System for the electroporation of particles

Abstract

A system for cell bioprocessing and cell therapy manufacturing can include a series of microfluidic modules that enable continuous-flow, end-to-end cell bioprocessing. Each module can perform a different technology, and the modules can be coupled to each other to perform various unit operations in the cell bioprocessing or cell therapy manufacturing chain to enable direct processing of blood or blood product samples. The system can automatically and continuously process samples into genetically modified lymphocytes or T cells for cell therapy. The technologies performed by each module in the system can include any combination of microfluidic acoustophoresis, microfluidic acoustophoretic medium exchange or cell washing, and continuous-flow microfluidic electrotransfection. The modules performing these microfluidic technologies can be interconnected with plastic tubing or custom manifolds.

Description

Technical Field

[0001] <Cross - Reference to Related Applications> This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 939,191, filed on November 22, 2019, entitled "END - TO - END CELL THERAPY BIOPROCESSING DEVICE FOR CONTINUOUS - FLOW ENRICHMENT, WASHING, AND ELECTROTRANSFECTION OF TARGET CELLS", which is hereby incorporated by reference in its entirety.

Background Art

[0002] The use of genetically engineered T cells is adoptable for treating various blood cancers and leads to the first cell - therapy - based treatment approved by the FDA. The success of cell - therapy - based treatments has led to the research of new cell therapies and an increasing demand for cell therapy manufacturing. The current manufacturing pipeline mostly relies on old - fashioned cell bioprocessing equipment designed for research use rather than large - scale manufacturing. As a result, the processing time is long (e.g., generally several weeks), and the treatment can be very expensive, with the cost to patients sometimes reaching up to about $400,000 per treatment.

Summary of the Invention

Problems to be Solved by the Invention

[0003] The present disclosure describes systems and methods for cell bioprocessing and manufacturing for cell therapy. In some implementations, the techniques of the present disclosure can rationalize and automate cell bioprocessing or manufacturing processes for cell therapy using a certain combination of microfluidics-based technologies. The systems and methods of the present disclosure can utilize several steps including concentration of target cells from blood or blood products, cell washing or media exchange, and gene delivery by electroporation, in the manufacturing process. In some implementations, since all of these processing steps can be performed in a continuous flow, a fully automated process can process about one billion cells in a few hours without human intervention.

[0004] In some implementations, a system for cell bioprocessing and manufacturing for cell therapy can be mainly constructed from microfluidic modules and can enable continuous flow end-to-end cell bioprocessing. Conventionally, microfluidic solutions may be limited by low processing capabilities. However, the techniques disclosed herein overcome these limitations using parallelization and mesoscale geometries (e.g., microchannels with a width of 1 mm), thereby enabling the manufacture of cell therapy at the clinical scale. The present disclosure also describes feedback sensors for automatically and precisely controlling important parameters such as flow rate and magnitude of the electric field. This feedback sensor can help ensure that the final product is consistent through the processing of large samples that can contain more than 500,000 cells. Accordingly, the present disclosure provides techniques that can result in improved automation, reduction of touch labor on-site, improved processing capabilities, and more precise control of the processes used for cell bioprocessing and manufacturing for cell therapy.

Means for Solving the Problems

[0005] At least one aspect of the present disclosure is directed to a system. The system can include an inlet channel that receives a flow of a target fluid containing target particles. The system can include an acoustophoresis device that receives the flow of the target fluid containing the target particles from the inlet channel and moves the target particles from the flow of the target fluid to a flow of a buffer fluid containing cargo particles. The system can include an electroporation device that receives the flow of the buffer fluid containing the target particles and the cargo particles. The electroporation device can apply an electric field to the flow of the buffer fluid to cause some of the cargo particles to absorb some of the target particles in the flow of the buffer fluid. The system can include an outlet channel that supplies an output flow of the buffer fluid from the electroporation device.

[0006] In some implementations, the acoustophoresis device can include a central channel that receives the flow of the buffer fluid containing the cargo particles from a source of the buffer fluid and receives the flow of the target fluid from the inlet channel. In some implementations, the acoustophoresis device can include a piezoelectric element connected to the central channel that moves the target particles from the flow of the target fluid to the flow of the buffer fluid in a second channel. In some implementations, the electroporation device can include a central channel that receives the flow of the buffer fluid exiting the acoustophoresis device and the flow of a conductive buffer exiting the second channel. In some implementations, the electroporation device can include an electrode that supplies a current and is electrically connected to a portion of the central channel.

[0007] In some embodiments, the target particles in the flow of the input fluid may be lymphocytes. In some embodiments, the flow of the input fluid further includes unwanted particles including at least one of red blood cells, granulocytes, or monocytes. In some embodiments, the system can include a second inlet channel that receives a flow of the input fluid including the target particles and the unwanted particles. In some embodiments, the system receives a flow of the input fluid including the target particles and the unwanted particles from the second inlet channel, and includes an acoustic separation device that separates the flow of the input fluid into a flow of the target fluid including the target particles and a flow of the unwanted fluid including the unwanted particles. In some embodiments, the flow of the unwanted fluid can be transported to a waste reservoir via one or more channels. In some embodiments, the flow of the target fluid exiting the acoustic separation device is transported into the target reservoir via a second channel. In some embodiments, the system can include a pump that transports the flow of the target fluid from the target reservoir to the acoustic levitation device via the inlet channel.

[0008] In some embodiments, the system can include a first pump that transports the flow of the target fluid from the input reservoir to the acoustic levitation device via the inlet channel. In some embodiments, the system can include a second pump that transports a flow of the buffer fluid including the target particles and the cargo particles exiting the acoustic levitation device to the electroporation device via an intermediate channel. In some embodiments, the system can include one or more temporary storage reservoirs between two or more of the inlet channel, the acoustic levitation device, the electroporation device, or the outlet channel. In some embodiments, the system can include a separation device that receives a flow of the output buffer fluid from the output channel and separates the concentrated target particles in the flow of the output buffer fluid from the unwanted particles in the flow of the output buffer fluid. In some embodiments, the system can include an output reservoir that receives the concentrated target particles from the separation device.

[0009] In some embodiments, the connection between at least the inlet channel, the acoustophoresis device, the electro-poration device or the outlet channel comprises at least one of a polyvinyl chloride tube or a silicone tube. In some embodiments, the system can comprise one or more fluid capacitors connected to at least one connection, and the fluid capacitor is configured to adjust the flow rate of the fluid within the system. In some embodiments, the system is configured to send a signal representing the value of the density of target particles or unwanted particles in at least one of the flow of the target fluid, the flow of the buffer fluid, or the flow of the output buffer fluid to a controller device, and can comprise one or more sensors. In some embodiments, the system can comprise one or more flow sensors that send a signal representing the flow rate or conductivity of the fluid flowing through at least one of the inlet channel, the acoustophoresis device, the electro-poration device or the outlet channel to a controller device.

[0010] At least one other aspect of the present disclosure is directed to a system. The system can include a sensor upstream of an electroporation device that measures the conductivity of a fluid flowing into the electroporation device. The system can include an electrical signal generator that generates a voltage within the electroporation device. The system can include a controller device that includes one or more processors connected to a memory. The controller device can identify the magnitude of a desired electric field to be induced in a fluid flowing through the electroporation device. The controller device can receive from the sensor the conductivity of the fluid flowing into the electroporation device. The controller device can determine, based on the conductivity and the voltage generated by the electrical signal generator, the magnitude of the expected electric field in the fluid as the fluid flows through the electroporation device. The controller can calculate an adjustment voltage for the electrical signal generator based on the magnitude of the expected electric field and the magnitude of the desired electric field. The controller can supply a signal representing the adjustment voltage to the electrical signal generator to cause the electrical signal generator to generate a second voltage within the electroporation device.

[0011] In some implementations, the system can include a conductivity probe that measures a second conductivity of the fluid as the fluid flows through the electroporation device. In some implementations, the system can include a current sensor that measures a current passing through the fluid as the fluid flows through the electroporation device. In some implementations, the system can include an optical sensor that measures a width of a central portion of the fluid as the fluid passes through the electroporation device. In some implementations, the controller device can determine the magnitude of the expected electric field based on the second conductivity, the current, and the width of the central portion of the fluid.

[0012] In some implementations, the fluid flowing through the electroporation device includes a first fluid from a first fluid input and a second fluid from a second fluid input. In some implementations, the controller device can calculate an adjusted flow rate for at least one of the first fluid or the second fluid based on a second conductivity, a current, and a width of a central portion of the fluid. In some implementations, the controller device supplies a second signal representing the adjusted flow rate to a pump that controls the flow of the first fluid or the second fluid, so that the first fluid or the second fluid can flow at a second flow rate.

[0013] In some implementations, the fluid flowing through the electroporation device can include a first fluid from a first fluid input and a second fluid from a second fluid input. In some implementations, the electroporation device can include a second sensor that determines a first flow rate of the first fluid and a second flow rate of the second fluid at an input side of the electroporation device. In some implementations, the controller device can calculate an adjusted flow rate for at least one of the first fluid or the second fluid based on at least one of the first flow rate or the second flow rate. In some implementations, the controller device supplies a second signal representing the adjusted flow rate to a pump that controls the first flow rate of the first fluid or the second flow rate of the second fluid, so that the first fluid or the second fluid can flow at a third flow rate.

[0014] Yet another aspect of the present disclosure is directed to a method. The method can be implemented by a controller device having one or more processors and a memory. The method can include identifying a magnitude of a desired electric field to be induced in a fluid flowing through an electroporation device. The method can include receiving, from a first sensor, a conductivity of the fluid when the fluid flows into the electroporation device. The method can include determining a magnitude of an expected electric field in the fluid when the fluid flows through the electroporation device based on the conductivity and a voltage generated by an electrical signal generator. The method can include calculating an adjustment voltage for the electrical signal generator based on the magnitude of the expected electric field and the magnitude of the desired electric field. The method can include supplying a signal representing the adjustment voltage to the electrical signal generator to cause the electrical signal generator to generate a second voltage within the electroporation device.

[0015] In some implementations, the method can include receiving, from one or more second sensors that detect signals from the fluid within the electroporation device, a second conductivity of the fluid, a current flowing through the fluid, and a width of a central portion of the fluid. In some implementations, the method can include determining a magnitude of the expected electric field based on the second conductivity, the current, and the width of the central portion of the fluid. In some implementations, the fluid flowing into the electroporation device is received from a first fluid flow and a second fluid flow. In some implementations, the method can include receiving, from at least one flow rate sensor, a flow rate of the first fluid in the first fluid flow and a flow rate of the second fluid in the second fluid flow. In some implementations, the method can include calculating an adjusted flow rate for at least one of the first fluid flow or the second fluid flow. In some implementations, the method can include supplying a signal representing the adjusted flow rate to a pump that controls the first fluid flow or the second fluid flow to cause the first fluid flow or the second fluid flow to flow at a second flow rate.

[0016] These and other aspects and implementations will be discussed in detail below. The above information and the following detailed description include examples that illustrate various aspects and implementations, and also provide an overview or framework for understanding the nature and characteristics of the aspects and implementations recited in the claims. The drawings illustrate various aspects and implementations to enhance understanding and are incorporated herein and form part of this specification. It will be readily understood that aspects can be combined with each other, and that features described in the context of one aspect of the invention can be combined with other aspects. Aspects can be implemented in any convenient form.

