Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
  • C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/14011—Baculoviridae
  • C12N2710/14041—Use of virus, viral particle or viral elements as a vector
  • C12N2710/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vectore

Definitions

• the present disclosure relates to methods of introducing foreign nucleic acids into cells, such as by transfection and transduction utilizing acoustic waves. Also included are cells and associated compositions produced by such methods. Such methods and compositions may be useful in cell therapy applications
• Transfection and transduction are processes for intentionally introducing nucleic acids (either DNA or RNA) into cells.
• Transduction is performed using a viral vector such as a bacteriophage or other virus.
• Viruses such as adenovirus, lentivirus, or paramyxovirus are commonly used.
• Transfection refers to the introduction of the nucleic acids into a cell using non-viral methods. .
• the present disclosure relates, in various embodiments, to processes for introducing foreign nucleic acids (DNA/RNA) into cells, such as by performing transfection or transduction of cells using acoustic waves.
• the acoustic waves are used to bring the cells and the nucleic acids together, such that the nucleic acids can be transferred to the cells.
• An acoustic device can be used for this purpose, and such devices are described herein. Also included are cells and associated compositions produced by such methods.
• the nucleic acids may be naked, or they may be in a viral vector.
• the cells and nucleic acids are placed in an acoustophoretic device, for example by placing them in a bag that is inserted into the acoustophoretic device, or by flowing a fluid mixture containing both the cells and the nucleic acids through the acoustophoretic device.
• the acoustophoretic device comprises: an acoustic chamber in which the cells and the nucleic acids are placed; and an ultrasonic transducer and a reflector opposite the ultrasonic transducer, the ultrasonic transducer including a piezoelectric material that can be driven to create a multi-dimensional acoustic standing wave in the acoustic chamber.
• the ultrasonic transducer is driven to create an acoustic standing wave.
• the acoustic standing wave may be a multi-dimensional acoustic standing wave, a planar standing wave, or a combination of both.
• the nucleic acids are in a viral vector. Transduction can occur when the virus attaches itself to the target cell and injects the nucleic acid into the target cell.
• pores are opened in the cell membranes of the cells prior to co-locating the cells with the nucleic acids. Transfection can occur when the nucleic acid enters the cell, for example via the pores. The pores can be opened by electroporation, sonoporation, or by exposure to calcium phosphate.
• the cells and the nucleic acids may be suspended in a fluid.
• fluids can include cell culture media, water, saline solution, and the like.
• 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, peripheral blood mononuclear cells (PBMCs), algae, plant cells, or bacteria.
• CHO Chinese hamster ovary
• NS0 hybridoma cells NS0 hybridoma cells
• BHK baby hamster kidney
• human cells regulatory T-cells
• helper T-cells cytotoxic T-cells
• memory T-cells memory T-cells
• effector T-cells gamma delta T-cells
• Jurkat T-cells CAR-T cells
• B cells B cells
• NK cells peripheral blood mononuclear cells
• the ultrasonic transducer may be driven for a time period of about 5 minutes to about 15 minutes, although this time period can vary as desirable.
• the ultrasonic transducer may be driven at a frequency of about 0.5 MHz to about 20 MHz.
• the frequency of the multi-dimensional acoustic standing wave is varied in a sweep pattern to move the cells relative to the nucleic acids.
• the piezoelectric material of the ultrasonic transducer may be lead zirconate titanate (PZT) or lithium niobate.
• the acoustophoretic device may further comprise a cooling unit for cooling the ultrasonic transducer.
• Also disclosed herein are methods for causing transduction of cells.
• the cells and a viral vector comprising nucleic acids are placed in an acoustophoretic device comprising: an acoustic chamber; and an ultrasonic transducer including a piezoelectric material that can be driven to create a multi-dimensional acoustic standing wave, a planar acoustic standing wave, or a combination of planar and multidimensional acoustic standing waves in the acoustic chamber.
• the ultrasonic transducer is driven to create the multi-dimensional acoustic standing wave, planar acoustic standing wave, or combination of multidimensional and planar acoustic standing waves.
• the cells and the viral vector are co-located by the acoustic standing wave to cause transduction of the cells.
• the acoustic standing wave can be a multi-dimensional acoustic standing wave, a planar acoustic standing wave, or a combination of planar and multidimensional acoustic standing waves.
• the pores can be opened before or after the cells are placed in the acoustophoretic device.
• the ultrasonic transducer is then driven to create the acoustic standing wave.
• the cells and the nucleic acids are co-located by the acoustic standing wave to cause transfection of the cells.
• FIG. 1 is a diagram illustrating a method / process of the present disclosure, in which the efficiency of viral transduction is enhanced.
• a cell culture is combined with a viral vector tagged with green fluorescent protein (GFP) and exposed to acoustic processing, where the multi-dimensional acoustic standing wave brings the cells and viruses into close proximity with each other, enhancing reaction efficiency. After washing and overnight incubation, GFP is expressed in the cells, thus showing transduction has occurred.
• GFP green fluorescent protein
• FIG. 2A is an exploded perspective view of an example acoustophoretic device according to the present disclosure including 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 acoustophoretic device that can be used to practice the methods / processes of the present disclosure.
• a disposable container such as a plastic bag, contains fluid mixture with two particle types that are caused to interact with each other in a separate acoustophoretic device containing one or more ultrasonic transducers.
• FIG. 4 is a cross-sectional diagram of a conventional ultrasonic transducer.
• FIG. 5 is a cross-sectional diagram of an ultrasonic transducer of the present disclosure. An air gap is present within the transducer, and no backing layer or wear plate are present.
• FIG. 6 is a cross-sectional diagram of an ultrasonic transducer of the present disclosure. An air gap is present within the transducer, and a backing layer and wear plate are present.
