WO2024220694A2

WO2024220694A2 - System, devices and protocols for automated transfection

Info

Publication number: WO2024220694A2
Application number: PCT/US2024/025227
Authority: WO
Inventors: Bethany Grant; Rameech MCCORMACK; Ross BEIGHLEY; Sasha SMILJANIC; Ezekiel Parnow; Andy Ziegler
Original assignee: Kytopen Corp
Current assignee: Kytopen Corp
Priority date: 2023-04-19
Filing date: 2024-04-18
Publication date: 2024-10-24
Anticipated expiration: 2025-10-19
Also published as: WO2024220694A3

Abstract

Devices, systems, and kits for automated transfection are provided. Systems include flow cells with a first electrode, a second electrode, and an active zone therebetween and electrical discharge manifolds that integrate the flow cells into robotic systems and workflows. Software for such systems is also described.

Description

SYSTEM, DEVICES AND PROTOCOLS FOR AUTOMATED TRANSFECTION

BACKGROUND OF THE INVENTION

Immunotherapy is currently at the cutting edge of both basic scientific research and pharmaceutically driven clinical application. This trend is in part due to the recent strides in targeted gene modification and the expanded use of CRISPR/Cas complex editing for therapeutic development. In order to identify genetic modifications of therapeutic interest, research organizations often have to screen thousands of genetic variants, which can include modification of an endogenous gene or insertion of an engineered gene. This drug discovery process is laborious, typically requiring significant manual labor within the laboratory, creating an industry-wide bottleneck due to the lack of adequate high-throughput technologies.

Current high-throughput gene transfer methods typically require the use of viral delivery (e.g., lentiviral vectors), in which viral particles infect a cell and transduce the genetic modification of interest. While a viral methodology can be applied to high-throughput automated systems, there are limitations in the production that extend timelines for research efforts: viral vectors have to be cloned, transfected into a viral production line, and then viral particles must be purified. This process can take research organizations months, significantly affecting their timelines for platform development while simultaneously increasing the cost of drug discovery. Additionally, the use of viral transduction for gene transfer presents risks of immunogenicity and cytotoxicity and is not amenable to genetic modification for all cell types, since some cells (such as specific immune cell subsets) are resistant to viral infection. Therefore, within the biotechnology industry there is an unmet need to have a high-throughput automated system for gene transfer that does not rely on viral delivery mechanisms.

Definitions

Where values are described as ranges, it will be understood that such disclosure includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated.

The term “about,” as used herein, refers to ±10% of a recited value.

The term “average flow velocity,” as used herein refers to a velocity of a flowing liquid (e.g., in a channel or lumen of a flow cell electrode or active zone) determined from the quotient of the volumetric flow rate (Q, units of m³/s) of the liquid (e.g., from a fluid delivery source, e.g., a pump) divided by the cross-sectional area (A, units of m²), e.g., of the channel or lumen in which the liquid flows, thus the average flow velocity (u) has units of m/s.

The term “plurality,” as used herein, refers to more than one.

The term “conductivity,” as used herein, refers to electrical conductivity, i.e., the ability of electrically charged particles (e.g., ions) to move through a medium, e.g., ions of a salt in the flowing liquid, e.g., buffer ions.

The term “substantially uniform,” as used herein, refers to +/- 5% variance. The term “cross-sectional area,” unless otherwise specified, refers to the transverse cross- sectional area (e.g., along the plane perpendicular to the longitudinal axis or direction of flow).

The term “cross-section,” unless otherwise specified, refers to the transverse cross-section (e.g., along the plane perpendicular to the longitudinal axis or direction of flow).

The term “fluidically connected,” as used herein, refers to a direct connection between at least two device elements, e.g., an electro-mechanical device, a reservoir, etc., that allows for fluid to move between such device elements without passing through an intervening element.

The term “fluidic communication,” as used herein, refers to an indirect connection between at least two device elements, e.g., an active zone, a reservoir, etc., that allows for fluid to move between such device elements, e.g., through an intervening element, (e.g., through intervening tubing, an intervening channel, etc.).

The term “entry zone,” as used herein, comprises a portion of the devices of the invention through which a fluid and a plurality of cells suspended in the fluid may pass prior to electro-mechanical transfection in the active zone. An entry zone may further comprise an additional reservoir in fluidic communication with the active zone of the devices of the invention. When an electric potential difference is applied to a first and second electrode of the devices of the invention, the electric field that may be generated within an entry zone of the devices of the invention is not high enough to cause cell poration to occur.

The term “recovery zone,” as used herein, comprises a portion of the devices of the invention through which a fluid and a plurality of cells suspended in the fluid may pass or reside after electromechanical transfection in the active zone. A recovery zone may include a portion (e.g., a lumen, tube, channel, reservoir, etc.) of the device downstream of the active zone (e.g., immediately downstream, e.g., proximal to the second outlet). A recovery zone may further comprise an additional reservoir in fluidic communication with the active zone.