[0017] The accompanying drawings are not intended to be drawn to scale. Like reference numerals and designations in the various drawings indicate like elements. For clarity, not all components may be labeled with reference numerals in all of the drawings. By referring to the following description used in conjunction with the accompanying drawings, the above and other objects, aspects, features, and advantages of the present disclosure will become more apparent and be more deeply understood.

Brief Description of the Drawings

[0018]

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[0019] Since the concepts described are not limited to any particular implementation, the various concepts introduced above and described in further detail below can be implemented in any of a number of manners. Examples of specific implementations and applications are provided primarily for purposes of illustration.

[0020] The present disclosure describes systems and methods for cell bioprocessing and manufacturing for cell therapy. In some implementations, the techniques of the present disclosure can rationalize and automate the manufacturing process for cell therapy by using a combination of microfluidics-based technologies. The systems and methods of the present disclosure can employ several steps including concentration of target cells from blood or blood products, cell washing or media exchange, and gene delivery by electroporation, in a cell bioprocessing or manufacturing process for cell therapy. In some implementations, all of these processing steps can be performed in a continuous flow, so that a fully automated process can process about one billion cells in a few hours without human intervention.

[0021] In some embodiments, a system for cell bioprocessing and cell therapy manufacturing can be primarily constructed from microfluidic modules and can enable continuous-flow end-to-end cell bioprocessing. For example, a series of modules each performing a different technique can be interconnected to perform various unit operations involved in a series of cell therapy manufacturing, thereby directly processing a blood or blood product sample. In some embodiments, the sample can be a leukopak obtained by leukapheresis. The system can automatically and continuously process the sample to convert it into genetically engineered lymphocytes or T cells for cell therapy. In some embodiments, the techniques implemented by each module within the system can include any combination of microfluidic acoustophoresis, microfluidic acoustophoretic medium exchange or cell washing, and continuous-flow microfluidic electroporation. Modules implementing these microfluidic techniques may be interconnected as part of an integrated automated system with plastic tubing (e.g., silicone or vinyl tubing) or a custom manifold.

[0022] FIG. 1 shows an exemplary process flow diagram of a manufacturing method 100 for cell therapy. The process flow diagram 100 includes three stages. In the first stage 105, a blood sample can be concentrated for lymphocytes (or other types of cells such as T cells in other implementations) by acoustic phoresis. For example, a blood preparation can be introduced into a module configured to concentrate the sample for lymphocytes using acoustic separation. Unwanted cells can be discarded, and the concentrated sample can be introduced into the second stage 110. The second stage 110 can be implemented, for example, using a medium exchange device. In the second stage 110, within the medium exchange module, the target cells can be transferred into the electroporation medium by acoustic phoresis. Acoustic phoresis can be used to move cells into a low-conductivity electroporation buffer containing the cargo, and in the second stage 1100, the cargo can include nucleic acids, proteins, or a combination of these substances. In some implementations, the cargo can include mRNA. Then, the medium containing the target cells can be delivered to the third stage 115. In some implementations, the third stage 115 can be implemented using an electroporation device. In the third stage 115, the cells can be electroporated within a continuous flow type electroporation device.

[0023] The sequences of stages 105, 110, and 115 shown in FIG. 1 are merely exemplary. In some other implementations, the modules implementing the first stage 105, the second stage 110, and the third stage 115 can be configured in various orders or permutations, or can be configured as various serial and parallel networks and interconnected to automate the desired workflow. For example, in some implementations, acoustic enrichment of the target cells can be performed in the last stage, and medium exchange and electroporation can be performed in the stages before that. In addition to the stages shown in FIG. 1, the present disclosure provides an infrastructure for maintaining a continuous flow from one stage to the next, a holding reservoir that is agitated to keep the cells in suspension during processing steps at the front end of the device, a controller, sensors and feedback control, and pumps, all of which will be described further later. In some implementations, the entire system may be closed to maintain sterility.

[0024] FIG. 2 shows an exemplary module 200 that can be used to implement the first stage 105 of the process flow 100 of FIG. 1. In module 200, acoustic streaming can be used to concentrate lymphocytes and drastically reduce red blood cells from a sample containing blood or a blood product. Module 200 comprises a set of microchannels having one inlet 205 and three outlets 210a, 210b and 215. In some implementations, the wall of the inlet channel 205 may be connected to an ultrasonic oscillator such as a piezoelectric element, and by electrically driving the piezoelectric element, when some cells flow through the channel, these cells can be excited to move towards the central flow in the axial direction of the channel. Since the migration speed of cells can depend on the size, density of the cells, and the relative compressibility with respect to the surrounding medium, if there are differences in the intrinsic properties of the cells, some types of cells may move faster than other types of cells and be collected at the central outlet 215, while the remaining cells may be collected at the lateral outlets 210a and 210b. In the example of FIG. 2, red blood cells, granulocytes and monocytes can be concentrated at the central outlet 215, and lymphocytes can be concentrated at the lateral outlets 210a and 210b. The cells and medium flowing into the central outlet 215 may be unwanted products, and these unwanted products can be discarded. The cells and medium flowing into the lateral outlets 210a and 210b can be collected and introduced into subsequent stages of the system (e.g., stage 110 of FIG. 1). In some implementations, the separation process within module 200 can be further facilitated by modifying the medium, adding particles, or incorporating the cells into other states / phenotypes or aggregates.

[0025] FIG. 3 shows an exemplary module 300 that can be used to implement the second stage of the process flow of FIG. 1. In module 300, acoustic streaming can be used to move cells from one medium to another. In particular, module 300 can move target cells from an initial medium (e.g., plasma or cell culture medium) into an electroporation medium that is compatible with an upstream microfluidic electroporation module. Module 300 can include three inlets, including a central inlet 310 and two side inlets 315a and 315b. Module 300 can include three outlets, including a central outlet 320 and two side outlets 325a and 325b. A central channel 330 can connect the three inlets to the three outlets. A piezoelectric element 335 may be connected to the central channel 330.

[0026] In module 300, three parallel flows can be established in a laminar flow pattern, and each flow corresponds to one of the two side inlets 315a or 315b or the central inlet 310. The mixing of these flows within the central channel 330 can be governed by diffusion and dispersion. The acoustic radiation field generated by the piezoelectric element 335 can be used to manipulate particles with respect to the flow within the central channel 330, and thus can be used to move particles from one flow to another. The central channel 330 may be a microchannel fabricated from a rigid substrate such as silicon, glass, or quartz, or a polymer with a high acoustic impedance such as polystyrene. The cross-section of the central channel 330 may be rectangular, and the dimensions of its width and height may range from 100 μm to 1000 μm. The length of the central channel may range from 5 mm to 200 mm. In the configuration shown in FIG. 3, the central fluid flow can have a density greater than or equal to the density of the side flows. In some implementations, additives can be used to adjust the density of the central flow to obtain the required density difference.

[0027] Under the influence of the acoustic radiation field generated by the piezoelectric element 335, mainly the culture medium can stay in the respective culture medium flows, but the cells introduced into the side outlets 315a and 315b move to the central flow. The central flow containing the cells can be collected from the central outlet 320. The culture medium flowing into the side outlets 325a and 325b can be discarded as waste. Therefore, the target cells come out of the initial culture medium and move into the culture medium introduced through the central inlet 310, and this culture medium can be collected for introduction into subsequent stages (for example, the third stage 115 in FIG. 1).

[0028] FIG. 4 shows an exemplary module 400 that can be used to implement the third stage of the process flow of FIG. 1. In module 400, cells can be electroporated in a continuous flow. Module 400 can be provided with three inlets, including a central inlet 410 and two side inlets 415a and 415b. Module 400 can be provided with three outlets, including a central outlet 420 and two side outlets 425a and 425b. The central channel 430 can connect the three inlets to the three outlets. A set of electrodes 435a and 435b may be connected to the central channel 430. In some implementations, cells and cargo can be introduced into the central flow in a state of being taken into a low-conductivity culture medium through the central inlet 410. The flow of the high-conductivity culture medium in contact with the stimulation electrodes 435a and 435b can be introduced through the side inlets 415a and 415b and can be located on the side of the central flow. With this configuration, while exposing the cells to a large electric field, it is possible to keep the cells from directly contacting the electrodes 435a and 435b.

[0029] In some embodiments, module 400 can apply a pulsed electric field to target cells in a continuous flow to temporarily permeabilize the target cells, thereby making it easier to perform cargo uptake and genetic manipulation. In some embodiments, central channel 430 may be a microchannel made of a rigid plastic (e.g., cyclic olefin copolymer, Kapton®, polystyrene, Ultem®, etc.) and can support a sheath flow or parallel flow configuration including three parallel laminar flows. In some embodiments, the dimensions of the channels within module 400 may range from a width of 500 μm to 3 mm, a length of 1 cm to 5 cm, and a height of 125 μm to 500 μm.

[0030] Electrodes 435a and 435b may be coplanar rectangular electrodes patterned on the floor of central channel 430. The dimensions of electrodes 435a and 435b may be a width of 100 μm to 250 μm and a length of 8 μm to 45 mm. In some embodiments, electrodes 435a and 435b can be connected to a power source via connection to soldering pads. In some embodiments, electrodes 435a and 435b may be located 50 μm to 300 μm away from the walls of central channel 430. Electrodes 435a and 435b may be formed from an electrochemically stable material such as platinum.

[0031] The central fluid flow within the central channel 430 can contain cells and cargo suspended in a low-conductivity electroporation buffer (e.g., 0.01 - 0.1 S / m) that can be introduced via the central inlet 410. The lateral flow within the central channel 430 can include a high-conductivity cell culture buffer (e.g., 1 - 2 S / m). The parameters related to electroporation can be the ratio of the conductivity of the lateral flow to the conductivity of the central flow. In some implementations, this ratio can be 20 or more. For example, when the conductivity of the lateral flow ranges from 20 - 40 S / m, the conductivity of the central flow can range from 1 - 2 S / m. By adjusting the relative flow rate of the central flow to the lateral flow within the central channel 430, the electrodes 435a and 435b can be made to contact only the lateral flow. In this configuration, the central flow may affect the electrical resistance of the circuit, and as a result, when a voltage is applied to the electrodes 435a and 435b, most of the voltage drops across the ends of the central flow. In some implementations, the applied voltage can take the form of a sine curve with a period ranging from 10 ns to 10 ms. In some implementations, the applied voltage can take the form of a pulse train with a pulse width ranging from 10 ns to 10 ms. In some implementations, the magnitude of the applied voltage can vary to generate an electric field ranging from about 2 - 600 kV / m across the ends of the central flow with a pulse width ranging from 10 ns to 10 ms. Samples containing transfected cells can be collected via the central outlet 420, and the media exiting from the lateral outlets 425a and 425b can be treated as unwanted products and discarded.