• FIG. 7 is a graph of electrical impedance amplitude versus frequency for a square transducer driven at different frequencies.
• FIG. 8 illustrates the trapping line configurations for seven of the resonance frequencies (minima of electrical impedance amplitudes) of FIG. 7 from the direction orthogonal to fluid flow.
• FIG. 9 is a computer simulation of the acoustic pressure amplitude (right-hand scale in Pa) and transducer out of plane displacement (left-hand scale in meters).
• the text at the top of the left-hand scale reads“x1 O 7 ”.
• the text at the top of the left-hand scale by the upward-pointing triangle reads“1 .473x1 O 6 ”.
• the text at the bottom of the left-hand scale by the downward-pointing triangle reads“1 .4612x10 10 ”.
• the text at the top of the right-hand scale reads“x10 6 ”.
• the text at the top of the right-hand scale by the upward- pointing triangle reads“1 .1 129x10 6 ”.
• the text at the bottom of the right-hand scale by the downward-pointing triangle reads “7.357”.
• the triangles show the maximum and minimum values depicted in this figure for the given scale.
• the horizontal axis is the location within the chamber along the X-axis, in inches
• the vertical axis is the location within the chamber along the Y-axis, in inches.
• FIG. 10 shows the In-Plane and Out-of-Plane displacement of a crystal where composite waves are present.
• FIG. 11 is a picture of a plastic bag in which T-cells and viruses are interacting with each other.
• FIG. 12 is a graph showing the ratio of transduction efficiency (acoustics / no acoustics) versus transduction time for three experiments with different run times (30, 60, and 90 minutes).
• the modifier "about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context. When used in the context of a range, the modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the range of “from about 2 to about 10” also discloses the range “from 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 1 1 %, and “about 1 " may mean from 0.9-1 .1.
• the terms “upper” and “lower” are relative to each other in location, i.e. an upper component is located at a higher elevation than a lower component in a given orientation, but these terms can change if the device is flipped.
• the terms “inlet” and “outlet” are relative to a fluid flowing through them with respect to a given structure, e.g. a fluid flows through the inlet into the structure and flows through the outlet out of the structure.
• upstream and “downstream” are relative to the direction in which a fluid flows through various components, i.e. the flow fluids through an upstream component prior to flowing through the downstream component. It should be noted that in a loop, a first component can be described as being both upstream of and downstream of a second component.
• top and bottom are used to refer to surfaces where the top is always higher than the bottom/base relative to an absolute reference, i.e. the surface of the earth.
• upwards and “downwards” are also relative to an absolute reference; upwards is always against the gravity of the earth.
• the present application refers to "the same order of magnitude.” Two numbers are of the 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 acoustophoretic devices discussed herein may operate in a multimode or planar mode or a combination of both.
• Multimode refers to generation of acoustic waves by an acoustic transducer that create acoustic forces in three dimensions.
• the multimode acoustic waves which may be ultrasonic, can be generated by a single acoustic transducer, and are sometimes referred to herein as multi-dimensional or three- dimensional acoustic standing waves.
• Planar mode refers to generation of acoustic waves by an acoustic transducer that create acoustic forces substantially in one dimension, e.g. along the direction of propagation.
• Such acoustic waves, which may be ultrasonic, that are generated in planar mode are sometimes referred to herein as one- dimensional acoustic standing waves.
• the acoustic transducers may comprise a piezoelectric material, such as lead zirconate titanate (PZT) or lithium niobate. Such acoustic transducers can be electrically excited to generate planar or multimode acoustic waves.
• the three-dimensional acoustic forces generated by multimode acoustic waves include radial or lateral forces that are unaligned with a direction of acoustic wave propagation.
• the lateral forces may act in two dimensions.
• the lateral forces are in addition to the axial forces in multimode acoustic waves, which are substantially aligned with the direction of acoustic wave propagation.
• the lateral forces can be of the same order of magnitude as the axial forces for such multimode acoustic waves.
• the acoustic transducer excited in multimode operation may exhibit a standing wave on its surface, thereby generating a multimode acoustic wave.
• the standing wave on the surface of the transducer may be related to the mode of operation of the multimode acoustic wave.
• the surface of the transducer When an acoustic transducer is electrically excited to generate planar acoustic waves, the surface of the transducer may exhibit a piston-like action, thereby generating a one-dimensional acoustic standing wave.
• multimode acoustic waves exhibit significantly greater particle trapping activity on a continuous basis with the same input power.
• One or more acoustic transducers may be used to generate combinations of planar and multi- dimensional acoustic standing waves.
• two acoustic transducers may be arranged to oppose each other to generate a standing wave.
• one of the acoustic transducers may be passive and act as a reflector of an incident wave.
• 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.
• acoustophoresis is used for separation of materials, and may represent a low-power, no-pressure-drop, no-clog, solid-state approach to particle separation from fluid dispersions.
• the scattering of the acoustic field off the particles results in a three-dimensional acoustic radiation force, which acts as a three-dimensional trapping field.
• the acoustic radiation force is proportional to the particle volume (e.g., the cube of the radius) when the particle is small relative to the wavelength.
• the acoustic radiation force is proportional to frequency and the acoustic contrast factor.
• the acoustic radiation force scales with acoustic energy (e.g., the square of the acoustic pressure amplitude).
• acoustic energy e.g., the square of the acoustic pressure amplitude
• the sinusoidal spatial variation of the force is what drives the particles to the stable positions within the standing waves.
• the acoustic radiation force exerted on the particles is stronger than the combined effect of fluid drag force and buoyancy/gravitational force, the particle is trapped within the acoustic standing wave field.