The term “active zone,” as used herein, refers to a portion of a device that is disposed between first and second electrodes, and in fluidic communication with, and downstream of, the entry zone (e.g., downstream of a first outlet). The electric field is delivered to the fluid in the active zone.

The term “transfection,” as used herein, refers to a process by which payloads can be introduced into cells utilizing means other than viral delivery methods (which are referred to specifically as “transduction”), such as biological, chemical, electrical, mechanical, or physical methods.

The term “electroporation,” as used herein, refers to a process utilizing applied electric fields to reversibly create small pores in cell membranes through which payloads can be introduced into cells (e.g., as a method of transfection).

The term “electro-mechanical transfection,” as used herein, refers to a transfection process by which payloads can be introduced to cells utilizing a combination of an applied electric field and a mechanical mechanism. This delivery method has the potential to decrease and/or stabilize the overall electric field exposure of the cells in the active zone, thereby enhancing cell viability and/or transfection efficiency, or both. The devices of the invention are configured to transfect cells via electro-mechanical transfection rather than by electroporation alone. Methods of the invention allow the optimum combination of electrical energy (e.g., electric field strength) and mechanical energy (e.g., flow rate) to be determined for a given cell type.

The term “transfection profile,” refers to a combination of parameters applied during a transfection process (e.g., an electro-mechanical transfection process) in a device or system of the invention. A transfection profile may include: a flow rate; a voltage range; a duty cycle; a frequency; a voltage waveform; etc.

The term “payload,” refers to a material to be transfected into cells, e.g., a genetic payload. Exemplary payloads are charged molecules, uncharged, molecules, DNA, RNA, CRISPR-Cas9, proteins, polymers, ribonucleoprotein (RNP), Dextran, etc. Exemplary payloads are those described in US Publication No. 2020-0131500 A1 , US Publication No. 2022-0243164 A1 , and US Publication No. 2022- 0347468 A1 , which are incorporated by reference. Cells to be transfected may include eukaryotic cells, plant cells, prokaryotic cells, or synthetic cells. Exemplary cells for transfection with the devices, systems, and method described herein include primary cells, cells from a cell line, adherent cells, unstimulated cells, stimulated cells, activated cells, stem cells, blood cells, Chinese hamster ovary (CHO) cells, immune cells (e.g., adaptive immune cells and/or innate immune cells), red blood cells, or peripheral blood mononuclear cells (PBMCs). Cells may be antigen presenting cells (APCs), monocytes, T-cells, B- cells, dendritic cells, macrophages, neutrophils, natural killer (NK) cells, Jurkat cells, THP-1 cells, human embryonic kidney (HEK-293) cells, embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), or hematopoietic stem cells (HSCs), primary human NK cells, primary human induced pluripotent stem cells (iPSCs), primary human macrophages, or primary human monocytes. Exemplary cells for transfection are those described in US Publication No. 2020-0131500 A1 , US Publication No. 2022-0243164 A1 , and US Publication No. 2022-0347468 A1 , which are incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a flow diagram for a process achieved using a system of the invention.

FIGs. 2A-2B show embodiments of an electrical discharge manifold (EDM) of the invention in the open (FIG. 2A) and closed (FIG. 2B) states with a flow cell of the invention.

FIG. 3 shows a system of the invention including an electrical discharge manifold, a shuttle, and a stage for holding 96-well plates.

FIGs. 4A-4B show schematics of an EDM with fixed spring electrodes from a side view (FIG. 4A) and top view (FIG. 4B) in a system with a stage and shuttle.

FIG. 5 shows a flow diagram for a series of processes carried out in a robotic system including systems and devices of the invention.

FIG. 6 shows a side-view schematic of an EDM with fixed springs. FIG. 6 shows how contamination of fixed electrodes can occur during insertion and removal.

FIG. 7 shows a schematic of an EDM of the invention.

FIG. 8 shows a schematic of an EDM of the invention.

FIG. 9 shows a schematic of an EDM of the invention.

FIG. 10 shows a schematic of an EDM of the invention.

FIG. 11 shows a 3D representation of an EDM of the invention. FIG. 12 shows a 3D representation of an EDM of the invention.

FIG. 13 shows a 3D representation of an EDM of the invention.

FIG. 14 shows a 3D representation of an EDM of the invention.

FIG. 15 shows 3D representations of blade electrodes of the invention.

FIG. 16 shows side and top views of blade electrodes in an EDM interacting with a flow cell.

FIG. 17 shows an RC circuit without a flow cell in place. The RC components are separated from the voltage source and ground.

FIG. 18 shows an RC circuit with a flow cell in place. The addition of the flow cell makes a complete RC circuit.

FIG. 19 shows a schematic of an arrangement of electrical components that may be used for detecting the electrical contact between an electrode and a transfection assisting consumable (e.g., a flow cell).

FIG. 20 shows a graphic user interface (GUI) for simulating and visualizing transfection profiles.

FIG. 21 shows a graphic user interface for comparing transfection profiles.