[0032] In some embodiments, the microfluidic modules or devices described herein (e.g., module 200, module 300, module 400, etc.) can be interconnected with each other to form a process flow similar to the process flow system 500 described herein in connection with FIGS. 5A, 5B, and 5C. The microfluidic modules described herein may be disposed within a layer or substrate. For example, the devices or channels for microfluidics may each be disposed within a single substrate sheet, thereby forming one layer. In some embodiments, for one or more of the microfluidic modules or devices described herein, it is also possible to use different substrates, and each substrate may be connected via a tube, another microfluidic substrate channel, or other fluid connection means. Further, a microfluidic valve can be embedded in one or more microfluidic substrates forming the above layer together with any of the other microfluidic components or mechanisms described herein (e.g., pumps, sensors, reservoirs, waste bottles, pinch valves, etc.) to control the flow of fluid as it flows through microfluidic channels similar to the components described in connection with system 500.

[0033] In some embodiments, a microfluidic device can form part of a process flow layer, and this microfluidic device can also scale by multiplexing microfluidic channels across multiple parallel layers. Each layer in the process flow can include one or more microfluidic modules or devices (e.g., module 200, module 300, module 400, etc.), microfluidic channels for transporting fluid between the microfluidic modules or devices, and other microfluidic devices (e.g., fluid capacitors, fluid reservoirs, valves, pumps, any other microfluidic mechanisms described herein, etc.). It will be understood that any layer belonging to the microfluidic device can include any number of ports (e.g., inlet ports, outlet ports, etc.) for introducing or extracting fluid from a particular layer at any stage in the process flow. The ports can include one or more connectors (e.g., threaded connectors, snap connectors, friction fit connectors, press fit connectors, etc.) that can be connected to other fluid conduits such as fluid conduits from one or more reservoirs. Thus, in embodiments having multiple layers consisting of microfluidic devices or mechanisms, the fluid emerging from the fluid source can be supplied to multiple layers by multiplexing the fluid conduits from the fluid source to the inlet ports of each layer.

[0034] In some embodiments, ports of one layer may be connected to ports of another layer, thereby enabling fluid to flow between the layers. By using multiple layers, a laminate of microfluidic layers and devices can be formed. In some embodiments, the microfluidic layer laminate can have components similar to other layers in the microfluidic laminate, thereby enabling a process flow defined by a microfluidic device (e.g., module 200, module 300, module 400, etc.) to be defined in a parallel network of microfluidic devices. Parallel layers can define parallel microfluidic portions belonging to a microfluidic path (e.g., a path or channel for microfluidics, as described later in this specification together with FIGS. 5A, 5B, and 5C) by a design in which the layers are arranged horizontally or by adding vertically arranged layers belonging to a microchannel network for steps such as cell separation or transduction. In other words, a process flow system or a component for microfluidics can be expanded or scaled using one or more layers belonging to a microfluidic device, and one or more layers belonging to a microfluidic device can be arranged in a horizontally arranged pattern (e.g., a continuously arranged pattern, etc.), a parallelly arranged pattern, or any combination thereof.

[0035] Figures 5A, 5B, and 5C illustrate a portion of a block diagram of a system 500 for implementing a process flow similar to the process flow 100 shown in FIG. 1. System 500 shows the components necessary for connection and support in addition to the microfluidic device. In some implementations, at least some of the microfluidic devices shown in FIGS. 5A, 5B, and 5C may correspond to the modules of FIGS. 2-4 that can be used to implement the steps of the process flow 100 of FIG. 1. Microfluidic devices such as examples of each of the modules 200, 300, and 400 of FIGS. 2-4, as well as valves, sensors, and staging reservoirs, are shown in blue. FIG. 5A includes a legend indicating the symbols used for dampeners and pinch valves within the system 500 shown in FIGS. 5A, 5B, and 5C. The circles may represent peristaltic pumps that can move fluid within the system 500 and can be used to maintain the correct flow rate.

[0036] Referring now to FIG. 5A, at the front end of system 500, a user can place target cells in the medium into a temporary storage reservoir 505. The reservoir 505 can have a stirring mechanism, such as a magnetically driven impeller, for keeping the cells in a suspended state. In some implementations, the stirring mechanism may be configured to gently stir the medium to such an extent that the cells are not damaged. The reservoir 500 at the front end of system 500 may be connected via a tube to the central inlet of an acoustic streaming rapid medium exchange device illustrated as module 300 in FIG. 5A. Module 300 of FIG. 5A may be an example of the module 300 shown in FIG. 3. The peristaltic pump 510 can move the flow through module 300 by deforming the tube. Another reservoir 515 containing the cargo to be transfected (e.g., mRNA) suspended or dissolved in an electroporation buffer may be connected to the side inlet of module 300. Unwanted products exiting module 300 can be collected in reservoir 520. Another peristaltic pump 525 can move the flow from reservoir 515 to enter the side flow inlet of module 300. As described above, due to the acoustic field within module 300, it is possible to move the cells from the side flow into the central flow, and the cells can exit from the central outlet while suspended together with the cargo in the electroporation medium. At the outlet of module 300, the side flow can be collected as waste in reservoir 520. The central outlet of the rapid medium exchange module 300 may be connected via a tube to the inlet of a holding reservoir 530, and the holding reservoir 530 can have a stirring mechanism for keeping the cells in a suspended state.

[0037] Referring now to FIG. 5B, the outlet of the temporary reservoir 530 illustrated in FIG. 5A may be connected by a tube to a flow-through conductivity sensor 535, and this flow-through conductivity sensor 535 may be connected to the central inlet of a flow electroporation device shown as module 400 in FIG. 5B. The module 400 of FIG. 5B may be an embodiment of the module 400 shown in FIG. 4. The peristaltic pump 540 can move the flow of the cell suspension from the outlet of the temporary reservoir 530, through the conductivity probe 535, and through the central flow of the electroporation module 400. Another external reservoir 545 containing a high-conductivity cell culture medium (e.g., TexMACS) is connected via a tube to the side inlet of the module 400. The flow through each side inlet is driven by another set of peristaltic pumps 550 and 555. When the cells pass through the module 400, a voltage pulse is applied to transfect the cells. The fluid exiting the side outlet of the module 400 can be collected as waste in the reservoir 560. The fluid exiting the central outlet module 400 contains a large amount of transfected cells, and this fluid can flow into another temporary reservoir 565, which can have a stirring mechanism useful for maintaining the cells in a suspended state.

[0038] Referring now to FIG. 5C, the cell suspension exiting the temporary reservoir 565 shown in FIG. 5B can be moved by a final peristaltic pump 570 acting on the tube connecting the reservoir 565 and the module 200 to enter a single inlet in the microfluidic acoustic afresis module shown as the module 200 in FIG. 5C. The module 200 in FIG. 5C may be an embodiment of the module 200 shown in FIG. 2. When the module 200 is actuated acoustically, the cell suspension can be concentrated for lymphocytes at the outlet of the module 200. The sample at the outlet of the module 200 is collected in the reservoir 575 as the final product (e.g., transfected lymphocytes). The fluid exiting from the waste outlet of this module 200 can be collected in the final waste reservoir 580. Modules (e.g., modules 200, 300, and 400, etc.), pumps (e.g., pumps 510, 525, 540, 550, 555, 570, etc.) can each be controlled by one or more signals received from the controller 1005 as described herein.

[0039] In some implementations, the connections between the components of the system 500 can be made using various types of polymer tubes, for example, a PVC tube with an inner diameter of 1.12 mm (0.44 inches) that can be supplied by a peristaltic pump, Tygon tubes with inner diameters of 3.18 mm (1 / 8 inch) and 4.23 mm (1 / 6 inch) that can be used between components, and thinner (e.g., inner diameter 0.28 mm (0.011 inches) to 0.64 mm (0.025 inches)) silicone tubes for connecting to the modules 200, 300, and 400. An adapter can also be used to switch between tubes of different sizes as needed.

[0040] In some implementations, as described above, the flow through the entire system 500 can be moved by a series of fluid pumps. In some implementations, these pumps may be peristaltic pumps. Since the flow within a peristaltic pump is inherently pulsating, a steady flow may be required for modules 200, 300, and 400 to function properly. Therefore, a suitable fluid capacitor can be introduced behind each pump to smooth out fluctuations in the flow rate. The nominal flow rates generated by these pumps are shown in FIGS. 5A, 5B, and 5C for illustrative purposes, but it should be understood that these flow rates may be different in other implementations and that these flow rates may change as the system 500 operates. In some implementations, the pumps can move fluid and samples from the reservoir into the system 500. Thus, by using valves, the input line used for priming and setting up the system 500 and other lines used to flow samples within the system 500 can be switched. In some implementations, flow sensors can be placed at various locations within the fluid path (e.g., the inlets and outlets of modules 200, 300, and 400) and used for feedback control of the pumps (e.g., by a controller 1005 as described herein in connection with FIG. 10).

[0041] As shown in FIGS. 5A, 5B, and 5C, there may be storage reservoirs between each of the modules 200, 300, and 400. These reservoirs can provide ballast to make the system 500 robust against unexpected differences in flow rate between the output of one module and the input of the next module. In some implementations, by using a stirring mechanism such as a magnetically driven impeller, cells can be kept in a suspended state and prevented from settling within these storage reservoirs. To maintain the system 500 at a constant temperature while various components (e.g., acoustophoresis-based components) are generating heat, in some implementations, a shared heat sink or individual heat sinks for each of the modules 200, 300, and 400 can be used. Such heat sinks can also be combined with a closed-loop thermoelectric cooling system.

[0042] In some implementations, sensors may be incorporated into the system 500 to enable malfunction detection and inquiries to the system 500 regarding feedback control mechanisms or feedforward control mechanisms. For example, the sensors may be incorporated as components of the overall system or directly into the modules 200, 300, and 400. Possible sensors that can be incorporated into the system 500 include flow rate, conductivity probes, visual measurement of the flow width during sheath flow, and flow sensors for controlling current measurement values. In some implementations, optical sensors can also be used to evaluate the quality of the sheath flow used in the system 500, and this sheath flow can be used to adjust the flow rate as needed to generate an appropriate and stable flow. In some implementations, optical sensors can be used to calibrate and adjust the acoustophoresis module, and within the acoustophoretic module, the optimal drive frequency for the piezoelectric components can be automatically determined by observing the concentration of cells contained in one of the outlet fractions.

[0043] To estimate one or both of the cell concentration or cell density in real time, an absorbance or impedance sensor can also be incorporated into the system 500. This sensor can provide information regarding the processing capabilities of the system 500 and can also be used to determine where in the system 500 losses might be occurring when the cell recovery rate is low. By using the information obtained from the measured cell concentration in closed-loop control, the flow rate at the inlets or outlets of the modules 200, 300, and 400 can be adjusted, the acoustic power or acoustic frequency can be adjusted, or the automatic addition of reagents to the system 500 can be adjusted.

[0044] In some implementations, the sensor can be added to any of the modules 200, 300, and 400 or at a location within the system 500 between these modules to indicate the operational quality or efficiency of the functions performed by these modules. For example, the sensor can be added to module 200 or at a location within the system downstream of module 200 to indicate or detect the efficiency of acoustic phoretic separation of cells occurring within module 200. Such a sensor can be configured to identify the cells in the fluid either during or after the fluid has passed through module 200. In some implementations, the sensor can be added to module 400 or at a location downstream of module 400 to indicate the efficiency of transfection that results from the operation of module 400. Such a sensor can measure how much cargo has been introduced into the cells of the fluid sample by the electrotransfection operation performed by module 400. In some implementations, the sensor can be incorporated into the system 500 to monitor cell viability.