• the acoustic standing waves create localized regions of high and low pressure. Particles are pushed to the standing wave nodes or antinodes depending on their compressibility and density relative to the surrounding fluid. Particles of higher density and compressibility move to the nodes in the standing waves, while secondary phases of lower density move to the antinodes.
• the force exerted on the particles also depends on their size, with larger particles experiencing larger forces. The magnitude of the force depends on the particle density and compressibility relative to the fluid medium, and increases with the particle volume.
• biological cells can be considered as particles. Most biological cell types present a 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.
• the axial acoustic radiation force (ARF) drives the cells towards the standing wave pressure nodes.
• the axial component of the acoustic radiation force drives the cells, with a positive contrast factor, to the pressure nodes, whereas cells or other particles with a negative contrast factor are driven to the pressure anti-nodes.
• the radial or lateral component of the acoustic radiation force is the force that traps the cells.
• the radial or lateral component of the ARF is larger than the combined effect of fluid drag force and gravitational force.
• the ultrasonic transducer(s) generates a multi-dimensional standing wave in the fluid that exerts a lateral force on the suspended particles to accompany the axial force.
• the technology disclosed in this application provides for a lateral force to be of the same order of magnitude as the axial force.
• the device use both transducers that produce multi-dimensional acoustic standing waves and transducers that produce planar acoustic standing waves.
• the lateral force component of the total acoustic radiation force (ARF) generated by the ultrasonic transducer(s) of the present disclosure is significant and is sufficient to overcome the fluid drag force at linear velocities of up to 1 cm/s, and to create tightly 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 using such acoustophoretic devices containing ultrasonic transducers to perform transfection or transduction.
• the acoustophoretic devices are used to bring cells together with nucleic acids (either DNA or RNA, or both) in a localized volume. This permits the nucleic acids to be transferred into the cells.
• Current transduction processes can have relatively high cost, low efficiency, and poor ability to be scaled up for commercialization.
• the methods described herein can reduce cost, increase efficiency, and have a scalable platform for commercialization.
• the cells and the carrier are placed in the acoustic chamber of an acoustophoretic device. Generally, they are suspended in a fluid to form a fluid mixture.
• the acoustophoretic device contains an acoustic chamber that has an ultrasonic transducer and a reflector opposite the ultrasonic transducer (e.g. on opposite walls of the chamber).
• the ultrasonic transducer includes a piezoelectric material that can be driven to create an acoustic standing wave within the acoustic chamber, for example a multi-dimensional acoustic standing wave and/or a planar acoustic standing wave.
• the acoustic standing wave has locales of higher pressure and locales of lower pressure.
• the lower pressure locales trap and retain the cells.
• the nucleic acids are flowed into the acoustic chamber with the retained cells. This action causes the cells and the nucleic acids to be co-located so the nucleic acids can enter the cells.
• pores are opened in the cell membranes of the cells, to permit naked nucleic acids to enter the cells.
• the nucleic acids are part of a viral vector.
• the viral vector enters the cells or inserts the nucleic acids into the cells. Specific viral vectors that may be used for performing transduction in the present disclosure include adenovirus, lentivirus, or paramyxovirus.
• the acoustophoretic force created by the acoustic standing wave on the cells and/or the nucleic acids can be sufficient to overcome the fluid drag force exerted by the moving fluid on these particles.
• the acoustophoretic force can act as a mechanism that traps the cells and/or the nucleic acids in the acoustic field.
• the acoustophoretic force can drive the cells and/or the nucleic acids to the stable locations of reduced or minimum acoustophoretic force amplitudes. These locations of reduced or minimum acoustophoretic force amplitudes can be the nodes of a standing acoustic wave.
• the collection of cells and/or nucleic acids at the nodes grows steadily. Within some period of time, which can be minutes or less depending on their concentration, the collection of cells and/or nucleic acids can assume the shape of a beam-like collection of disks. Each disk can be spaced by a half wavelength of the acoustic field.
• the acoustic standing wave traps the cells and/or the nucleic acids and co-locates them, improving the efficiency of the transfection / transduction reaction.
• a number of different mechanisms may be implemented for these embodiments.
• the cells and the nucleic acids may have similar acoustic contrast factors, such that both types of particles can be driven to the nodes or anti-nodes of the standing wave. This mechanism brings the cells and the nucleic acids in close spatial proximity with each other more efficiently than reliance on Brownian motion (as with conventional stirring).
• the cells and the nucleic acids are trapped in a small three-dimensional volume created by the multi-dimensional acoustic standing wave, relative to the size of the acoustic chamber.
• the cells and the nucleic acids either both have a positive acoustic contrast factor, or both have a negative acoustic contrast factor. Put another way, their acoustic contrast factors have the same sign.
• the nucleic acids can be naked or in a viral vector.
• one of the two types of particles may be driven to the nodes, while the other type of particles is driven to the anti-nodes.
• the nodes and anti-nodes are sufficiently close to each other that the cells and the nucleic acids can react with each other.
• the cells or the nucleic acids have a positive acoustic contrast factor
• the other set i.e. nucleic acids or the cells
• their acoustic contrast factors have opposite signs.
• the particles with a positive contrast factor are driven to the nodes, and the particles with a negative contrast factor are driven to the anti-nodes.
• Some relevant factors for this reaction mechanism include the sizes of the cells and the carrier, and the frequency at which the ultrasonic transducer is operated.
• the cells and/or the nucleic acids continue to be captured and concentrated, they can attain a size and weight such that gravitational settling will occur, wherein the clusters of particles will fall out of the acoustic standing wave to the bottom of the acoustic chamber. New collections of particles can then be trapped and reacted within the acoustic field generated by the acoustic standing waves.