FIG. 22 shows a graphic user interface for assigning transfection profiles to an experimental plate.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides devices and systems that collectively perform continuous flow electromechanical transfection of suspensions via a robotic system or apparatus of which can be executed manually or automatically. The system may include components that pre-process prior to transfection and or post-process specimens after transfection. Systems of the invention may include components that are used for separating, sequestering, diluting, concentrating and/or resuspending one or more targeted specimens of interest that are prepared for transfection via robotic system or fluid transfer device. Systems of the invention may include components for separating, sequestering, diluting, concentrating and/or resuspending one or more payload of interest that will be integrated into the one or more specimen of interest via a robotic system or fluid transfer device. Systems of the invention may include components by which one or more suspended payloads to be integrated and the suspended specimen to be modified are placed in a particular sequence on a robotic system with or without the assistance of the robotic system or fluid transfer device. Systems of the invention may include components by which the robotic system displaces a suspension in a specified manner to achieve a specific biological response from either the specimen or payload. Systems of the invention may include components that enable transfection to be completed with the assistance of a robotic system or fluid transfer device, the device of which delivers sufficient energy to modify the specimen, e.g., flow cells. Devices and/or systems of the invention may or may not be a component of a robotic system. A system of device of the invention may enable transfection on a robotic system due to its ability to act as a reservoir for suspensions of either the specimen to be modified and/or the target to be integrated. Systems and/or devices of the invention may include devices or vessels that mechanically mate with, transport liquids/suspensions to/from, mix liquids/suspensions for, or transfer energy for transfection to a flow cell, as described. Such processes can occur pre- and or post-transfection. Systems may include a component that receives specimens that have undergone transfection, e.g., via a robotic and/or fluid transfer system, for incubation, culturing, analysis, diluting, concentrating, separating, sequestering or any combination of the aforementioned. Systems and/or components thereof may be configured to process specimens in a manner deemed a “Closed System” with regards to biological manufacturing. Systems may include components that communicate/interface with an energy supply, e.g., as a mating device, such as an electrical discharge manifold as described herein. The system may operate in sequence or parallel according to specific protocols via software and or a user interface to produce modified specimens. At least one of these devices used in the production which utilizes continuous flow electrochemical transfection.

Electrical Discharge Manifolds

Systems of the invention may include a device that interacts with a vessel for performing transfection, e.g., a flow cell, e.g., a consumable/disposable flow cell, to transfer energy from a voltage supply to the targeted specimen and payload by mechanical, chemical, electrical, or any specific combination of the principles. Such a device is an electric discharge manifold.

An Electrical Discharge Manifold (EDM) may be configured such that flow cells are inserted therein in order make electrical contact between electrodes in the flow cells and the source and drain contacts, e.g., blade or spring electrodes, in the EDM. A “Close” signal may be sent to the EDM from a controller to achieve contact. The state of the EDM switches from an open state to a closed state which allows the EDM to mechanically contact the flow cells which enable electrical connection and energy delivery to the specimen of interest. In addition, the blade electrodes may also align the flow cells during the transfection. The pressure applied to a flow cell electrode may be determined by the source or drain contact’s (e.g., blade electrode’s) mechanical properties and any springs attached to the arm of the EDM. The electrodes may slide on the surface of the flow cell electrode to clear debris or contamination for better electrical contact. Post transfection, the EDM opens to release the flow cell.

An EDM may be positioned on the deck of a robotic system to complete the transfection (see, e.g., FIG. 3). The flow cell is inserted into the EDM. The EDM mechanically closes to make electrical contact with the flow cell. A suspension of a specimen and a payload is delivered to the flow cell and energized while being displaced through the flow cell, and a specimen in the suspension is transfected by the payload. The specimen may be transferred to a well of a plate (e.g., a 96-well plate) during the displacement of the suspension from the flow cell. Post transfection, the EDM opens, the flow cell(s) are released from the EDM. A shuttle may shift the plate location to another column or row, and the process may be repeated for additional modification of specimens. Alternatively, the EDM may be moved while the plate remains stationary.

EDMs of the invention may include blade electrodes as source and drain contacts for electrically coupling a power source to flow cells. Exemplary blade electrodes are shown in FIGs. 2A, 2B, 15, and 16.

The blade electrodes may be flat sheets of metal with holes in them for mounting. The blades may conform to the surface of the mount, e.g., to allow some degree of curvature as they extend from the mount to where they mate with the flow cell electrodes. Blade electrodes may be affixed to a mount by a retaining bar. The retaining bar attaches the blade electrode to a back plate. When the mount moves toward the flow cells and brings the blade electrodes into contact with the flow cell electrodes, the blade electrodes may deform around the flow cell electrodes, making contact with all flow cell electrodes delivered to the EDM. The contact force may be sufficient to maintain electrical connection of the drain and source blade electrodes to the flow cell electrodes and may not permanently deform any system components (e.g. plastic deformation of the blade electrodes, unseating of the flow cells, or failure of the supporting elements). Blade electrodes may be any suitable material, e.g., a metal, e.g., stainless steel; however other materials may be considered. Suitable materials are conductive, are chemical resistant to withstand cleaning and sterilization requirements (e.g., wiping with an ethanol and/or bleach solution), and/or have properties and geometry to be flexible for this configuration. Other materials may include conductive polymers (e.g., carbon-doped polymeric material), provide they meet the above conditions. Other suitable metals may include gold, silver, platinum, nickel, titanium, copper, etc.