[0045] All of these sensors can provide outputs in real time and can also be connected to a control system such as the controller 1005 to function as a feedback control mechanism or a feedforward control mechanism. For example, based on the output of such a sensor, adjustable parameters of any of the modules 200, 300, and 400 (such as the flow rate of a fluid, the ratio of a fluid sample, an applied voltage, or an electric field, etc.) can be controlled. Therefore, real-time information regarding electrotransfer efficiency, separation efficiency, or cell viability can be incorporated into the control data to change the operating characteristics of the system 500 during cell processing.

[0046] In some implementations, the control of individual components of the system 500, such as the modules 200, 300, and 400, and the design parameters of the individual components can be selected according to the characteristics of the entire system 500. For example, instead of selecting the parameters of the components of the system independently for each individual component, they can also be selected to be interdependent. Therefore, in order to improve the performance of the entire system 500, some components can also be designed or controlled to operate at a performance (such as flow rate) lower than the maximum performance achievable by that component. In some implementations, design parameters that can be selected in this way can include characteristics that may not be adjustable after the fabrication of the system 500, such as dimensional features (such as the height of the channel, the width of the channel, the cross-sectional area of the channel, the cross-sectional shape of the channel, etc.). Such parameters can be selected according to a routine or algorithm that improves the overall operation of the system 500, even if the selection of the parameters causes the individual operating performance of one or more of the components of the system 500 to fall below the optimal value or decrease.

[0047] It should also be understood that the adjustable parameters regarding each component can likewise be selected or controlled. Regarding the adjustable parameters, the selection of appropriate values may change over time even during the operation of the system 500. In order to achieve an improvement in the overall performance of the system 500 (e.g., an improvement in total throughout, an improvement in cell viability, an improvement in cell separation efficiency, etc.), the adjustable parameters may be selected such that the processing capacity of a certain component may decrease. In one example, the parameters of the electroporation component such as the module 400 can be selected to decrease the processing capacity of the electroporation component in order to improve the performance of another component (e.g., the module 200 used for cell separation) or the overall performance of the system 500. In some implementations, rather than selecting the parameters of each component of the system 500 independently of each other, such adjustable parameters can be selected together with fixed or non-adjustable design parameters by using a routine or algorithm that can incorporate machine learning to improve the operation of the system 500 on an overall scale.

[0048] As described herein, the components, channels, or stages of the process flow system 500 illustrated in FIGS. 5A, 5B, and 5C can each be provided in one or more layers of a microfluidic device. For example, these layers may be arranged in parallel, where the microfluidic devices (e.g., module 200, module 300, module 400, etc.) are each replicated to span parallel microfluidic layers, and the flow of input fluid is supplied by multiplexing the flow of fluid across one or more of the parallel layers (e.g., using valves, nodes, or other fluid connections, etc.). In some implementations, the microfluidic channels can be multiplexed between different components (e.g., modules 200, 300, and 400, any other microfluidic features or components described herein, etc.) on a single layer. Using the pumps described above in conjunction with FIGS. 5A, 5B, and 5C herein, the flow of fluid can be moved through one or more of the parallel layers or through one or more of the laterally arranged layers. In some implementations, each microfluidic layer can have a unique pump that moves the flow of fluid based on the state of the particular layer (e.g., the state of the particular layer provided to a controller such as controller 1005 by a sensor, etc.).

[0049] It will be appreciated that sensors such as those described hereinabove, such as the sensors, can be incorporated into each microfluidic layer, disposed within the system 500 of FIGS. 5A, 5B, and 5C. Stated another way, some or all of the process flow system 500 described hereinabove can be embedded or formed as part of a single microfluidic layer. By replicating the layers in parallel or laterally, the process flow 500 can be scaled without affecting the processing capabilities of the system. Using the signals supplied to and from a controller device (e.g., the controller 1005 described later herein in conjunction with FIG. 10), the flow of fluid in each layer can be independently manipulated and monitored. Thus, the functions of the controller 1005 can be used to monitor and control the processing of the scaled process flow system 500 described herein.

[0050] The multi-layer process flow configuration has several significant advantages. One advantage of a microfluidic continuous flow system (e.g., system 500 described herein, etc.) from the perspective of shortening the process time and improving safety is that the intermediate product can be transferred to the next operation immediately after the process step is completed. This function can shorten the process cycle time and improve safety by avoiding cell damage caused by being unnecessarily exposed to high cell force multiple times. Therefore, the continuous flow process system of the continuous flow process method brings significant advantages to cell processing technology. However, there may be situations where it may be beneficial to run the process in batch mode (e.g., performing the process in one batch and moving the batch to the subsequent stage immediately after the entire batch is completed, etc.). If necessary, by temporarily placing the product of a certain step in a reservoir and then transferring it to a subsequent batch process, both continuous processing and batch processing can be adopted within a single system. In some implementations, such a reservoir for batch processing can be arranged between separate horizontally arranged layers that each define a stage in the batch processing part, or between horizontally arranged laminates consisting of parallel layers. That is, both the scaling of the continuous flow system and the batch processing system can be scaled using a parallel layer arrangement pattern and a horizontally arranged layer arrangement pattern.

[0051] A plurality of microfluidic devices (e.g., modules 200, 300, 400, any other microfluidics described herein, etc.) may be connected on a single layer via one or more microfluidic multiplexers. The microfluidic multiplexer can also be used in one layer to define multiple paths between networks of interconnected microfluidic channels, or can be used to define one or a portion or path of the process flow system 500 described above together with FIGS. 5A, 5B, and 5C herein. The microfluidic multiplexer can comprise one or more knot points, inlets, and outlets, and by using the microfluidic multiplexer, the path of fluid can be determined across all of one or more layers from a plurality of microfluidic channels.

[0052] Figure 6 shows a graph 600 that depicts the electric field experienced by the cells within module 400 of Figure 4. Generally, the electric field delivered to the cells can be affected by the relative conductivity of the lateral flow solution with respect to the central flow solution, and the relative flow rate of the lateral flow with respect to the central flow, where the relative flow rate of the lateral flow with respect to the central flow can determine the width of the lateral flow with respect to the central flow. The higher the conductivity ratio, the greater the voltage drop across the ends in the central flow, and the greater the magnitude of the electric field can be. The higher the flow rate ratio, the narrower the central flow can be made, shortening the distance over which the voltage drops across the ends of the cells, and increasing the magnitude of the electric field. Graph 600 shows the magnitude of the electric field experienced by the cells when the applied voltage is 65V in one implementation of an electroporation module having a channel width of 1.5 mm. The red dotted line in graph 600 indicates an exemplary flow rate ratio at which module 400 can operate, and the boxed frame indicates the range of electric field magnitudes that may be useful for delivering mRNA to primary human T cells. Graph 600 does not account for the effects of diffusion, which can potentially add spatial non-uniformity to the electric field. This can thereby form a framework for one possible feedforward control mechanism, in which the conductivity is measured and the flow rate is adjusted to obtain a desired electric field. Such a control mechanism will be described further later.

[0053] Figures 7A - 7D illustrate block diagrams of exemplary control systems for controlling the electric field experienced by cells within module 400 of FIG. 4. The magnitude of the electric field applied to the cells within module 400 can be an important parameter. The magnitude of the electric field can depend on the applied voltage, the ratio of the conductivity of the lateral flow to the conductivity of the central fluid flow containing the cells within module 400, and the width of the central fluid flow containing the cells, which can depend on the ratio of the flow rate of the lateral flow to the flow rate of the central flow. In some implementations, the applied voltage can be commanded by the user of module 400 and can be well controlled. The flow rate ratio can also be commanded by the user and can be well controlled if a feedback control mechanism using a flow sensor is implemented. However, in some implementations, the conductivity of the sample flow can vary depending on the sample preparation, the amount and type of cargo used, and the cell donor. As shown in FIGS. 7A - 7D, several different mechanisms can be used to control the electric field applied to the cells within module 400. Any of the control mechanisms illustrated in FIGS. 7A - 7D can be implemented by controller 1005, which will be described later in this specification in conjunction with FIG. 10.

[0054] Referring to FIG. 7A, control system 700 is illustrated. Control system 700 is a feed - forward control system that can measure the conductivity of the suspension of cells and cargo upstream of module 400. When using control system 700, the ratio of the flow rate of the lateral flow to the flow rate of the central flow can be adjusted in response to changes in conductivity to obtain a desired (e.g., predetermined) electric field. Referring to FIG. 7B, control system 720 is illustrated. Similar to control system 700 of FIG. 7A, control system 720 of FIG. 7B is also a feed - forward control system that can measure the conductivity of the suspension of cells and cargo upstream of module 400. When using control system 720, instead of the flow rates of the lateral flow and the central flow as illustrated for control system 700 of FIG. 7A, the applied voltage is adjusted to obtain a desired electric field.

[0055] Referring to FIG. 7C, a control system 740 is illustrated. The control system 740 is a feedback control system, rather than a feedforward control system as shown in FIGS. 7A and 7B. When using the control system 700, the applied electric field can be calculated using the width of the central flow, the conductivity of the solution, and the measured current within the module 400, and the flow rate ratio can be adjusted to reach a desired set point. FIG. 7D shows a control system 760 that similarly implements a feedback control system. When using the control system 760, the applied electric field is calculated using the width of the central flow, the conductivity of the solution, and the measured current within the module 400, and the applied voltage (rather than the flow rate ratio) is adjusted to reach a desired set point.

[0056] To account for changes in the magnitude of the electric field associated with changes in the conductivity of the sample, feedforward control or feedback control (or both) of the electric field can be implemented by the systems of FIGS. 7A-7D (e.g., using a controller 1005 as illustrated in FIG. 10). In some implementations, the conductivity can be measured upstream of the module 400. In a feedforward control system, this information can be used to adjust the flow rate ratio between the lateral flow and the central flow to narrow or widen the central flow containing the cells, or to adjust the applied voltage, or both. In some implementations, the width of the central flow can be directly measured using an optical sensor, and the current flowing through the module 400 can be measured while an electric field waveform is being applied to the cells. In some implementations, a conductivity sensor can be placed at the lateral outlet of the module 400, and the measured conductivity at the outlet can be used to estimate the width of the central flow. By combining this information with the measured value of the conductivity of the solution, the spatial average of the magnitude of the electric field delivered to the cells can be calculated.

[0057] In some implementation forms, the measured value of the electric field can be used for feedback control. For example, one or both of the lateral or central flow ratio or the applied voltage can be adjusted to approach the setpoint of the desired magnitude of the electric field. In some implementation forms, the sensor electrodes may be incorporated into the module 400. For example, such sensor electrodes may be implemented as thin film electrodes disposed on the floor of the channel. Using such sensor electrodes, the applied electric field can be directly measured. In some implementation forms, the feedback control mechanism and the feedforward control mechanism can be used in a tandem manner.