• an acoustophoretic device is provided with an acoustic chamber that includes an ultrasonic transducer that includes a piezoelectric material that can be driven to create an acoustic standing wave within the acoustic chamber, for example a multi-dimensional acoustic standing wave and/or a planar acoustic standing wave.
• the acoustic standing wave has locales of higher pressure and locales of lower pressure.
• a recirculation loop is provided to the acoustic chamber that permits output fluid and particles to be reintroduced to the acoustic chamber.
• the lower pressure locales trap and retain the cells, but do not trap or retain the viral vector or nucleic acids, which can flow out of the acoustic chamber and be reintroduced via the recirculation loop.
• the cells are retained in the acoustic field generated by the acoustic standing wave, and the viral vectors are flowed into the field to permit the cells and viral vectors to interact. Increased numbers of interactions can be attained between the viral vectors and the cells by recirculating to the acoustic chamber the viral vectors that do not interact on a prior pass.
• an additional treatment can be performed on the cells that are retained in the acoustic field to permit pores to be formed in the cell membranes, as discussed in greater detail elsewhere herein.
• the nucleic acids are flowed into the acoustic chamber with the retained cells to permit the nucleic acids to interact with the cells and to permit the nucleic acids to enter the cells via the pores.
• the nucleic acids are not retained by the acoustic field, and are recirculated to the acoustic chamber to permit multiple passes at transfection to occur.
• acoustic streaming refers to the fluid flow that results within the acoustic chamber when the fluid absorbs the acoustic energy that is transmitted by the ultrasonic transducer (from the vibration of the ultrasonic transducer).
• the velocity of the fluid is induced by the oscillating acoustic waves generated by the ultrasonic transducer.
• acoustic streaming when acoustic streaming is generated, it results in circulatory motion or vortices that can cause stirring in the fluid mixture. This phenomenon is nonlinear, and can cause the cells and the nucleic acids to interact with each other.
• the cells and the nucleic acids are brought into proximity so that they can react with each other.
• the terms“interact” and“react” are used to indicate that a physical change occurs in the cells.
• the virus containing the nucleic acids may penetrate into the cell to cause transduction to occur.
• the virus can be, for example, a retrovirus such as a lentivirus.
• pores are opened in the cell membranes of the cells to permit nucleic acids to enter the cells.
• the cells could be subjected to electroporation, where the cells are exposed to an electric field to increase the permeability of the cell membrane.
• Sonoporation could also be used, where the cells are exposed to ultrasound to induce pore formation in the cell membrane.
• Calcium phosphate could also be used to cause transfection.
• the location at which the pore opening occurs is not important, and can occur in the acoustophoretic device upstream of the acoustic chamber in which the cells and the nucleic acids are mixed, or can occur outside of the acoustophoretic device.
• productive transfection and gene transfer require not simply the entry of DNA into cells and subsequent transcription from an appropriate promoter, but also a number of intracellular events that allow the DNA to move from the extracellular surface of the cell into and through the cytoplasm, and ultimately across the nuclear envelope and into the nucleus before any transcription can initiate.
• naked DNA either delivered through physical techniques or after disassembly of DNA-carrier complexes
• Plasmids then enter the nucleus either upon the mitotic disassembly of the nuclear envelope or through nuclear pore complexes in the absence of cell division, using a different set of proteins.
• Specific viral vectors that may be used for performing transduction in the present disclosure include adenovirus, lentivirus, or paramyxovirus. Retroviruses are characterized by their ability to retrotranscribe their RNA genome into a cDNA copy, which is then stably integrated into the host cell genome. Thus, the virus carries the nucleic acid into the cell.
• Retroviruses can be categorized as simple or complex (e.g., lentiviruses).
• the viral particles of both types contain two copies of positive-stranded RNA with an associated viral reverse transcriptase (RT) located within an internal core.
• RT viral reverse transcriptase
• structural and enzymatic proteins including the nucleocapsid (NC), capsid (CA), integrase (IN), and protease (PR).
• NC nucleocapsid
• CA capsid
• I integrase
• PR protease
• the inner core is surrounded by an outer protein layer comprised of the matrix (MA) protein, which is in turn encompassed by the envelope glycoprotein (ENV)-studded, host cell membrane-derived envelope.
• MA matrix
• EMV envelope glycoprotein
• Lentiviral and retroviral gene delivery systems exploit aspects of retrovirus replication to provide stable integration of the desired nucleic acid sequence. Whereas transfection of foreign nucleic acids results only in transient transgene expression, the activity of the viral integrase in retroviral and lentiviral-based systems allows for stable integration of the foreign transgene, which is then inherited and continuously expressed over repeated cell divisions.
• a key feature of both lentiviral and retroviral vectors is that they produce replication-defective, or self-inactivating, particles. This allows for delivery of the desired sequence, without continued viral replication in the target cells.
• a common way to validate that the foreign nucleic acids were successfully introduced into cells is to measure protein expression. This is typically performed by Western blot or immunostaining.
• Examples of cells that can be transfected / transducted 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, cytotoxic T-cells, memory T-cells, effector T-cells, gamma delta 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 may themselves be attached to other materials, such as beads.
• beads include polymer beads, magnetic beads, superparamagnetic beads, and microspheres. These can be used for biochemical reactions or for labeling purposes.
• additives can be included in the mixture of the cells and the nucleic acids.
• additives can include PolybreneTM and RetroNectinTM.
• PolybreneTM is a cationic polymer (polyhexadimethrine bromide) used to increase the efficiency of transduction of certain cells with retrovirus (and lentivirus) in cell culture, and is typically used in amounts of 10 pg/mL or less.