A blade electrode may have a flat edge to enable uniform actuation (clamping motion) across one or a plurality of flow cell electrodes. Preferably, the blades equally interact (with equivalent applied force) with the surface of the flow cell electrode (e.g., an electrode tube) for each of the multiple (e.g., 2-8) flow cells processed simultaneously. Alternatively, a blade electrode can have a curved edge that interacts with the flow cell electrode. Blade electrodes for this configuration may be 0.006 - 0.008 inches thick. The geometry and material properties determine the flexibility for ensuring connection to the flow cell electrodes. Combinations of thickness and width/length used for this application require sufficient bending flexibility (mechanical stiffness) to ensure contact with the flow cell tubes while ensuring conductivity. Blade electrodes may have a deflection of approximately up to 0.5x a flow cell electrode diameter, e.g., approximately ~ 0.032 inches. Blade electrodes may, in the “closed” position of the EDM, apply a force of up to approximately 2 Ib-f across, e.g., for contacting a single flow cell with four blade electrodes, force will be applied equally on each side of the flow cell electrodes. The force applied equally to each side of the flow cell electrodes determines the local strain experienced by the blade electrodes; the stiffness of the blade material determines the deflection experienced: Displacement is a function of force applied, material stiffness, and the geometry of the material, e.g., a blade electrode of stainless steel, with a 0.005 in thickness, mounted 1 inch from the contact location, deflecting by 0.032 inches would experience approximately 0.01 to 0.1 Ib-f. Blade electrodes may apply equal force across all flow cells in an EDM once the device is clamped. The blade electrodes are configured not to cross the center axis of the flow cell when they are inserted, and the blade electrodes are moved into contact with the flow cell electrodes.

Other electrode shapes are considered, e.g., spring electrodes such as those shown in FIG. 4. Such electrodes can have the same material properties as described herein for the blade electrodes. Alternatively, such electrodes may have greater or lesser stiffness. Such electrodes may be made of any electrode material described herein.

EDMs may include various mechanisms for clamping the flow cells. One example is the “Bear Trap” mechanism shown in FIG. 8. While the flexible electrodes on the EDM are separated, the tip of a flow cell extends downward until a vertical pusher is engaged (on right). The tip is relatively higher than electrodes at this point. Then the tip and mechanism are pushed downward together as flexible electrodes move toward tip electrodes in a downward arc. Advantageously, the top electrodes of the EDM are further apart when open, leaving more room for flow cells to pass. In addition, there is less motion in wires connecting the source of the electric field to the EDM electrodes. Another clamping mechanism is shown in FIG. 9, a “parallelogram” mechanism. Here, a flow cell tip extends downward with flexible electrodes apart until electrodes are roughly vertically aligned. Then, the tip and mechanism are pushed downward together as the flexible electrodes move toward tip electrodes and make contact. There can, depending on geometry, be some wiping between electrodes at the end of downward travel. Such a mechanism affords low forces for a given contact force and is relatively simple. In some mechanisms, e.g., those of FIG. 8 and FIG. 9, the actuator is a portion of a liquid handler. For example, the liquid handler may press down on a flow cell to activate the mechanism. Another mechanism is the “active mechanism” of FIG. 10. Here, a flow cell tip extends downward and stops at operational height/treatment position. Just before, a switch is activated, engaging a motor which drives pivoting arms containing flexible electrodes to close. After treatment, the tip moves upward, sliding against the flexible electrodes until the switch is deactivated, at which point arms swing away, e.g., powered by a spring. Such a mechanism is relatively simple to implement for this application. Another mechanism is that shown in FIG. 1 1 , with a cam to actuate the closing mechanism. Here, a motor rotates one way to close, and reversing rotation opens the EDM. The motor stalls at each extreme of travel. A spring controls force on electrodes. Another mechanism is that shown in FIG. 12, with a cam to actuate the closing mechanism. Here, a motor rotates one way to close, and reversing rotation opens the EDM. The motor stalls at each extreme of travel. The motor/cam determines the force on the electrodes. Another mechanism is that shown in FIG. 13, with a cam to actuate the closing mechanism. The cam has no stops and is closed by a spring. Here, a motor rotates one way only. The motor/cam determines the force on the electrodes. Such a mechanism may include pressure sensors. Another mechanism is that shown in FIG. 14, with a linkage and no stops. Here, a motor rotates one only and does not stall at each extreme of travel. The motor determines the force on the electrodes. Such a mechanism may include pressure sensors. The mechanism provides good mechanical advantage and there is no uncertainty in the position or motion.