[0058] To demonstrate the functionality of the systems and methods of the present disclosure, a leukopak sample (leukapheresis product) was introduced into a system similar to system 500 shown in FIGS. 5A, 5B, and 5C, and processed to produce lymphocytes that transiently express a fluorescent reporter protein known as mCherry. In the microfluidic electroporation module (e.g., module 400 of FIG. 4), since cells and cargo may need to be suspended in a low-conductivity medium (e.g., a conductivity about one-twentieth that of the medium used for sheath flow), a module similar to module 300 was used to move the target cells into the electroporation medium by acoustic phoresis before performing electroporation. This reduction in conductivity cannot be achieved simply by diluting the starting cell sample in a low-conductivity electroporation medium because components in the blood product interfere with electroporation. As illustrated in graph 800 of FIG. 8, even in a commercial bulk electroporation device that does not explicitly require a low-conductivity medium, the direct dilution approach may require a sample-to-diluent ratio of at least 1:100 to enable electroporation. However, diluting above 1:100 may reduce the target cell density to 0.25 - 0.35 M cells / mL. At a flow rate of 1 mL / min, it takes several days to process 1 billion cells, and thus, in some cases, the processing capacity for patient samples for cell therapy may become unacceptable. Therefore, an active media exchange step of resuspending the cells in the electroporation medium may be helpful. Conventionally, this media exchange step is achieved by a batch process using centrifugation, which requires a significant amount of on-site labor. To address this technical challenge, the present disclosure provides module 300 shown in FIG. 3 for automatically and continuously performing the above exchange using acoustic phoresis.

[0059] Using the process flow and parameters of system 500 shown in FIGS. 5A, 5B, and 5C, a leukopak sample containing approximately 500 million primary human lymphocytes was processed. All components and tubing were sterilized using an autoclave or ethylene oxide and assembled in a biosafety cabinet for aseptic operation. Before introduction into system 500, the leukopak sample was centrifuged and the cells were resuspended in TexMACS to a 1:2 dilution to reduce the weight density of the cell solution, maintain an appropriate cell concentration, shorten the processing time, and reduce the amount of mRNA required. The target cells were transferred into an electroporation buffer containing mRNA encoding mCherry (32 μg / ml) using the rapid media exchange module 300 shown in FIG. 3, electroporated (three 250 μs pulses at 165 kV / m) using the microfluidic electroporation module 400 shown in FIG. 4, and finally concentrated for lymphocytes using the microfluidic acoustic afresis module 200 shown in FIG. 2. The entire process was completed in approximately 3.5 hours by an automated continuous flow. As illustrated in graph 900 of FIG. 9A, the transfection efficiency was greater than 75%, which was higher than the transfection efficiency of a control sample transfected using an electroporation process. Furthermore, as illustrated in graph 910 of FIG. 9B, there was no measurable decrease in viability relative to the input sample, and as illustrated in graph 920 of FIG. 9C, the lymphocyte parent population was concentrated from approximately 56% to approximately 76%.

[0060] Referring now to FIG. 10, a block diagram of an exemplary system 1000 for controlling the flow rate of a fluid flowing through a system similar to system 500 illustrated in FIGS. 5A, 5B, and 5C or the electric field received by this fluid is illustrated. System 1000 can be used to implement the control systems illustrated in FIGS. 7A, 7B, 7C, and 7D. System 1000 can include at least one controller 1005, one or more sensors 1040, one or more pumps 1050, and one or more electrical signal generators 1055. Controller 1005 can include at least one electric field identification device 1010, at least one sensor data receiving device 1015, at least one predicted electric field calculator 1020, at least one adjusted voltage calculator 1025, at least one adjusted flow rate calculator 1030, and at least one signal supply device 1035.

[0061] The components of system 1000 (e.g., controller 1005, sensor 1040, pump 1050, etc.) can be implemented respectively using hardware components or a combination of hardware components and software of a computer system (e.g., computer system 1200 detailed together with FIG. 12 herein, any other computer system described herein, etc.). The components of controller 1005 (e.g., electrical signal generator 1055, electric field identification device 1010, sensor data receiving device 1015, predicted electric field calculator 1020, adjusted voltage calculator 1025, adjusted flow rate calculator 1030, signal supply device 1035, etc.) can each implement any of the functions detailed herein. Controller 1005 or its components can perform any of the acts described above herein in connection with FIGS. 7A, 7B, 7C, and 7D.

[0062] The controller 1005 can include at least one processor and memory, such as a processing circuit. The memory can store processor-executable instructions that, when executed by the processor, cause the processor to perform one or more of the operations described herein. The processor can include a microprocessor, an application-specific integrated circuit (ASIC), a field-programmable gate array (FPGA), or the like, or a combination thereof. The memory can include, but is not limited to, an electronic, optical, magnetic, or any other storage device or transmission device capable of providing program instructions to the processor. The memory can further include a floppy disk, a CD-ROM, a DVD, a magnetic disk, a memory chip, an ASIC, an FPGA, a read-only memory (ROM), a random access memory (RAM), an electrically erasable and programmable ROM (EEPROM), an erasable and programmable ROM (EPROM), a flash memory, an optical medium, or any other suitable memory from which the processor can read instructions. The instructions can include code in any suitable computer programming language. The controller 1005 can include any or all of the above components and can perform any or all of the functions of the computer system 1200 described in conjunction with FIG. 12.

[0063] Sensor 1040 (which may also be referred to as "sensor 1040") may be one or more sensors capable of transmitting or receiving data from controller 1005 or components of controller 1005. The sensor may include, or be similar to, the through-flow conductivity sensor 535, flow sensor, optical sensor, or other types of sensors described herein, along with FIGS. 5A-5C. For example, sensor 1040 may be any of the sensors incorporated into system 500 or other similar flow systems to enable interrogation of the system 500 about malfunctions and feedback or feedforward control mechanisms (e.g., by components of controller 1005, etc.). For example, the sensor may be incorporated as a component of the overall system, or may be directly incorporated into modules 200, 300, and 400, or any other module, reservoir, valve, or flow structure described herein. Possible sensors that may be incorporated into system 500 or other similar flow systems include flow rate, conductivity probes, visual measurement of the width of the flow during sheath flow, and flow sensors for controlling current measurements. In some implementations, it is also possible to use an optical sensor to evaluate the quality of the sheath flow used in system 500, which can be used to adjust the flow rate as needed to generate an appropriate and stable flow. In some implementations, it is possible to use an optical sensor to calibrate and adjust the acoustophoresis module, and the optimal drive frequency for the piezoelectric component can be automatically determined by observing the concentration of cells included in one of the outlet fractions.

[0064] Sensor 1040 can be similarly incorporated into system 500 or other similar flow systems for estimating one or both of cell concentration or cell density in real time, and can include one or more absorbance or impedance sensors. This sensor can provide information about the processing capabilities of system 500 to the components of controller 1005, and can also be used to determine where losses may be occurring within system 500 or other similar flow systems when cell recovery is low. By using the information obtained from the measured cell concentration in closed-loop control, the flow rate at the inlets or outlets of modules 200, 300, and 400 can be adjusted, the acoustic power or acoustic frequency can be adjusted, or the automatic addition of reagents to system 500 or other similar flow systems can be adjusted.

[0065] By adding sensor 1040 into any of the modules described in this specification (such as modules 200, 300, and 400, etc.), or at a location between these modules within a fluid system (such as system 500, etc.), the operational quality or efficiency of the flow system and the modules within the flow system can be displayed. For example, by adding sensor 1040 to a separation module (such as module 200, etc.), or at a location downstream of the separation module within the system, the efficiency of acoustic phoretic separation of cells occurring within the separation module can be displayed or detected. Such a sensor can be configured to identify cells in the fluid while the fluid is passing through the separation module or after passing through the separation module. In some implementations, by adding the sensor to an electroporation module (such as module 400, etc.), or at a location downstream of the electroporation module, the efficiency of transfection resulting from the operation of the electroporation module can also be displayed. Such a sensor 1040 can measure how much cargo has been introduced into the cells of the fluid sample by the electroporation operation performed by module 400. In some implementations, by incorporating the sensor into system 500, the cell viability can be monitored. Sensor 1040 can include a conductivity probe that measures the conductivity of the fluid as the fluid flows through an electroporation device such as module 400. Sensor 1040 can include a current sensor that measures the current passing through the fluid as the fluid flows through an electroporation device such as module 400. Sensor 1040 can include an optical sensor that measures the width of the central portion of the fluid as the fluid flows through an electroporation device such as module 400.

[0066] Sensor 1040 can provide output in real time to controller 1005 or components within controller 1005 via one or more communication interfaces. Information received from sensor 1040 can be used as part of a feedback control mechanism or a feedforward control mechanism. For example, based on the output of such a sensor, adjustable parameters of any of modules 200, 300, and 400 (such as fluid flow rate, ratio of fluid samples, applied voltage, or electric field, etc.) can be controlled. Thus, real-time information regarding electrotransfer efficiency, separation efficiency, or cell viability can be incorporated into control data to modify the operating characteristics of system 500 or a similar fluid system during cell processing.

[0067] By means of pump 1050, fluid can flow through one or more pipes or channels within a fluid flow system such as system 500 described herein in connection with FIGS. 5A - 5C. Pump 1050 can include, for example, pumps 510, 525, 540, 550, 555, or 570 described above herein in connection with FIGS. 5A - 5C. Pump 1050 can be a peristaltic pump or any other type of pump described herein. By using pump 1050, fluid can be transported throughout a fluid system similar to system 500 illustrated in FIGS. 5A - 5C. Pump 1050 may be connected to one or more fluid connectors, fluid reservoirs, fluid capacitors, or other fluids. The rate at which pump 1050 transports fluid can be managed by a signal received from a controller such as controller 1005 (or its components). For example, a signal from controller 1005 can cause one or more of pump 1050 to flow fluid at a desired flow rate. The desired flow rate may be indicated by a signal received from controller 1005. The signal can be a voltage signal that operates a peristaltic pump or another type of signal that can adjust or control the rate at which one or more of pump 1050 can transport fluid.

[0068] The electrical signal generator 1055 may be part of the module 400 described above in this specification in connection with FIG. 4. For example, the electrical signal generator 1055 can generate a desired voltage between the electrodes 435A and 435B described above in this specification. The desired voltage can be indicated by a signal received from the controller 1005 (or a component of the controller 1005). The electrical signal generator 1055 may be a voltage source such as a direct current (DC) voltage source or an alternating current (AC) voltage source. In some implementations, the electrical signal generator 1055 may be a current source such as a DC source or an AC source. The electrical signal generator 1055 may be electrically connected to one or more electrodes such as the electrodes 435A and 435B. The electrical signal generator 1055 can create a voltage difference between one or more electrodes to form an electric field. Similar to the case of the module 400 in FIG. 4, when the electrodes are connected to one or more fluid channels, the electrical signal generator 1055 can generate an electric field in the fluid flowing through the module. In some implementations, the voltage applied by the electrical signal generator can take the form of a sine curve with a period ranging from 10 ns to 10 ms. In some implementations, the voltage applied by the electrical signal generator can take the form of a pulse train with a pulse width ranging from 10 ns to 10 ms. In some implementations, the magnitude of the voltage applied by the electrical signal generator 1055 can be varied to generate an electric field ranging from about 2 to 600 kV / m between the two ends of the central flow with a pulse width ranging from 10 ns to 10 ms. However, it should be understood that there can also be other types of electric field pulses, voltage magnitudes, and electric field magnitudes.