• RetroNectinTM is a polypeptide consisting of three functional domains derived from human fibronectin protein (C-domain, H-doman, and CS-1 site).
• RetroNectinTM enhances the efficiency of retroviral- and lentiviral- mediated transduction in hematopoietic cells including hematopoietic stem cells and terminally differentiated cells such as primary T cells and macrophages.
• Other additives can include lipofectin, lipofectamine, and cationic peptides such as protamine. These additives can be used to enhance transduction if appropriate.
• the frequency of the multi- dimensional acoustic standing wave determines the diameter of the particles that can be trapped by the acoustic standing 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.
• cells are labeled with green fluorescent protein (GFP).
• GFP green fluorescent protein
• the methods of the present disclosure can be carried out in a continuous process, wherein a fluid mixture containing the cells and the nucleic acids suspended in a host fluid is flowed through the acoustophoretic device.
• the nucleic acids can be naked, or they can 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 for continuous processing.
• FIG. 2B is a view of the device 200 in a fully assembled condition.
• the acoustophoretic device can be built such that each component is modular, and can be changed or switched out separately from each other. Thus, when new revisions or modifications are made to a given component, the component can be replaced while the remainder of the device stays the same.
• the device includes an ultrasonic transducer 220 and a reflector 250 on opposite walls of an acoustic chamber 210. It is noted that the reflector 250 may be made of a transparent material, such that the interior of the flow chamber 210 can be seen.
• the ultrasonic transducer is proximate a first wall of the acoustic chamber.
• the reflector is proximate a second wall of the acoustic chamber or can make up the second wall of the acoustic chamber.
• a cooling unit 260 can be located between the ultrasonic transducer 220 and the flow chamber 210. As illustrated here, the cooling unit 260 includes an independent flow path that is separate from the flow path through the acoustic chamber.
• a coolant inlet 262 permits the ingress of a cooling fluid into the cooling unit. The coolant and waste heat exit the cooling unit through a coolant outlet 264.
• the coolant that flows through the cooling unit can be any appropriate fluid.
• 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 electric short-circuits.
• the cooling unit can be in the form of a thermoelectric generator, which converts heat flux (i.e. temperature differences) into electrical energy using the Seebeck effect, thus removing heat from the flow chamber. Put another way, electricity can be generated from undesired waste heat while operating the acoustophoretic device.
• the cooling unit can be used to cool the ultrasonic transducer, which can be particularly advantageous when the device is to be run continuously with repeated processing and recirculation for an extended period of time (e.g., perfusion).
• the cooling unit can also be used to cool the fluid running through the acoustic chamber 210.
• the cells should be maintained around room temperature ( ⁇ 20°C), and at most around 28°C. This is because when cells experience higher temperatures, their metabolic rates increase. Without a cooling unit, however, the temperature of the cells flowing through the acoustic chamber can rise as high as 34°C.
• the acoustic chamber 210 is illustrated 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, recirculation outlet, bleed / harvest outlet) may be included as desired.
• the interior volume 216 can be considered as being bounded by the ultrasonic transducer 220, the cooling unit 260, the acoustic chamber 210, and the reflector 250.
• the flow direction of the acoustophoretic device 200 can be oriented in a direction other than horizontal.
• the fluid flow can be vertical either upward or downward or at some angle relative to vertical or horizontal.
• More than one transducer can be included in the system.
• FIG. 3 illustrates another acoustophoretic device 300 which can be used to practice the methods and processes of the present disclosure.
• the system includes the acoustophoretic device 300 and a substantially acoustically transparent container 310. These two components are separable from each other.
• the container 310 of the acoustophoretic device is generally formed from a substantially acoustically transparent material such as plastic, glass, polycarbonate, low- density polyethylene, and high-density polyethylene (all at an appropriate thickness).
• the container may be formed from any material suitable for allowing the passage of the acoustic standing wave(s) of the present disclosure therethrough.
• the container may be in the form of a bottle or a bag. The difference between these forms lies in their composition and structure.
• a bottle is more rigid than a bag. When empty, a bag is generally unable to support itself, while a bottle is able to stand upright.
• the container 310 shown here is a high-density polyethylene bag.
• Container 310 generally has an upper end 312 and a lower end 314, and an interior volume in which the fluid mixture (containing the first particles and second particles in a host fluid) is located.
• the acoustophoretic device 300 is defined by at least one wall 332, and usually a plurality of walls, which form its sides.
• the acoustophoretic device may be in the shape of a cylinder, or in a rectangle (as depicted).
• the wall(s) are solid.
• An opening 326 is present in an upper end of the acoustophoretic device, for receiving the container 310 therethrough.
• the acoustophoretic device 300 is separable from the container 310, so that the container can be either disposable or reusable, depending upon the desired application of the acoustophoretic device.
• the base of the acoustophoretic device 300 is solid.
• the acoustophoretic device 300 includes at least one ultrasonic transducer 330 on a wall 334.
• the ultrasonic transducer 330 has a piezoelectric material driven by a voltage signal to create an acoustic standing wave. Cables 332 are illustrated for transmitting power and control information to the ultrasonic transducer 330.
• a reflector 340 may be present, and is located on the wall 336 opposite the ultrasonic transducer 330. The standing wave is thus generated through initial waves radiated from the transducer and reflected waves from the reflector. In some embodiments, a separate reflector is not necessary.
• a chamber wall or release boundary such as may be provided by ambient air, may be used to reflect the incident waves and create the standing waves. It is to be understood that various transducer and reflector combinations may be used.
• the planar and/or multi-dimensional acoustic standing wave(s) are generated within the container, and are used to cause interaction of the particles within the container 310. It should be noted that there is no contact between the ultrasonic transducer and the fluid mixture within the container 310.