RC Circuit to Confirm Electrical Contact

The invention also provides circuits for determining proper contact between detachable components, e.g., a flow cell, and a power source. FIG. 17 shows an RC component of the system separated from the voltage source and ground. This represents the circuit without the detachable component (e.g., flow cell) inserted: two separate halves of a whole RC circuit. FIG. 18 shows a detachable component (e.g., flow cell) with impedance Z connected to the voltage source and ground; it also makes contact with the resistor and capacitor as pictured. This represents the circuit with the detachable component (e.g., flow cell) inserted. With such circuits, systems of the invention may confirm the appropriate integration between an electromechanical transfection system and a flow cell. Such circuits may include a low pass or high pass resistor-capacitor circuit that is able to receive electrical signals with magnitudes of 1 -10,000 V. Capacitors may range in capacitance from 1 pF-1 mF. Resistors of such circuits may range in resistance from 0.0001 -100E^A6 Ohms. The confirmation circuit may be disposed serially with respect to the electromechanical system and the flow cell. The confirmation circuit may be disposed in parallel with respect to the electromechanical system and the detachable component (e.g., flow cell). The circuit may include 1 or more capacitors, e.g., a plurality of capacitors, e.g., 1 -10 capacitors. The circuit may include 1 or more resistors, e.g., a plurality of capacitors, e.g., 1 -10 capacitors. The circuit may include one or more switches, e.g., to isolate a confirmed detachable component (e.g., flow cell) from the circuit while a second detachable component (e.g., flow cell) is confirmed, and so on. The circuit may be configured to test one or a plurality of detachable components, e.g., flow cells (e.g., 1 -8).

The RC circuit detailed herein takes advantage of the insertion of the detachable component (e.g., flow cell) to bridge the circuit to the voltage source and ground, moving from the FIG. 17 (open/empty) state to the FIG. 18 (closed/inserted) state. It does this without the need for any actively moving parts. This is because the electrodes of the flow cell that were designed to interface with the voltage source and ground also bridge the gap between the two halves of the circuits above.

Advantageously, there is no interference with any transfection process because the flow cell is empty during the “check.” This means that the impedance of Z is arbitrarily large. Normally, one would expect this to mean that no current could flow. But because of the resistor and capacitor in parallel, there is the potential for current if the applied voltage frequency is high enough. The RC circuit can test the contacts over specific frequency ranges corresponding to transfection processes, e.g., from 1 -100,000 Hz all the way up to 1 -1000 MHz or even multiple GHz, depending on the capabilities of the source.

Because there is an arbitrarily large Z, there is no flow through the flow cell. It functions as an open switch. Because it has no active components during the process described, it could electrically interface with the rest of the circuit in any configuration (in parallel or serially) without affecting the process. The RC circuit can test the contacts without having to draw fluid into the flow cell.

In the FIGs. 17 and 18, one RC circuit corresponds to one cell, but one RC circuit could be made to serve for as many cells/contacts as necessary, e.g., by placing multiple “open circuit junctions” in a row. An RC circuit of the invention can accommodate an arbitrarily large amount of flow cells + contacts. Alternatively, each set of flow cell contacts may have its own RC circuit.

Graphical User Interfaces and Design Software

The invention provides software (machine-readable memory) configured to allow users to plan, visualize and execute automated transfection processes with the systems of the invention, e.g., via a graphical user interface (GUI). The combination of the systems of the invention and the software has the potential to lead to the development of a variety of commercial products related to the discovery and scale-up of curative medicines.

The current state of the cell editing field is rapidly developing, particularly in non-viral electroporation-based transfection procedures. Existing software solutions are limited in their ability to provide seamless scalability from discovery phase work to production systems. Software for planning and executing transfection processes with systems of the invention addresses this limitation by providing unique methods for users to control the profile settings in their experiments, thereby allowing them to test and discover what unique settings can be used to optimize the process to edit cells for the eventual development of curative medicines. The software is user-friendly, designed for ease of use, and allows users control of the parameters in which they run experiments. By making the software accessible to researchers at all levels of experience, the pace of discovery in this field is accelerated, providing a powerful tool to help advance the development of curative medicines. Specific parameters to be varied are any of those described herein.

The machine-readable memory may include code for a Profile Designer subcomponent. Examples of components of a GUI of the invention are shown in FIGs. 20-22. The Profile Designer subcomponent of the software is particularly significant as it allows users to generate experimental and, one day, manufacturing profiles for use with the system. This feature affords customizable and scalable manufacturing processes that will enable the production of novel curative medicines. With the ability to generate customized experimental profiles, the system has the potential to streamline the drug discovery process, reducing the time and resources required for the development of new treatments. This could ultimately lead to the production of more effective and affordable therapies. The invention allows for scalable and customizable solutions for the discovery and manufacturing of curative medicines.

The software of the invention enables users with little workflow knowledge of electro-mechanical transfection to do experimental exploration and optimization, allowing novice users to make meaningful changes to the transfection processes that can be carried out on systems of the invention.