[0069] Here, the functions of the controller 1005 will be described. The electric field identification device 1010 can identify the magnitude of a desired electric field to be induced in a fluid flowing through an electroporation device. Identifying the desired electric field can include receiving the desired electric field from one or more external sources such as user input via an input interface, from a configuration file loaded into the memory of the controller 1005, or from internal settings (e.g., hardware settings such as jumpers). In some implementations, the desired electric field can be stored as a variable in one or more data structures included in the memory of the controller 1005. In a fluid system having more than one electroporation device (e.g., more than one module 400, etc.), the electric field identification device 1010 can identify the value of the magnitude of the desired electric field for each electroporation device. In such an implementation, the values of the magnitudes of the desired electromagnetic fields can each be stored in one or more data structures associated with the identifier of the electroporation device corresponding to the value of the magnitude of that electromagnetic field.

[0070] The sensor data receiving device 1015 can receive sensor values including the conductivity or flow rate of a fluid flowing into or through an electroporation device from the sensor 1040. The sensor data receiving device 1015 can receive, among other things, one or more signals from the sensor 1040 representing the conductivity of the fluid, the flow rate of the fluid, or numerical values of other sensor data. In some implementations, the sensor data receiving device 1015 can send a ping or query one or more of the sensors 1040 based on a predetermined criterion, in response to user input, based on a periodic schedule or another type of sensor interrogation procedure. In response to the query, the sensor can transmit or transfer sensor information including numerical values representing physical characteristics of the fluid (e.g., the width of the central channel, the current flowing through the fluid, the flow rate of the fluid in a pipe or channel, the flow rate through any of the modules 200, 300, or 400 described herein, etc.) or other characteristics of the fluid system (e.g., system 500, etc.) via one or more communication interfaces. The sensor information received by the sensor data receiving device 1015 from the sensor 1040 can be stored in one or more data structures included in the memory of the controller 1005. The sensor information can be stored together with various identifiers, such as, among other things, a timestamp corresponding to the time when the sensor measurement was acquired or received, or an identifier of the sensor 1040 that supplied the sensor measurement.

[0071] The predicted electric field calculator 1020 can determine the magnitude of the predicted electric field in a fluid when the fluid flows through an electroporation device. The predicted electric field can be calculated based on the conductivity of the fluid flow in the electroporation device and the voltage generated by the electrical signal generator. For example, using Ohm's law to calculate the electric field strength, the magnitude of the predicted electric field received by the fluid flowing through the electroporation device (e.g., module 400 described herein in connection with FIG. 4, etc.) can be determined. As shown in FIG. 4, module 400 can receive, as inputs, a central flow and two lateral flows. In a non-turbulent flow, the fluid received from each input channel into the central channel 430 does not mix, but instead, the flow shape corresponding to the flow when these fluids are input into module 400 can be maintained. The conductivity of each fluid may be different. For example, the cell solution in the electroporation buffer (e.g., the electroporation buffer in the central channel, etc.) may have a lower conductivity than the buffer solution supplied by the lateral channels.

[0072] When a voltage potential is applied to the central channel, different voltage drops may occur between both ends of the width of each fluid flow (e.g., the flow of the fluid received from the central inlet 410, the buffer solution received by the lateral inlets 415a and 415b, etc.), and thus, each fluid flow may receive a different electric field. The magnitude of the electric field can be calculated using the electric field equation

Equation

[0073] Thus, in some implementations, the calculation of the electric field, which can know the voltage supplied by the electrical signal generator 1055, will be based on the width of the central flow (or in some implementations, the width of one or more other channels) within the electroporation device. As described herein, the width of the central flow when flowing through the central channel 430 can be a function of the ratio of the flow rate of the lateral channels to the flow rate of the central flow. Thus, the width of the central flow can be estimated using the ratio of the flow rate of the lateral channels to the flow rate of the central flow. Together with the known voltage between the electrodes 415a and 415b, using the width of the central flow in the above equation, the magnitude of the expected electric field can be calculated. As described above herein, the flow rate of the lateral channels and the flow rate of the central flow can be received, for example, from one or more of the sensors 1040. In some implementations, the voltage drop experienced by the fluid in the central chamber can vary based on the relative conductivity of the fluid received from the lateral channels 415a and 415b and the fluid received from the central channel 420. If the conductivity of the fluid entering the central channel is known (e.g., when received from one or more of the sensors 1040), the voltage drop across the ends of the central flow can be calculated using the voltage division formula. Then, using the voltage drop together with the estimated width of the central flow in the above equation, the magnitude of the expected electric field experienced by the central flow within the module 400 can be calculated.

[0074] In some implementations, the predicted electric field calculator 1020 can determine the magnitude of the predicted electric field based on the conductivity of the fluid flowing through the electric field, the current generating the electric field, and the width of the central flow of the fluid. For example, the module 400 includes a central portion of the fluid introduced into the central channel 430 via the central inlet 410. In some implementations, one or more of the sensors 1040 (e.g., an optical sensor, etc.) can provide values corresponding to the width of the central flow, the conductivity of the central and lateral flows, and the current flowing through the central and lateral flows. By using these values, the predicted electric field strength can be calculated. For example, the voltage drop between the central flow and the lateral flow can be calculated by dividing the amount of current flowing through the flow by the value of the conductivity of each flow. When the value of the width received from the optical sensor is used in the above formula, the electric field strength received by the central flow when flowing through the module 400 can be calculated. The predicted electric field received by the central channel (or other fluid flowing through the module 400) can be stored in one or more data structures in the memory of the controller 1005.

[0075] The adjustment voltage calculator 1025 can calculate an adjustment voltage for the electrical signal generator based on the magnitude of the predicted electric field and the magnitude of the desired electric field. The adjustment voltage calculator 1025 can calculate the difference (e.g., by subtraction, etc.) between the magnitude of the electric field received by the fluid flowing through the module 400 and the magnitude of the desired electric field. In some implementations, the adjustment voltage calculator 1025 can calculate the percentage difference between the magnitude of the predicted electric field and the magnitude of the desired electric field. Since the electric field is proportional to the voltage drop across the ends of the central flow within the module 400, the adjustment voltage calculator 1025 can calculate the adjustment voltage by multiplying the percentage difference by the current voltage setting of the electrical signal generator 1055. For example, if the desired electric field strength is 200% of the predicted (e.g., estimated) electric field strength in the central flow, the adjustment voltage calculator 1025 can calculate the adjustment voltage as 2.00 × VC (where VC is the current voltage setting value of the electrical signal generator). In some implementations, the controller can adjust the voltage within a range of a set of operating conditions, such as the operating conditions illustrated in FIG. 6.

[0076] The adjustment flow calculator 1030 can calculate an adjusted flow rate for one or more fluid flows entering an electroporation device such as module 400 based on the value of conductivity, the current within the electroporation device, and the width of the central portion of the fluid. In some implementations, the adjustment flow calculator 1030 can calculate an adjusted flow rate for at least one of the first fluid or the second fluid based on at least one of the first flow rate or the second flow rate. The adjustment flow calculator 1030 can determine an adjusted flow rate for one or more of the central flow or the lateral flow received by the central channel 430 of the module 400. As described herein, the width of the central channel can be inversely proportional to the electric field strength received by the central channel. Further, the width of the central channel can be a function of the ratio of the flow rate of the lateral flow to the flow rate of the central flow. Thus, in some implementations, the adjustment flow calculator 1030 can calculate an adjusted flow rate for the lateral flow by increasing or decreasing the flow rate of the lateral flow to change the width of the central flow. For example, if the magnitude of the predicted electric field is less than the magnitude of the desired electric field, the adjustment flow calculator 1030 can increase the flow rate of the lateral flow. In some implementations, the adjustment flow calculator 1030 can decrease the flow rate of the central flow to increase the magnitude of the predicted electric field. Similarly, if the magnitude of the predicted electric field is greater than the magnitude of the desired electric field, the adjustment flow calculator 1030 can decrease the flow rate of the lateral flow. In some implementations, the adjustment flow calculator 1030 can increase the flow rate of the central flow to decrease the predicted electric field strength.

[0077] In some implementation forms, the adjustment flow calculator 1030 can adjust both the flow rate of the central flow and the flow rate of the lateral flow (for example, decreasing the flow rate of the central flow to increase the flow rate of the lateral flow, increasing the flow rate of the central flow to decrease the flow rate of the lateral flow, increasing both the flow rate of the central flow and the flow rate of the lateral flow, decreasing both the flow rate of the central flow and the flow rate of the lateral flow, etc.). The adjustment value of the flow rate calculated by the adjustment flow calculator 1030 can be stored, for example, in one or more data structures in the memory of the controller 1005. In some implementation forms, each adjusted flow rate value can be stored in a state associated with the identifier of the pump that controls the adjusted flow rate. It should be understood that the value of the flow rate adjusted by the adjustment flow calculator 1030 corresponds to the value stored in the memory of the controller 1005, which is used to determine the speed or frequency at which the pump 1050 transfers fluid within a fluid system (such as system 500, etc.).

[0078] The signal supply device 1035 can supply one or more signals representing an adjustment voltage to the electrical signal generator 1055. Due to the signal, the electrical signal generator 1055 can generate a voltage within the electroporation device. For example, the signal can include an instruction to increase or decrease the magnitude of the signal or electrical signal generated by the electrical signal generator. In some implementation forms, the signal can be an analog signal representing a value proportional to the value of the adjustment voltage calculated by the adjustment voltage calculator 1025. In some implementation forms, the signal can be a digital signal representing a value proportional to the value of the adjustment voltage calculated by the adjustment voltage calculator 1025. Since the adjustment value is calculated by the adjustment voltage calculator 1025 or the adjustment flow calculator 1030, the signal generated and transmitted by the signal supply device 1035 can be transmitted in real time. In some implementation forms, the signal supply device 1035 can control the frequency at which the electrical signal generator 1055 generates electrical pulses via the central channel 430 of the module 400.

[0079] The signal supply device 1035 supplies a second signal representing an adjusted flow rate to a pump that controls the flow of a first fluid (e.g., a fluid in a lateral flow, etc.) or a second fluid (e.g., a fluid in a central flow), so that the first fluid or the second fluid can flow at the second adjusted flow rate. For example, the signal supply device 1035 can access the memory of the controller 1005 to search for the values of the adjusted flow rates of the lateral flow and the central flow respectively. In some implementations, when the value of the adjusted flow rate is generated by the adjusted flow rate calculator 1030, the signal supply device 1035 can search for the value of the adjusted flow rate. The signal supply device 1035 can send the signal to one or more pumps 1050 to operate the pumps 1050 and transfer the fluid through the central channel 430 of the module 400 at the adjusted flow rate calculated by the adjusted flow rate calculator 1030. In some implementations, the signal may be an analog signal representing a value proportional to the value of the adjusted flow rate (e.g., the value of the adjusted flow rate of one or more of the lateral flow or the central flow, etc.) calculated by the adjusted flow rate calculator 1030. In some implementations, the signal may be a digital signal representing a value proportional to the value of the adjusted flow rate calculated by the adjusted flow rate calculator 1030. In some implementations, the signal supply device 1035 can control the pump 1050 (e.g., send a signal for periodically operating the pump 1050, etc.). In such an implementation, the frequency at which the signal supply device 1035 sends the signal to the pump 1050 to operate the pump 1050 may be based on the value of the adjusted flow rate.