• the acoustophoretic device includes a plurality of ultrasonic transducers 330 located on a common wall 334 opposite the wall 336 on which the reflector 340 is located. Alternatively, the ultrasonic transducers can be located opposite each other, with no reflector being present. Additionally, the acoustophoretic device 300 may include a viewing window 324 in another wall 338. As illustrated here, when a viewing window is provided, it can be in a wall adjacent the walls upon which the ultrasonic transducer(s) and reflector are located, such that the lower end 314 of the container 310 can be viewed through the viewing window 324 in the separation chamber 320. In other embodiments, the viewing window can take the place of the reflector.
• a fluid such as water
• a fluid may be placed in the interstitial space 305 between the container 310 and the acoustophoretic 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 can be any fluid, though it should have an acoustic impedance value that allows for good transmission of the acoustic standing wave(s), and preferably a low acoustic attenuation.
• 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 in turn are better suited for trapping smaller particles like viruses.
• the ultrasonic transducer can be driven for a time period of about 5 minutes to about 15 minutes. This is a considerably shorter time period than, for example, conventional viral transduction processes where the cell culture and viral vector are incubated together for about 30 minutes to about 120 minutes. Such lengthy incubation periods are due to the reaction between cells and viruses only occurring when Brownian motion brings them in proximity to each other.
• the ultrasonic transducer can be driven for as long a time period as desired, for example up to 120 minutes or longer.
• FIG. 4 is a cross-sectional diagram of a conventional ultrasonic transducer.
• This transducer has a wear plate 50 at a bottom end, epoxy layer 52, ceramic piezoelectric element 54 (made of, e.g. Lead Zirconate Titanate (PZT) or lithium niobate), an epoxy layer 56, and a backing layer 58.
• PZT Lead Zirconate Titanate
• an epoxy layer 56 On either side of the ceramic piezoelectric element, there is an electrode: a positive electrode 61 and a negative electrode 63.
• the epoxy layer 56 attaches backing layer 58 to the piezoelectric element 54.
• a housing 60 which may be made out of, for example, aluminum.
• the housing is used as the ground electrode.
• An electrical adapter 62 provides connection for wires to pass through the housing and connect to leads (not shown) which attach to the piezoelectric element 54.
• backing layers are designed to add damping and to create a broadband transducer with uniform displacement across a wide range of frequency and are designed to suppress excitation of particular vibrational eigen-modes of the piezoelectric element.
• Wear plates are usually 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 of the present disclosure, which is used in the acoustic filtering device of the present disclosure.
• Transducer 81 is shaped as a square, and has an aluminum housing 82.
• the aluminum housing has a top end and a bottom end.
• the transducer housing may also be composed of plastics, such as medical grade HDPE or other metals.
• the piezoelectric element is a mass of perovskite ceramic, each consisting of a small, tetravalent metal ion, usually titanium or zirconium, in a lattice of larger, divalent metal ions, usually lead or barium, and O 2 ions.
• a PZT (lead zirconate titanate) piezoelectric element 86 defines the bottom end of the transducer, and is exposed from the exterior of the bottom end of the housing.
• the piezoelectric element is supported on its perimeter by a small elastic layer 98, e.g. epoxy, silicone or similar material, located between the piezoelectric element and the housing. Put another way, no wear plate or backing material is present.
• a layer of plastic or other material separating the piezoelectric element from the fluid in which the acoustic standing wave is being generated.
• the piezoelectric material / element / crystal has an exterior surface (which is exposed) and an interior surface as well.
• Screws 88 attach an aluminum top plate 82a of the housing to the 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 electrode 90 and a negative electrode 92, which are separated by an insulating material 94.
• the electrodes can be made from any conductive material, such as silver or nickel. Electrical power is provided to the piezoelectric element 86 through the electrodes on the piezoelectric element. Note that the piezoelectric element 86 has no backing layer or epoxy layer. Put another way, there is an interior volume or an air gap 87 in the transducer between aluminum top plate 82a and the piezoelectric element 86 (i.e. the air gap is completely empty).
• a minimal backing 58 and/or wear plate 50 may be provided in some embodiments, as seen in FIG. 6.
• the transducer design can affect performance of the system.
• a typical transducer is a layered structure with the ceramic piezoelectric element bonded to a backing layer and a wear plate. Because the transducer is loaded with the high mechanical impedance presented by the standing wave, the traditional 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 not be appropriate. Rather, in one embodiment of the present disclosure the transducers, there is no wear plate or backing, allowing the piezoelectric element to vibrate in one of its eigenmodes with a high Q-factor, or in a combination of several eigenmodes. The vibrating ceramic piezoelectric element/disk is directly exposed to the fluid flowing through the fluid cell.
• Removing the backing also permits the ceramic piezoelectric element to vibrate at higher order modes of vibration with little damping (e.g. higher order modal displacement).
• the piezoelectric element vibrates with a more uniform displacement, like a piston.
• Removing the backing allows the piezoelectric element to vibrate in a non-uniform displacement mode.
• the higher order the mode shape of the piezoelectric element the more nodal lines the piezoelectric element has.
• the higher order modal displacement of the piezoelectric element creates more trapping lines, although the correlation of trapping line to node is not necessarily one to one, and driving the piezoelectric element at a higher frequency will not necessarily produce more trapping lines.
• the piezoelectric element may 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 higher order mode shape and maintains 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 having holes through the layer, such that the lattice follows the nodes of the vibrating piezoelectric element in a particular higher order vibration mode, providing support at node locations while allowing the rest of the piezoelectric element to vibrate freely.
• the goal of the lattice work or acoustically transparent material is to provide support without lowering the Q-factor of the piezoelectric element or interfering with the excitation of a particular mode shape.