The software enables users to visualize and compare transfection profiles (e.g., electromechanical transfection profiles). Visualization of transfection profiles allows users to internalize the electromechanical signal being delivered to individual cells. Giving users insight into the actual signals allows them to understand the meaningful differences between profiles and select the optimum experiments.

The software enables users to build experimental plate setups by assigning transfection profiles. There is great difficulty preparing a given plate for automated transfection experimentation. By providing an interactive GUI, we remove several barriers to entry: bookkeeping, settings assignment, randomization.

The invention includes a single-workflow series of graphical user interfaces (GUIs) that aid users in the process of planning, executing, and inspecting a transfection experiment. These GUIs are specifically designed to introduce and guide users through the transfection workflow. The software may include a GUI/workflow for simulating and visualizing electromechanical profiles during experimental planning stages that allows users to control the individual settings of transfection in order to optimize its effect with their specific biology. The invention may include GUI/workflow features for comparing multiple electromechanical profiles in real-time. The invention may include GUI/workflow features for assigning such profiles to individual wells of a multi-well (e.g., 96-well) plate, or a series of plates. The invention may include a feature for assigning such profiles to multiple wells of a multi-well plate. The invention may include a feature for assigning volumes to wells of a multi-well plate. The invention may include a feature for assigning experimental labels to wells of a multi-well plate. The invention may include a feature for running such experimental setups on automated robotics (e.g., a liquid handling robot, e.g., in combination with an EDM and sample stage of the invention, e.g., all configured to move relative to each other according to a script provided by the software). The invention may include a feature for reviewing completed automated experiments. Systems and Kits

One or more electro-mechanical transfection devices of the invention may be combined with various external components, e.g., power supplies, pumps, reservoirs (e.g., bags), controllers, reagents, liquids, and/or samples in the form of a system. In some embodiments, a system of the invention includes a plurality of devices of the invention and a source of electrical potential that is releasably connected to the first and second electrodes of the device(s) of the invention. In this configuration, the flow cells are connected to the source of electrical potential, a first electrode is energized, and a second electrode is held at ground. This creates a localized electric field in the active zone, thus transfecting the cells that pass through the device(s). Electro-mechanical systems incorporating flow cells may induce reversible poration of the cells that pass through the device and system of the invention. For example, systems of the invention may induce substantially non-thermal reversible poration.

In some cases, the releasable connection to the first and second electrodes may include any practical electro-mechanical connection that can maintain consistent electrical contact between the source of electrical potential and the first and second electrodes. Example electrical connections include, but are not limited to clamps, clips, e.g., alligator clips, springs, e.g., a leaf spring, an external sheath or sleeve, wire brushes, flexible conductors, pogo pins, mechanical connections, inductive connections, or a combination thereof. Other types of electrical connections are known in the art. A device of the invention can be installed into an opening in the conducting grid such that the first and second electrodes of the flow cells can contact the conducting grid. In particular, the conducting grid includes spring loaded electrodes, e.g., electrodes connected to a spring, such that when a device of the invention is installed into an opening of the conducting grid, the spring-loaded electrodes displace and compress the spring (which further provides a restoring force against the first and second electrodes of the flow cells), thus ensuring electrical contact between the flow cells and the source of electrical potential.

The source of electrical potential is configured to deliver an applied voltage to one or more electrode(s) in order to provide an electrical potential difference between the electrodes and thus establish a uniform electric field in the active zone. In some cases, such as in a two-electrode circuit, the applied voltage is delivered to a first electrode, and a second electrode is held at ground. Without wishing to be bound by any particular theory, an applied voltage delivered to the electrode is delivered at a particular amplitude, a particular frequency, a particular pulse shape, a particular duration, a particular number of pulses applied, and a particular duty cycle. These parameters, coupled to the geometry of the active zone, will deliver a particular electric field within the active zone that will be experienced by the cells suspended in a fluid. The electrical parameters described herein may be optimized for a particular cell line and/or composition being delivered to a particular cell line. The application of the electrical potential to the electrodes of devices(s) of the invention may be initiated and/or controlled by a controller, e.g., a computer with programming, operatively coupled to the source of electrical potential.

Along with the electrical potential parameters described herein, the geometry of flow cells, e.g., the shape and dimensions of the cross-section of the active zone, control the shape and intensity of the resulting electric field within the active zone. Typically, flow cells with an active zone that has a uniform cross section will exhibit a uniform electric field along its length. In order to modulate the resulting electric field in the active zone, the active zone may include a plurality of different hydraulic diameters and/or different cross-section shapes along its length. As a non-limiting example, flow cells may include a plurality of serially-connected active zones, each of the plurality of active zones having a circular crosssection of a different hydraulic diameter, e.g., each has a different diameter. In this configuration, the different diameter circular cross-sections of the active zone each act as an independent active zone, and each will induce a different electric field at every change in dimension with an identical applied voltage, e.g., a constant DC voltage.