[0080] Referring now to FIG. 11, there is illustrated a flow diagram of an exemplary method 1100 for controlling the flow rate of a fluid flowing through a system similar to the system illustrated in FIGS. 5A, 5B, and 5C or the electric field received by this fluid. This method can be implemented, for example, by a controller device (e.g., controller 1005, computer system 1200, etc.). To explain the overview of method 1100, method 1100 can include identifying the magnitude of a desired electric field (block 1102), receiving the conductivity of the fluid flow through an electroporation device (e.g., module 400, etc.) (block 1104), determining the magnitude of the predicted electric field in the fluid flowing through the electroporation device (block 1106), calculating an adjustment voltage for an electrical signal generator (block 1108), and supplying a signal representing the adjustment voltage to the electrical signal generator (block 1110).

[0081] To describe method 1100 in more detail, method 1100 can include identifying the magnitude of a desired electric field (block 1102). Identifying the desired electric field can include receiving the desired electric field from one or more external sources such as user input via an input interface, from a configuration file loaded into the memory of the controller, or from internal settings (e.g., hardware settings such as jumpers, etc.). In some implementations, the desired electric field can be stored as a variable in one or more data structures within the memory of the controller. In a fluid system having more than one electroporation device (e.g., more than one module 400, etc.), the controller can identify the value of the magnitude of the desired electric field for each electroporation device. In such an implementation, the values of the magnitudes of the desired electromagnetic fields can each be stored in one or more data structures associated with the identifier of the electroporation device to which the value of the magnitude of that electromagnetic field corresponds.

[0082] Method 1100 can include receiving (block 1104) the conductivity of a fluid flow through an electroporation device (such as module 400, etc.). The controller can receive, among other things, one or more signals from sensors (such as sensor 1040, etc.) representing numerical values of the conductivity of the fluid, the flow rate of the fluid, or other sensor data. In some implementations, the controller can send a ping or query one or more of the sensors according to a predetermined criterion, in response to user input, based on a periodic schedule or another type of sensor interrogation procedure. In response to the query, the sensor can transmit or transfer sensor information including numerical values representing physical characteristics of the fluid (such as the width of the central channel, the current flowing through the fluid, the flow rate of the fluid in a pipe or channel, the flow rate through any of modules 200, 300, or 400 described herein, etc.) or other characteristics of the fluid system (such as system 500, etc.) via one or more communication interfaces. The sensor information received by the controller from the sensor can be stored in one or more data structures within the controller's memory. The sensor information can be stored together with various identifiers, such as, among other things, a timestamp corresponding to the time when the sensor measurement was obtained or received, or an identifier of the sensor that supplied the sensor measurement.

[0083] Method 1100 can include determining the magnitude of an expected electric field in a fluid flowing through an electroporation device (block 1106). The expected electric field can be calculated based on the conductivity of the fluid flow within the electroporation device and the voltage generated by an electrical signal generator. For example, Ohm's law can be used to calculate the electric field strength to determine the magnitude of the expected electric field experienced by the fluid flowing through an electroporation device (e.g., module 400 described herein in connection with FIG. 4, etc.). As shown in FIG. 4, module 400 can receive, as inputs, a central flow and two lateral flows. In a non-turbulent flow, the fluid received from each input channel into the central channel 430 does not mix, but instead can maintain the flow shape corresponding to the flow when these fluids are introduced into module 400. The conductivity of each fluid can be different. For example, the cell solution in an electroporation buffer (e.g., an electroporation buffer within the central channel, etc.) can have a lower conductivity than the buffer solution supplied by the lateral channels.

[0084] When a voltage potential is applied to the central channel, different voltage drops can occur across the ends of the width of each fluid flow (e.g., the flow of fluid received from the central inlet 410, the buffer solution received by the lateral inlets 415a and 415b, etc.), and thus each fluid flow can be subject to a different electric field. The magnitude of the electric field can be calculated using the electric field equation

Equation

[0085] Thus, in some implementations, the voltage supplied by an electrical signal generator (e.g., electrical signal generator 1055, etc.) can be known, and the calculation of the electric field will be based on the width of the central flow (or in some implementations, the width of one or more other channels) within the electroporation device. As described herein, the width of the central flow when flowing through the central channel 430 can be a function of the ratio of the flow rate of the lateral channels to the flow rate of the central flow. Thus, the width of the central flow can be estimated using the ratio of the flow rate of the lateral channels to the flow rate of the central flow. Together with the known voltage between the electrodes 415a and 415b, using the width of the central flow in the above equation, the magnitude of the expected electric field can be calculated. As described above herein, the flow rate of the lateral channels and the flow rate of the central flow can be received, for example, from one or more sensors communicating with the controller. In some implementations, the voltage drop received by the fluid within the central chamber can vary based on the relative conductivity of the fluid received from the lateral channels 415a and 415b and the fluid received from the central channel 420. If the conductivity of the fluid entering the central channel is known (e.g., when received from one or more of the sensors 1040), the voltage drop across the ends of the central flow can be calculated using the voltage division formula. Then, together with the estimated width of the central flow, using the voltage drop in the above equation, the magnitude of the expected electric field received by the central flow within the module 400 can be calculated.

[0086] In some implementations, the controller can determine the magnitude of the expected electric field based on the conductivity of the fluid flowing through the electric field, the current generating the electric field, and the width of the central flow of the fluid. For example, module 400 includes a central portion of the fluid introduced into central channel 430 via central inlet 410. In some implementations, one or more sensors (e.g., an optical sensor, etc.) can provide values corresponding to the width of the central flow, the conductivity of the central and lateral flows, and the current flowing through the central and lateral flows. By using these values, the expected electric field strength can be calculated. For example, the voltage drop across the two ends of the central flow and the voltage drop across the two ends of the lateral flow can be calculated by dividing the amount of current flowing through the flow by the value of the conductivity of each flow. When the value of the width received from the optical sensor is used together with the above formula, the electric field strength received by the central flow when flowing through module 400 can be calculated. The expected electric field received by the central channel (or other fluid flowing through module 400) can be stored in one or more data structures in the memory of the controller.

[0087] Method 1100 can include calculating an adjustment voltage for an electrical signal generator (block 1108). The controller can calculate the adjustment voltage for the electrical signal generator based on the magnitude of the predicted electric field and the magnitude of the desired electric field. The controller can calculate the difference (e.g., by subtraction, etc.) between the magnitude of the electric field experienced by the fluid flowing through module 400 and the magnitude of the desired electric field. In some implementations, the controller can calculate the percentage difference between the magnitude of the predicted electric field and the magnitude of the desired electric field. Since the electric field is proportional to the voltage drop across the ends of the central flow within module 400, the controller can calculate the adjustment voltage by multiplying the percentage difference by the current voltage setting of the controller. For example, if the desired electric field strength is 200% of the predicted (e.g., estimated) electric field strength in the central flow, the controller can calculate the adjustment voltage as 2.00 × VC, where VC is the current voltage setting of the electrical signal generator. In some implementations, the controller can adjust the voltage within a range of a set of operating conditions, such as the operating conditions illustrated in FIG. 6.

[0088] The controller can calculate an adjusted flow rate for one or more fluid flows entering an electroporation device, such as module 400, based on the value of the conductivity, the current within the electroporation device, and the width of the central portion of the fluid. In some implementations, the controller can calculate an adjusted flow rate for at least one of the first fluid or the second fluid based on at least one of the first flow rate or the second flow rate. The controller can determine an adjusted flow rate for one or more of the central flow or the lateral flow received by the central channel 430 of module 400. As described herein, the width of the central channel can be inversely proportional to the electric field strength received by the central channel. Additionally, the width of the central channel can be a function of the ratio of the flow rate of the lateral flow to the flow rate of the central flow. Thus, in some implementations, the controller can calculate an adjusted flow rate for the lateral flow by increasing or decreasing the flow rate of the lateral flow to change the width of the central flow. For example, if the magnitude of the predicted electric field is less than the magnitude of the desired electric field, the controller can increase the flow rate of the lateral flow. In some implementations, the controller can decrease the flow rate of the central flow to increase the magnitude of the predicted electric field. Similarly, if the magnitude of the predicted electric field is greater than the magnitude of the desired electric field, the controller can decrease the flow rate of the lateral flow. In some implementations, the controller can increase the flow rate of the central flow to decrease the predicted electric field strength.

[0089] Method 1100 can include supplying one or more signals representing an adjustment voltage to an electrical signal generator (block 1110). By means of the signal, the electrical signal generator (e.g., electrical signal generator 1055, etc.) can generate a voltage within the electroporation device. For example, the signal can include an instruction for increasing or decreasing the magnitude of a signal generated by the electrical signal generator or an electrical signal. In some implementations, the signal can be an analog signal representing a value proportional to the value of the adjustment voltage calculated by the controller at block 1108. In some implementations, the signal can be a digital signal representing a value proportional to the value of the adjustment voltage. In some implementations, the controller can control the frequency at which the electrical signal generator generates electrical pulses via the central channel 430 of module 400.

[0090] In some implementations, the controller can supply a second signal representing an adjustment flow rate to a pump that controls the flow of a first fluid (e.g., a fluid in a lateral flow, etc.) or a second fluid (e.g., a fluid in a central flow) to flow the first fluid or the second fluid at a second adjustment flow rate. When the value of the adjustment flow rate is generated at block 1108, the controller can obtain the value of the adjustment flow rate. The controller can send the signal to one or more pumps (e.g., pump 1050, etc.) to operate the pumps and transfer the fluid through the central channel 430 of module 400 at the adjustment flow rate. In some implementations, the signal can be an analog signal representing a value proportional to the value of the adjustment flow rate (e.g., the value of the adjustment flow rate of one or more of the lateral flow or the central flow, etc.). In some implementations, the signal can be a digital signal representing a value proportional to the value of the adjustment flow rate. In some implementations, the controller can control the pump (e.g., by sending a signal to periodically operate the pump, etc.). In such an implementation, the frequency at which the controller sends the signal to the pump can be based on the value of the adjustment flow rate.

[0091] FIG. 12 shows a schematic architecture of an exemplary computer system 1200 that may be employed to implement any of the computer systems described herein according to some implementations. The computer system 1200 can be used within a control system similar to the system 1000 described herein in conjunction with FIG. 10. The computer system 1200 is capable of controlling one or more other devices 1230, and the one or more other devices 1230 can include one or more pumps (e.g., pump 1050, any other pump described herein, etc.), one or more electrical signal generators (e.g., electrical signal generator 1055, any other electrical signal generator described herein, etc.), or any other type of device or system that can be controlled using one or more signals. The computer system 1200 of FIG. 12 includes one or more processors 1220 communicatively connected to a memory 1225, one or more communication interfaces 1205, and one or more output devices 1210 (e.g., one or more display units) and one or more input devices 1215. The processor 1220 can be included in any of the computer devices described herein.

