 inch. In order to obtain sufficient acoustic capture power, the power consumption per transducer was 10 W per 1 inch x 1 inch dimensional transducer cross-sectional area and per inch of acoustic standing wave span. A 4-inch span in an intermediate scale system consumes 40 W per 1-inch x 1-inch square transducer. The larger 1 "x 2.5" rectangular transducer consumes 100W in an intermediate scale system. An array of three 1 "x 1" square transducers consumes a total of 120W, and an array of two 1 "x 2.5" rectangular transducers consumes about 200W. The size, shape, number and position of the transducers can be changed as needed to generate the desired multidimensional acoustic standing wave pattern.

The size, shape and thickness of the transducer determine the displacement of the transducer at different excitation frequencies, which in turn affects the separation efficiency. Typically, the transducer operates at a frequency close to the thickness resonance frequency (half wavelength). The displacement gradient of the transducer usually provides more capture positions for cells / biomolecules. Higher-order mode displacements generate a three-dimensional acoustic standing wave with a strong gradient in the acoustic field in all directions, which produces equally strong acoustic radiation in all directions, and the number of capture lines identifies the transducer. When correlated with the mode shape of, it results in multiple capture lines.

In order to investigate the effect of the displacement profile of the transducer on the acoustic capture force and the separation efficiency, a 1 inch × 1 inch square transducer was used, and the experiment was repeated 10 times under the same conditions except for the excitation frequency. As the excitation frequency, 10 consecutive acoustic resonance frequencies represented by the circled numbers 1 to 9 in FIG. 7 and the letter A were used. The conditions were an experiment time of 30 minutes, a SAE-30 oil droplet of about 5 microns having an oil concentration of 1000 ppm, a flow rate of 500 ml / min, and an applied power of 20 W. Oil droplets are used because oil is less dense than water and can be separated from water using acoustic electrophoresis.

FIG. 7 illustrates the amplitude of the measured electrical impedance of a square transducer as a function of the frequency near the resonance point of the 2.2 MHz transducer. The minimum points of the transducer's electrical impedance correspond to the acoustic resonance of the water column and represent the frequencies available for operation. There are additional resonance points at other frequencies where the multidimensional standing wave is excited. Numerical modeling has shown that these acoustic resonance frequencies significantly change the displacement profile of the transducer, which directly affects the acoustic standing wave and the resulting capture force. Since the transducer operates near its thickness-wise resonance point, the displacement of the electrode surface is essentially out of phase. The typical displacement of the transducer electrodes is not uniform and varies with excitation frequency. As an example, at one excitation frequency with one line of captured oil droplets, the displacement has one local maximum at the center of the electrode and a local minimum near the end of the transducer. At different excitation frequencies, the transducer profile has multiple maxima, resulting in multiple oil droplet capture lines. The higher the displacement pattern of the transducer, the stronger the catching force and the more stable catching lines are obtained for the trapped oil droplets.

As the oil-water emulsion passed through the transducer, oil droplet capture lines were observed and characterized. This property included observations and patterns of the number of capture lines across the fluid channel for 7 of the 10 resonant frequencies identified in FIG. 7, as shown in FIG. Different transducer displacement profiles can produce different (more) capture lines for standing waves, and higher displacement profile gradients generally produce higher capture forces and more capture lines. Will be done.

FIG. 9 is a numerical model showing a pressure field that matches the pattern of the nine capture lines. Since this numerical model is a two-dimensional model, only three capture lines are observed. In three dimensions perpendicular to the paper, there are two more sets of three capture lines.

The lateral force of the acoustic radiation generated by the transducer drives the transducer in a higher mode shape, as opposed to the vibrational form in which the crystals move effectively, such as a piston with uniform displacement. Can be increased by doing. In some implementations, the higher-order mode shape is the Bessel function. The acoustic pressure is proportional to the drive voltage of the transducer. Electric power is proportional to the square of the voltage. Transducers are usually thin piezoelectric plates with an electric field on the z-axis and a primary displacement on the z-axis. Transducers are typically coupled with air on one side (ie, the air gap within the transducer) and on the other side with a fluid mixture containing particles that interact with each other. The type of wave produced by the plate is known as a composite wave. Some of the composite waves in the piezoelectric plate are similar to the leaky symmetry (also called compression or decompression) Lamb waves. The piezoelectricity of the plate usually excites a symmetrical Lamb wave. Lamb waves are radiated into the water layer, causing leaks, resulting in acoustic standing waves in the water layer. Lamb waves exist in an infinitely thin plate on its surface in a stress-free state. Since the transducer of this embodiment is inherently finite, the actual mode displacement is more complex.