In some cases, flow cells may include a plurality of active zones fl uidically connected in series, with each active zone having either a uniform or non-uniform cross-section and each may have a different cross-section shape. Alternatively, a system of the invention may include a plurality of flow cells in a parallel configuration, with each flow cell operating independently of each other to increase the overall throughput of the electro-mechanical transfection.

In some cases, the amplitude of the applied voltage is from -3 kV to 3 kV, e.g., -3 kV to -0.1 kV, - 2 kV to -0.1 kV, -1 kV to -0.1 kV, -0.1 kV to -0.01 kV, 0.01 kV to 3 kV, e.g., 0.01 kV to 0.1 kV, 0.02 kV to 0.2 kV, 0.03 kV to 0.3 kV, 0.04 kV to 0.4 kV, 0.05 kV to 0.5 kV, 0.06 kV to 0.6 kV, 0.07 kV to 0.7 kV, 0.08 kV to 0.8 kV, 0.09 kV to 0.9 kV, 0.1 kV to l kV, 0.1 kV to 2.0 kV, 0.1 kV to 3 kV, 0.15 kV to 1 .5 kV, 0.2 kV to 2 kV, 0.25 kV to 2.5 kV, or 0.3 kV to 3 kV, e.g., 0.01 to 1 kV, 0.1 kV to 0.7 kV, or 0.2 to 0.6 kV, e.g., about 0.01 kV, 0.02 kV, 0.03 kV, 0.04 kV, 0.05 kV, 0.06 kV, 0.07 kV, 0.08 kV, 0.09 kV, 0.1 kV, 0.2 kV, 0.3 kV, 0.4 kV, 0.5 kV, 0.6 kV, 0.7 kV, 0.8 kV, 0.9 kV, 1 kV, 1 .1 kV, 1 .2 kV, 1 .3 kV, 1 .4 kV, 1 .5 kV, 1 .6 kV, 1 .7 kV, 1 .8 kV, 1 .9 kV, 2 kV, 2.1 kV, 2.2 kV, 2.3 kV, 2.4 kV, 2.5 kV, 2.6 kV, 2.7 kV, 2.8 kV, 2.9 kV, or 3 kV.

In some cases, the frequency of the applied voltage is from 1 Hz to 50,000 Hz, e.g., from 1 Hz to 1 ,000 Hz, 1 Hz to 500 Hz, 100 Hz to 500 Hz, 100 Hz to 5,000 Hz, 500 Hz to 10,000 Hz, 1000 Hz to 25,000 Hz, or from 5,000 Hz to 50,000 Hz, e.g., from 10 Hz to 1000 Hz, 10 Hz to 500 Hz, 500 Hz to 750 Hz, or 100 Hz to 500 Hz, e.g., from about 1 Hz, 2 Hz, 3 Hz, 4 Hz, 5 Hz, 6 Hz, 7 Hz, 8 Hz, 9 Hz, 10 Hz, 20 Hz, 30 Hz, 40 Hz, 50 Hz, 60 Hz, 70 Hz, 80 Hz, 90 Hz, 100 Hz, 1 10 Hz, 120 Hz, 130 Hz, 140 Hz, 150 Hz, 160 Hz, 170 Hz, 180 Hz, 190 Hz, 200 Hz, 210 Hz, 220 Hz, 230 Hz, 240 Hz, 250 Hz, 260 Hz, 270 Hz, 270 Hz, 280 Hz, 290 Hz 300 Hz, 310 Hz, 320 Hz, 330 Hz, 340 Hz, 350 Hz, 360 Hz, 370 Hz, 380 Hz, 390 Hz, 400 Hz, 410 Hz, 420 Hz, 430 Hz, 440 Hz, 450 Hz, 460 Hz, 470 Hz, 480 Hz, 490 Hz, 500 Hz, 510 Hz, 520 Hz, 530 Hz, 540 Hz, 550 Hz, 600 Hz, 700 Hz, 800 Hz, 900 Hz, 1 ,000 Hz, 2,000 Hz, 3,000 Hz, 4,000 Hz, 5,000 Hz, 6,000 Hz, 7,000 Hz, 8,000 Hz, 9,000 Hz, 10,000 Hz, 15,000 Hz, 20,000 Hz, 25,000 Hz, 30,000 Hz, 35,000 Hz, 40,000 Hz, 45,000 Hz, or 50,000 Hz.

In some embodiments, the shape of the applied pulse, e.g., waveform, can be a square wave, pulse, a bipolar wave, a sine wave, a ramp, an asymmetric bipolar wave, or arbitrary. Other voltage waveforms are known in the art. The chosen waveform can be applied at any practical voltage pattern including, but not limited to, high voltage-low voltage, low voltage-high voltage, direct current (DC), alternating current (AC), unipolar, positive (+) polarity only, negative (-) polarity only, (+)/(-) polarity, (-)/(+) polarity, or any superposition or combination thereof. A skilled artisan can appreciate that these pulse parameters will depend on the cell line any electrical characteristics of the composition being delivered to the cell.