• the piezoelectric element in direct contact with the fluid also contributes to the high Q-factor by avoiding the dampening and energy absorption effects of the epoxy layer and the wear plate.
• Other embodiments of the transducer(s) may have wear plates or a wear surface to prevent the PZT, which contains lead, contacting the host fluid. This may be desirable in, for example, biological applications such as separating blood, biopharmaceutical perfusion, or fed-batch filtration of mammalian cells. Such applications might use a wear layer such as chrome, electrolytic nickel, or electroless nickel. Chemical vapor deposition could also be used to apply a layer of poly(p-xylylene) (e.g. Parylene) or other polymer.
• poly(p-xylylene) e.g. Parylene
• Organic and biocompatible coatings such as silicone or polyurethane are also usable as a wear surface.
• Thin films such as a polyetheretherketone (PEEK) film, can also be used as a cover of the transducer surface exposed to the fluid with the advantage of being a biocompatible material.
• PEEK film is adhered to the face of the piezoelectric material using pressure sensitive adhesive (PSA).
• PSA pressure sensitive adhesive
• the ultrasonic transducer has a nominal 2 MHz resonance frequency. Each transducer can consume about 28 W of power for droplet trapping at a flow rate of 3 GPM (gallons per minute). This translates to an energy cost of 0.25 kW hr/ m 3 . This is an indication of the very low cost of energy of this technology. Desirably, each transducer is powered and controlled by its own amplifier. In other embodiments, the ultrasonic transducer uses a square piezoelectric element, for example with 1 "x1 " dimensions. Alternatively, the ultrasonic transducer can use a rectangular piezoelectric element, for example with 1 "x2.5" dimensions.
• Power dissipation per transducer was 10 W per 1 "x1 " transducer cross-sectional area and per inch of acoustic standing wave span in order to get sufficient acoustic trapping forces.
• each 1 "x1 " square transducer consumes 40 W.
• the larger 1 "x2.5" rectangular transducer uses 100W in an intermediate scale system.
• the array of three 1 "x1 " square transducers would consume a total of 120W and the array of two 1 "x2.5” transducers would consume about 200 W.
• Arrays of closely spaced transducers represent alternate potential embodiments of the technology. Transducer size, shape, number, and location can be varied as desired to generate desired multi-dimensional acoustic standing wave patterns.
• the size, shape, and thickness of the transducer determine the transducer displacement at different frequencies of excitation, which in turn affects separation efficiency.
• the transducer is operated at frequencies near the thickness resonance frequency (half wavelength).
• Gradients in transducer displacement typically result in more trapping locations for the cells/biomolecules.
• Higher order modal displacements generate three-dimensional acoustic standing waves with strong gradients in the acoustic field in all directions, thereby creating equally strong acoustic radiation forces in all directions, leading to multiple trapping lines, where the number of trapping lines correlate with the particular mode shape of the transducer.
• FIG. 7 shows the measured electrical impedance amplitude of a square transducer as a function of frequency in the vicinity of the 2.2 MHz transducer resonance.
• the minima in the transducer electrical impedance correspond to acoustic resonances of the water column and represent potential frequencies for operation. Additional resonances exist at other frequencies where multi-dimensional standing waves are excited.
• Numerical modeling has indicated that the transducer displacement profile varies significantly at these acoustic resonance frequencies, and thereby directly affects the acoustic standing wave and resulting trapping force. Since the transducer operates near its thickness resonance, the displacements of the electrode surfaces are essentially out of phase. The typical displacement of the transducer electrodes is not uniform and varies depending on frequency of excitation.
• the displacement has a single maximum in the middle of the electrode and minima near the transducer edges.
• the transducer profile has multiple maxima leading to multiple trapped lines of oil droplets. Higher order transducer displacement patterns result in higher trapping forces and multiple stable trapping lines for the captured oil droplets.
• the trapping lines of oil droplets were observed and characterized.
• the characterization involved the observation and pattern of the number of trapping lines across the fluid channel, as shown in FIG. 8, for seven of the ten resonance frequencies identified in FIG. 7.
• Different displacement profiles of the transducer can produce different (more) trapping lines in the standing waves, with more gradients in displacement profile generally creating higher trapping forces and more trapping lines.
• FIG. 9 is a numerical model showing a pressure field that matches the 9 trapping line pattern.
• the numerical model is a two-dimensional model; and therefore only three trapping lines are observed. Two more sets of three trapping lines exist in the third dimension perpendicular to the plane of the page.
• the lateral force of the acoustic radiation force generated by the transducer can be increased by driving the transducer in higher order mode shapes, as opposed to a form of vibration where the crystal effectively moves as a piston having a uniform displacement.
• the higher order mode shapes are Bessel functions.
• the acoustic pressure is proportional to the driving voltage of the transducer.
• the electrical power is proportional to the square of the voltage.
• the transducer is typically a thin piezoelectric plate, with electric field in the z-axis and primary displacement in the z-axis.
• the transducer is typically coupled on one side by air (i.e.
• the types of waves generated in the plate are known as composite waves.
• a subset of composite waves in the piezoelectric plate is similar to leaky symmetric (also referred to as compressional or extensional) Lamb waves.
• the piezoelectric nature of the plate typically results in the excitation of symmetric Lamb waves.
• the waves are leaky because they radiate into the water layer, which result in the generation of the acoustic standing waves in the water layer.
• Lamb waves exist in thin plates of infinite extent with stress free conditions on its surfaces. Because the transducers of this embodiment are finite in nature, the actual modal displacements are more complicated.
• FIG. 10 shows the typical variation of the in-plane displacement (x- displacement) and out-of-plane displacement (y-displacement) across the thickness of the plate, the in-plane displacement being an even function across the thickness of the plate and the out-of-plane displacement being an odd function.