FIG. 10 shows a typical change between the in-plane displacement (x displacement) and the out-of-plane displacement (y displacement) over the entire thickness of the plate, and the in-plane displacement is an even function over the entire thickness of the plate. Yes, the out-of-plane displacement is an odd function. Due to the finite size of the plate, the displacement component varies over the width and length of the plate. In general, the (m, n) mode is a displacement mode of a transducer having m undulations in the displacement of the transducer in the width direction and n undulations in the displacement in the length direction, as described with reference to FIG. It has a large change in thickness. The maximum number of m and n is a function of the dimensions of the piezoelectric material (eg, piezoelectric crystal) and the excitation frequency. There are also additional 3D modes that are not in the form (m, n).

The transducer is driven so that the piezoelectric element vibrates in a higher-order mode of the general formula (m, n), where m and n are independent numbers of 1 or more. Generally, the transducer oscillates in a mode higher than (2, 2). Higher-order mode produces more nodes and antinodes, resulting in a three-dimensional standing wave in the water layer, in an omnidirectional acoustic field not only in the direction of the standing wave but also in the lateral direction. It is characterized by a strong gradient. As a result, the acoustic gradient produces a strong lateral capture force.

In general, ultrasonic transducers may be driven by electrical signals that can be controlled based on voltage, current, phase angle, power, frequency, or any other electrical signal characteristic. In particular, the transducer drive signal may be based on voltage, current, magnetic, electromagnetic, capacitive, or other type of signal that the transducer responds to. In embodiments, the voltage signal driving the transducer can have a sine wave, square wave, sawtooth wave, pulse wave or triangle wave waveform and can have a frequency of 500 kHz to 10 MHz. The voltage signal can be driven by pulse width modulation to produce any desired waveform. The voltage signal can also have an amplitude modulation or frequency modulation start / stop function to eliminate streaming. In certain embodiments, the voltage signal can have a frequency of about 0.5 MHz to about 30 MHz, which is generated by the ultrasonic transducer.

Transducers are used to create a pressure field that produces acoustic radiation of the same magnitude in both the direction orthogonal to the direction of the standing wave and the direction of the standing wave. When the forces are on the order of magnitude, particles with a size of 0.1 to 300 microns move more effectively toward the "capture line", thus allowing cells and nucleic acids (bare or viral vectors). Both (whether inside or not) are placed next to each other, allowing them to react to each other.

In biological applications, all parts of the system (ie, bioreactors, acoustic filtering devices, tubes connecting them fluidly, etc.) can be separable from each other and disposable. By avoiding centrifuges and filtration devices, living cells can be better separated from the fluid without compromising cell viability. The transducer may also be driven to produce a rapid pressure change to prevent or clear clogging due to aggregation of living cells. The frequency of the transducer may also be varied for optimum effect on a given power.

The techniques and implementations described herein may be used for integrated, continuous and automated bioprocessing. Control can be distributed to some or all units involved in bioprocessing. Feedback from the unit can be provided to get an overview of the bioprocess and may be in the form of screen displays, control feedback, reports, status reports and other communication. Distributed processing provides a high degree of flexibility to achieve the desired process control, for example by coordinating steps between units and providing batch execution control.

Acoustic electrophoretic devices that utilize acoustic wave systems can be implemented with biocompatible materials and can include gamma-sterilizable, disposable components. The processing system also enables ultrasonic flow measurement, which is non-invasive and can operate on high viscosity fluids. The system can be implemented with a disposable sterile, sterile connector and a simple graphical user interface (GUI) for control. The electrophoretic device is scalable. For example, a relatively small unit can operate on a scale of 2L to 50L.

The methods, systems and devices described above are examples. Various configurations may omit, replace or add various procedures or components as needed. For example, in an alternative configuration, the methods may be performed in a different order than described, and various steps may be added, omitted, or combined. Also, the features described for a particular configuration may be combined in various other configurations. Aspects and elements of different configurations may be combined in a similar manner. Also, the technology is evolving and therefore many of the above elements are examples and do not limit the scope of the disclosure or claims.

Specific details are given in the description of this document to provide a complete understanding of the exemplary configuration (including embodiments). However, the configuration may be implemented without these specific details. For example, well-known processes, structures and techniques are shown without unnecessary details to avoid obscuring the configuration. This description provides only exemplary configurations and does not limit the scope, applicability or configuration of the claims. Rather, the configuration description described above provides a description for implementing the described technique. Various changes may be made to the function and arrangement of the elements without departing from the spirit or scope of the present disclosure.