[0092] In the computer system 1200 of FIG. 12, the memory 1225 can comprise any computer-readable storage medium and can store computer instructions such as processor-executable instructions for implementing the various functions of each system described herein, any data related to each system, any data generated by each system, or any data received (if any) via a communication interface or an input device. Referring again to the system 1200 of FIG. 12, the computer system 1200 can comprise, among other things, a memory 1225 for storing any of the information, variables, vectors, data structures or other computer-readable information described herein. Using the processor 1220 illustrated in FIG. 12, it is possible to execute the instructions stored in the memory 1225 and, by doing so, also to read from or write to the memory various information processed and / or generated by the execution of the instructions.

[0093] The processor 1220 of the computer system 1200 shown in FIG. 12 may also be communicatively connected to, or may control, a communication interface 1205 for transmitting or receiving various information in response to the execution of instructions. For example, the communication interface 1205 may be connected to a wired or wireless network, a bus, or other communication means, and thus the computer system 1200 may be enabled to transmit information to, or receive information from, other devices (e.g., other computer systems). Although not shown explicitly in the system of FIG. 12, one or more communication interfaces facilitate the flow of information between the components of the system 1200. In some implementations, the communication interface can be configured (e.g., by various hardware components or software components) to provide one or more interfaces (e.g., an application interface, a command-line interface, a website interface, etc.) as an access portal to at least some aspects of the computer system 1200. Examples of the communication interface 1205 include a user interface (e.g., a web page), a network interface, a network port, a command-line protocol, or any other type of communication interface that enables communication between a user and the computer system 1200.

[0094] The communication interface 1205 can comprise one or more sensor interfaces for transmitting and receiving information from any of the sensors described herein, including the sensor 1040 described herein in conjunction with FIG. 10. The communication interface 1205 can transmit or receive one or more signals for controlling parameters of other systems described herein, including system 500 or system 1000. Some exemplary parameters controllable by the computer system 1200 include the flow rate between one or more fluid nodes (e.g., controllable by sending a signal to one or more pumps to change the fluid flow rate), or the voltage generated by an electrical signal generator (e.g., electrical signal generator 1055, etc.) or any other controllable device described herein (e.g., a valve, etc.).

[0095] The output device 1210 of the computer system 1200 illustrated in FIG. 12 may be provided to, for example, observe or perceive various information in association with the execution of instructions. The input device 1215 may be provided to, for example, enable a user to manually adjust, make selections, input data, or interact with the processor during the execution of instructions in any of a variety of ways. Additional information regarding the schematic architecture of a computer system that can be employed in the various systems described herein is also provided herein.

[0096] Some implementations of the subject matter and operations described in this specification can be implemented in digital electronic circuitry, or in computer software embodied in a tangible medium, firmware, or hardware that includes the structures disclosed in this specification and their structural equivalents, or in a combination of one or more of them. Implementations of the subject matter described in this specification can be implemented as one or more computer programs, e.g., as one or more components of one or more computer program instructions, encoded on a computer storage medium for execution by, or to control the operation of, a data processing apparatus. The program instructions can be encoded in an artificially generated propagated signal, e.g., a machine-generated electrical, optical, or electromagnetic signal generated to encode information for transmission to an appropriate receiver-type device for execution by a data processing apparatus. The computer storage medium can be a computer-readable storage device, a computer-readable storage substrate, a random access or serial access memory array or device, or a combination of one or more of them, or incorporated in them. Further, the computer storage medium is not a propagated signal, but the computer storage medium can include the source or destination of computer program instructions encoded in an artificially generated propagated signal. The computer storage medium can be one or more separate physical components or media (e.g., multiple CDs, disks, or other storage devices, any other storage medium described in this specification, etc.), or incorporated in them.

[0097] In the drawings, the operations are illustrated in a particular order, but such operations need not be performed in the particular order or sequence shown, nor all illustrated operations need to be performed. The acts described in this specification can be performed in a different order.

[0098] The fact that the components of various systems are separated does not require them to be separated in all implementation forms, and the components of the described program can be incorporated into a single hardware product or software product.

[0099] Although several exemplary implementation forms have been described so far, the above are exemplary and not restrictive, and it is clear that they are given as examples. In particular, many of the examples presented in this specification include specific combinations of method acts or system elements, but these method acts and system elements can also be combined in other ways to achieve the same purpose. The acts, elements, and features discussed in relation to one implementation form are not intended to be excluded from similar roles in other multiple implementation forms.

[0100] The expressions and terms used in this specification are for the purpose of explanation and should not be regarded as restrictive. The use of "including", "comprising", "having", "containing", "involving", "characterized by" and their variants means including the things listed after these, their equivalents and further things, as well as alternative implementation forms consisting only of the things listed after these. In one implementation form, the systems and methods described in this specification consist of one, each, or all of the described elements, acts, or components.

[0101] As used in this specification, the terms "about" and "substantially" are understood by those skilled in the art and there are certain differences depending on the context in which these terms are used. If there is a use of a term that is not clear to those skilled in the art even considering the context in which the term is used, "about" means up to ±10% of that specific term.

[0102] Any reference in this specification to an implementation, element, or act of a system or method in the singular may include implementations that include a plurality of such elements, and any reference to any implementation, element, or act in the plural in this specification may include implementations that include only a single element. References in the singular or plural form are not intended to limit the systems or methods, their components, acts, or elements disclosed herein to a single configuration or a plurality of configurations. Any reference to an act or element being based on any information, act, or element may include implementations in which the act or element is based at least in part on any information, act, or element.

[0103] The implementations disclosed in this specification may also be combined with any other implementation or embodiment, and references to "an implementation", "some implementations", or "one implementation", etc. are not necessarily mutually exclusive, and are intended to indicate that the specific features, structures, or characteristics described in connection with that implementation may be included in at least one implementation or embodiment. Such terms as used in this specification do not necessarily all refer to the same implementation. Any implementation may be combined with any other implementation, comprehensively or exclusively, in any manner consistent with the aspects and implementations disclosed herein.

[0104] The indefinite article "a" or "an" as used in this specification and the claims should be understood to mean "at least one" unless explicitly stated otherwise.

[0105] References to "or" can be construed in an inclusive sense such that any term described using "or" can indicate any of the described single term, more than one of the described terms, and all of the described terms. For example, a reference to "at least one" of "A" and "B" can include a reference to only "A", only "B", and both "A" and "B". Such references used in conjunction with "comprising" or other open terms can include additional things.

[0106] If there are reference signs after the technical features included in the drawings, the detailed description or any claims, the reference signs are included to enhance the understanding of the drawings, the detailed description and the claims. Therefore, the scope of any element of a patent claim is never limited by the presence or absence of reference signs.

[0107] The systems and methods described herein can be embodied in other specific forms without departing from the features of these systems and methods. The above implementations are illustrative rather than limiting the described systems and methods. Therefore, the scope of the systems and methods described herein is indicated by the appended claims rather than the above description, and modifications that are equivalent in meaning and scope to the appended claims are also included in the appended claims.

Claims

1. An inlet channel that receives the flow of a target fluid containing target particles, An acoustic levitation device that receives the flow of the target fluid containing the target particles from the inlet channel and moves the target particles from the flow of the target fluid to the flow of a buffer fluid containing cargo particles, An electroporation device that is downstream of the acoustic levitation device, receives the flow of the buffer fluid containing the target particles and the cargo particles, applies an electric field to the flow of the buffer fluid, and causes a part of the cargo particles to be absorbed by a part of the target particles in the flow of the buffer fluid, A conductivity sensor connected to the inlet of the electroporation device, configured to measure the conductivity of the flow of the buffer liquid received by the electroporation device, and supply a signal indicating the conductivity to a controller device, An outlet channel that supplies the flow of the output buffer fluid from the electroporation device A system comprising.

2. The system according to claim 1, wherein the acoustic levitation device A central channel that receives the flow of the buffer fluid containing the cargo particles from a buffer fluid supply source and also receives the flow of the target fluid from the inlet channel, A piezoelectric element connected to the central channel that moves the target particles from the flow of the target fluid to the flow of the buffer fluid in a second channel A system comprising.

3. The system according to claim 1, wherein the electroporation device A central channel that receives the flow of the buffer fluid exiting the acoustic levitation device and the flow of the conductive buffer exiting the second channel, An electrode that is electrically connected to a part of the central channel and supplies an electric current A system comprising.

4. The system according to claim 1, further comprising that the target particles in the flow of the input fluid are lymphocytes, and the flow of the input fluid contains unwanted particles including at least one of red blood cells, granulocytes or monocytes.

5. The system according to claim 1, further comprising A second inlet channel that receives the flow of the input fluid containing the target particles and unwanted particles An acoustic separation device that receives the flow of the input fluid containing the target particles and the unwanted particles from the second inlet channel and separates the flow of the input fluid into a flow of a target fluid containing the target particles and a flow of an unwanted fluid containing the unwanted particles A system comprising the same. **Claim 6** The system according to claim 5, wherein the flow of the unwanted fluid is transported to a waste reservoir via one or more channels, and the flow of the target fluid exiting the acoustic separation device is transported into a target reservoir via a second channel, The system further comprises a pump that transports the flow of the target fluid from the target reservoir to the acoustic migration device via the inlet channel. **Claim 7** The system according to claim 1, further comprising a first pump that transports the flow of the target fluid from an input storage to the acoustic migration device via the inlet channel, and a second pump that transports the flow of the buffer fluid containing the target particles and the cargo particles exiting the acoustic migration device to the electroporation device via an intermediate channel A system comprising the same. **Claim 8** The system according to claim 1, comprising one or more temporary storage reservoirs between two or more of the inlet channel, the acoustic migration device, the electroporation device, or the outlet channel. **Claim 9** The system according to claim 1, further comprising a separation device that receives the flow of the output buffer fluid from the output channel and separates concentrated target particles in the flow of the output buffer fluid from unwanted particles in the flow of the output buffer fluid, and an output storage that receives the concentrated target particles from the separation device A system comprising the same. **Claim 10** The system according to claim 1, wherein at least the connection between the inlet channel, the acoustic migration device, the electroporation device, or the outlet channel comprises at least one of a polyvinyl chloride tube or a silicone tube. **Claim 11** The system according to claim 10, further comprising one or more fluid capacitors connected to at least one of the connections, the fluid capacitors being configured to regulate the flow rate of the fluid within the system. **Claim 12** The system according to claim 1, further comprising one or more sensors configured to send a signal representing a value of the density of the target particles or unwanted particles in at least one of the flow of the target fluid, the flow of the buffer fluid, or the flow of the output buffer fluid to a controller device.

13. The system according to claim 1, further comprising one or more flow sensors configured to send a signal representing the flow rate or conductivity of a fluid flowing through at least one of the inlet channel, the acoustophoresis device, the electroporation device, or the outlet channel to a controller device.

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