• the displacement components vary across the width and length of the plate.
• a (m,n) mode is a displacement mode of the transducer in which there are m undulations in transducer displacement in the width direction and n undulations in the length direction, and with the thickness variation as described in FIG. 10.
• the maximum number of m and n is a function of the dimension of the piezoelectric material (e.g., a piezoelectric crystal) and the frequency of excitation. Additional three-dimensional modes exist that are not of the form (m,n).
• the transducers are driven so that the piezoelectric element vibrates in higher order modes of the general formula (m, n), where m and n are independently 1 or greater.
• the transducers will vibrate in higher order modes than (2,2).
• Higher order modes will produce more nodes and antinodes, result in three-dimensional standing waves in the water layer, characterized by strong gradients in the acoustic field in all directions, not only in the direction of the standing waves, but also in the lateral directions.
• the acoustic gradients result in stronger trapping forces in the lateral direction.
• the ultrasonic transducer(s) may be driven by an electrical signal, which may be controlled based on voltage, current, phase angle, power, frequency or any other electrical signal characteristic.
• the driving signal for the transducer may be based on voltage, current, magnetism, electromagnetism, capacitive or any other type of signal to which the transducer is responsive.
• the voltage signal driving the transducer can have a sinusoidal, square, sawtooth, pulsed, or triangle waveform; and have a frequency of 500 kHz to 10 MHz.
• the voltage signal can be driven with pulse width modulation, which produces any desired waveform.
• the voltage signal can also have amplitude or frequency modulation start/stop capability to eliminate streaming.
• the voltage signal can have a frequency of about 0.5 MHz to about 30 MHz, so that such frequencies are produced by the ultrasonic transducer.
• the transducers are used to create a pressure field that generates acoustic radiation forces of the same order of magnitude both orthogonal to the standing wave direction and in the standing wave direction.
• forces are roughly the same order of magnitude, particles of size 0.1 microns to 300 microns will be moved more effectively towards "trapping lines", so that the cells and the nucleic acids (whether naked or in a viral vector) are co-located next to each other, permitting them to react with each other.
• the bioreactor i.e. , the bioreactor, acoustic filtering device, tubing fluidly connecting the same, etc.
• the transducers may also be driven to create rapid pressure changes to prevent or clear blockages due to agglomeration of biological cells.
• the frequency of the transducers may also be varied to obtain optimal effectiveness for a given power.
• Control can be distributed to some or all units involved in the bioprocessing.
• Feedback from units can be provided to permit overview of the bioprocess, which may be in the form of screen displays, control feedbacks, reporting, status reports and other information conveyance.
• Distributed processing permits a high degree of flexibility in achieving a desired process control, for example by coordinating steps among units and providing a batch executive control.
• the acoustophoretic devices utilizing an acoustic wave system can be implemented with biocompatible materials, and may include gamma sterilizable and single use components.
• the processing system also permits ultrasonic flow measurement, which is noninvasive, and is capable of operating with high viscosity fluids.
• the system can be implemented with single use sterile septic connectors and a simple graphical user interface (GUI) for control.
• GUI graphical user interface
• the acoustophoretic device is scalable. For example, a relatively small unit is capable of operation at 2 L to 50 L scale.
• a statement that a value exceeds (or is more than) a first threshold value is equivalent to a statement that the value meets or exceeds a second threshold value that is slightly greater than the first threshold value, e.g., the second threshold value being one value higher than the first threshold value in the resolution of a relevant system.
• a statement that a value is less than (or is within) a first threshold value is equivalent to a statement that the value is less than or equal to a second threshold value that is slightly lower than the first threshold value, e.g., the second threshold value being one value lower than the first threshold value in the resolution of the relevant system.
• configurations may be described as a process that is depicted as a flow diagram or 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. A process may have additional stages or functions not included in the figure.
• FIG. 11 is a picture of a plastic bag containing a fluid mixture with T-cells and viruses.
• the plastic bag was placed into an acoustophoretic device that was filled with water.
• a multi-dimensional acoustic standing wave was generated, causing the T-cells and viruses to interact with each other. This is visible as a series of beams of disks within the plastic bag.
• the BacMam® system uses baculoviruses for transduction, and was used for transduction of green fluorescent protein (GFP) into Jurkat T-cells. This system was used for various experiments. Five results are shown below. They were labeled Control, Process Control 1 , Process Control 2, Acoustics 3MFIz, and Acoustics 10MFIz.
• the results are listed in Table A below.
• the MOI is the multiplicity of infection, or the number of viral vector particles per cell.
• the GFP+ is the % of cells that expressed GFP. Table A.
• An acoustic chamber included an ultrasonic transducer and a reflector.
• a recirculation loop drew fluid from one end of the acoustic chamber and then circulated the fluid back through the other end into the acoustic chamber.
• the acoustic chamber had a volume of about 1 ml_, and the recirculation loop had a volume of about 3 ml_. Transduction tests were performed using the acoustic chamber.
• Each Acoustic test was also performed with a Control run and a Static Run.
• virus was added to cells in a Petri dish, and then immediately washed out.
• Static run virus was added to cells in a Petri dish for 90 minutes, then washed out.
• the MOI the number of viral vector particles per cell
• cell viability was measured by nucleocounter.
• Vi-Cell cell viability analyzer was also measured using a Vi-Cell cell viability analyzer.
• Table B indicates the parameters of each test.
• Table C indicates the results of each test run.
• Table D provides the Vi-Cell analysis.
• Table E provides the Nucleocounter analysis.
• the transduction gain is the Acoustic efficiency divided by the Static efficiency. Table B.

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