The description that the value exceeds (or exceeds) the first threshold corresponds to the description that the value satisfies or exceeds the second threshold slightly larger than the first threshold, for example, the second threshold. Corresponds to the description that the resolution of the associated system is one higher than the first threshold. The statement that the value is below (or is within) the first threshold corresponds to the statement that the value is less than or equal to the second threshold, which is slightly less than the first threshold, eg, Corresponds to the description that the second threshold is one lower than the first threshold in the resolution of the associated system.

The configuration may also be described as a process shown as a flow diagram or block diagram. Although each may describe an operation as a sequential process, many of the operations can be performed in parallel or simultaneously. Further, the order of operations may be rearranged. The process may have additional stages or functions not included in the figure.

The following examples are provided to illustrate the devices and processes of the present disclosure. The examples are merely exemplary and are not intended to limit this disclosure by the materials, conditions, or process parameters described therein.

Example 1:

FIG. 11 is a photograph of a plastic bag containing a fluid mixture of T cells and a virus. The plastic bag was placed in a water-filled acoustic electrophoresis device. When a multidimensional acoustic standing wave was generated, T cells and the virus interacted with each other. This is visible as a series of disc-shaped beams in a plastic bag.

Example 2:

The BacMam® system (Thermo Fisher Scientific) uses baculovirus for transduction and is used for transduction of green fluorescent protein (GFP) into jarcut T cells. did. Various experiments were performed using this system. The five results are shown below. They were labeled Control, Process Control 1, Process Control 2, Acoustic 3 MHz and Acoustic 10 MHz.

The control experiment, the process control 1 experiment, and the process control 2 experiment were not exposed to the acoustic standing wave.

In acoustic 3 MHz experiments, the use of acoustic standing waves with a nominal frequency of 3 Hz enhanced the interaction between T cells and the virus.

In acoustic 10 MHz experiments, the use of acoustic standing waves with a nominal frequency of 10 Hz enhanced the interaction between T cells and the virus.

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

When acoustics were used, comparable transduction efficiencies were obtained with 80% less virus particles per cell.

Example 3:

The acoustic chamber is equipped with an ultrasonic transducer and a reflector. The recirculation loop draws fluid from one end of the acoustic chamber and then returns the fluid through the other end to the acoustic chamber for circulation. The volume of the acoustic chamber was about 1 mL and the volume of the recirculation loop was about 3 mL. The transduction test was performed using the acoustic chamber.

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

Each acoustic test was also performed in controlled operation (Control Run) and static operation (Static Run). In controlled operation, the cells in the Petri dish were added with the virus and immediately washed. In static operation, the cells in the Petri dish were added with the virus for 90 minutes and then washed. The MOI (number of viral vector particles per cell) for these tests was 25. Cell viability was measured on the Nucleo counter for all operations. For each acoustic operation, cell viability was also measured using a Vi-Cell cell viability analyzer.

Table B shows the parameters of each test. Table C shows the results of each test run. Table D shows the Vi-Cell analysis. Table E shows the Nucleo counter analysis. In Table C, the transduction gain is the acoustic efficiency divided by the static efficiency.

As can be seen in Table C, the transduction efficiency was improved by using recirculation. By using sound in addition to recirculation, the transduction efficiency was further improved. Referring to Table D, the two operations using acoustics and recirculation (operations 1 and 2) are compared to the two operations using recirculation without acoustics (operations 3 and 4). , Showed that the total number of transduced viable cells was about 72% higher. Referring to Table E, the two operations using acoustics and recirculation (operations 1 and 2) are compared to the two operations using acoustics and recirculation (operations 3 and 4). , Showed that the total number of transduced viable cells was about 68% higher.

Example 4:

Three acoustic tests were performed using the acoustic chamber with recirculation described in Example 3 for 30 minutes, 60 minutes and 90 minutes of execution time. The sound was operated at a frequency of 2 MHz and a power of 3.5 W. Controlled and static operations were also performed as described in Example 3, but the operating time was 30, 60 or 90 minutes. Table F shows the parameters of each test. The results are shown in FIG. As can be seen there, as the operating time increased, the transduction efficiency improved compared to the case without sound.

Although some configuration examples have been described, various modifications, alternative configurations and equivalents may be used without departing from the spirit of the present disclosure. For example, the elements described above may be components of a larger system, in which case other structures or processes take precedence over the application of the invention, or otherwise modify the application of the invention. You may. Also, many actions may be performed before, during, or after the above factors are considered. Therefore, the above description does not limit the scope of claims.

Claims (26)

A method for introducing foreign nucleic acids into cells
The cells and nucleic acids are placed in an acoustic migration device including an acoustic chamber in which the cells and nucleic acids are arranged and an ultrasonic transducer containing a piezoelectric material that can be driven to generate an acoustic standing wave in the acoustic chamber. To place and
Including driving the ultrasonic transducer to generate a multidimensional acoustic standing wave.
A method in which at least the cell is retained by the acoustic standing wave and the nucleic acid is co-located with the cell to allow transduction of the foreign nucleic acid into the cell. In the method of claim 1,
The method, wherein the nucleic acid is in a viral vector. In the method of claim 1,
A method further comprising opening pores in the cell membrane of the cell prior to co-locating the cell with the nucleic acid. In the method of claim 3,
The method by which the pores are opened by electroporation, sonoporation or exposure to calcium phosphate. In the method of claim 1,
The electrophoretic device further comprises a recirculation loop coupled to the acoustic chamber.
A method in which one or more of the cells or nucleic acids are recirculated through the acoustic chamber. In the method of claim 1,
The cells 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, and the like. A method of gamma delta T cells, jarcat T cells, CAR-T cells, B cells or NK cells, or peripheral blood mononuclear cells (PBMCs), algae, plant cells or bacteria. In the method of claim 1,
The method, wherein the acoustic standing wave is a multidimensional acoustic standing wave, a plane standing wave, or a combination of a multidimensional acoustic standing wave and a plane standing wave. In the method of claim 1,
A method in which the ultrasonic transducer is driven at a frequency of about 0.5 MHz to about 20 MHz. In the method of claim 1,
A method in which the frequency of the acoustic standing wave varies in a sweep pattern for moving the cell relative to the nucleic acid. A method for inducing cell transduction,
The acoustic apparatus comprising an acoustic chamber in which the cells and a viral vector having a nucleic acid are arranged, and an ultrasonic transducer containing a piezoelectric material that can be driven to generate an acoustic standing wave in the acoustic chamber. Placing cells and the viral vector
Including driving the ultrasonic transducer to generate a multidimensional acoustic standing wave.
A method in which the cell and the viral vector are co-located by the acoustic standing wave to allow transduction of the cell. In the method of claim 10,
A method in which the cells and the viral vector are suspended in a fluid. In the method of claim 10,
The cells 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, and the like. A method of gamma delta T cells, jarcat T cells, CAR-T cells, B cells or NK cells, or peripheral blood mononuclear cells (PBMCs), algae, plant cells or bacteria. In the method of claim 10,
The electrophoretic device further comprises a recirculation loop coupled to the acoustic chamber.
A method in which one or more of the cells or nucleic acids are recirculated through the acoustic chamber. In the method of claim 10,
A method in which the ultrasonic transducer is driven at a frequency of about 0.5 MHz to about 20 MHz. In the method of claim 10,
A method in which the frequency of the acoustic standing wave varies with a sweep pattern for moving the cell against the viral vector. A method for inducing cell transfection
To make pores in the cell membrane of the cell,
The cells and nucleic acids are placed in an acoustic migration device including an acoustic chamber in which the cells and nucleic acids are arranged and an ultrasonic transducer containing a piezoelectric material that can be driven to generate an acoustic standing wave in the acoustic chamber. To place and
Including driving the ultrasonic transducer to generate a multidimensional acoustic standing wave.
A method in which the cell and the nucleic acid are co-located by the acoustic standing wave to cause transfection of the cell. In the method of claim 16,
The method by which the pores are opened by electroporation, sonoporation or exposure to calcium phosphate. In the method of claim 16,
A method in which the pores are opened before or after the cells are placed in the electrophoretic device. In the method of claim 16,
The electrophoretic device further comprises a recirculation loop coupled to the acoustic chamber.
A method in which one or more of the cells or nucleic acids are recirculated through the acoustic chamber. In the method of claim 16,
The cells 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, and the like. A method of gamma delta T cells, jarcat T cells, CAR-T cells, B cells or NK cells, or peripheral blood mononuclear cells (PBMCs), algae, plant cells or bacteria. In the method of claim 16,
The method, wherein the acoustic standing wave is a multidimensional acoustic standing wave, a plane standing wave, or a combination of a multidimensional acoustic standing wave and a plane standing wave. In the method of claim 16,
A method in which the ultrasonic transducer is driven at a frequency of about 0.5 MHz to about 20 MHz. In the method of claim 16,
A method in which the frequency of the acoustic standing wave varies in a sweep pattern for moving the cell relative to the nucleic acid. A cell produced by the method of claim 1. A cell produced by the method of claim 10. A cell produced by the method of claim 16.

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