Applied voltage pulses can be delivered to the active zone with durations from 0.01 ms to 1 ,000 ms, e.g., from 0.01 ms to 1 ms, 0.1 ms to 10 ms, 0.1 ms to 15 ms, 1 ms to 10 ms, 1 ms to 50 ms, 10 ms to 100 ms, 25 ms to 200 ms, 50 ms to 400 ms, 100 ms to 600 ms, 300 ms to 800 ms, or 500 ms to 1 ,000 ms, e.g., about 0.01 ms to 100 ms, 0.1 ms to 50 ms, or 1 ms to 10 ms, e.g., 0.01 ms, 0.02 ms, 0.03 ms, 0.04 ms, 0.05 ms, 0.06 ms, 0.07 ms, 0.08 ms, 0.09 ms, 0.1 ms, 0.2 ms, 0.3 ms, 0.4 ms, 0.5 ms, 0.6 ms, 0.7 ms, 0.8 ms, 0.9 ms, 1 ms, 2 ms, 3 ms, 4 ms, 5 ms, 6 ms, 7 ms, 8 ms, 9 ms, 10 ms, 11 ms, 12 ms, 13 ms, 14 ms, 15 ms, 20 ms, 30 ms, 40 ms, 50 ms, 60 ms, 70 ms, 80 ms, 90 ms, 100 ms, 150 ms, 200 ms, 250 ms, 300 ms, 350 ms, 400 ms, 450 ms, 500 ms, 550 ms, 600 ms, 650 ms, 700 ms, 750 ms, 800 ms, 850 ms, 900 ms, 950 ms, or 1 ,000 ms.

In some cases, the number of applied voltage pulses delivered can be 1 or more, e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 100 or more, e.g., 1 -4, 2-5, 3-6, 4-7, 5-8, 6-9, 7-10, 8-11 , 7-12, or 9-13, e.g., 0.01 to 1 ,000, e.g., from 1 to 10, 1 to 50, 5 to 10, 5 to 15, 10 to 100, 25 to 200, 50 to 400, 100 to 600, 300 to 800, or 500 to 1 ,000, e.g., 1 to 100, 1 to 50, or 1 to 10, e.g., about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1 ,000, or more than 1000.

In some instances, the number of applied voltage pulses delivered can be 1 or more. For example, in some instances, the number of applied voltage pulses delivered is from 1 ,000 to 1 ,000,000, e.g., from 1 ,000 to 10,000 (e.g., from 1 ,000 to 2,000, from 2,000 to 3,000, from 3,000 to 4,000, from 4,000 to 5,000, from 5,000 to 6,000, from 6,000 to 7,000, from 7,000 to 8,000, from 8,000 to 9,000, or from 9,000 to 10,000, e.g., 1 ,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000), from 10,000 to 100,000 (e.g., from 10,000 to 20,000, from 20,000 to 30,000, from 30,000 to 40,000, from 40,000 to 50,000, from 50,000 to 60,000, from 60,000 to 70,000, from 70,000 to 80,000, from 80,000 to 90,000, or from 90,000 to 100,000, e.g., 10,000, 25,000, 30,000, 40,000, 50,000, 60,000, 70,000, 75,000, 80,000, 90,000, or 100,000), or from 100,000 to 1 ,000,000 (e.g., from 100,000 to 200,000, from 200,000 to 300,000, from 300,000 to 400,000, from 400,000 to 500,000, from 500,000 to 600,000, from 600,000 to 700,000, from 700,000 to 800,000, from 800,000 to 900,000, or from 900,000 to 1 ,000,000, e.g., about 100,000, 200,000, 250,000, 300,000, 400,000, 500,000, 600,000, 700,000, 750,000, 800,000, 900,000, or 1 ,000,000).

The pulses of applied voltage can, in some instances, be delivered at a duty cycle of 1% to 100%, e.g., from 1 % to 10%, 2.5% to 20%, 5% to 40%, 10% to 60%, 30% to 80%, or 50% to 100%, e.g., 0.01 % to 100%, 0.1 % to 99%, 1 % to 97%, or 10% to 95%, e.g., about 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Flow cells, when the electrodes are connected to the source of electrical potential and energized, generate a localized electric field in the active zone that, in combination with mechanical energy (e.g., from the flow) transfect cells that pass through. In some cases, the electric field generated in the active zone has a magnitude from 2 V/cm to 50,000 V/cm, e.g., 2 V/cm to 1 ,000 V/cm, 100 V/cm to 1 ,000 V/cm, 100 V/cm to 5,000 V/cm, 400 V/cm to 2,000 V/cm, 400 to 1000 V/cm, 500 V/cm to 10,000 V/cm, 1000 V/cm to 25,000 V/cm, or from 5,000 V/cm to 50,000 V/cm, e.g., from 2 V/cm to 20,000 V/cm, 5 V/cm to 10,000 V/cm, or 100 V/cm to 1 ,000 V/cm, e.g., from about 2 V/cm, 3 V/cm, 4 V/cm, 5 V/cm, 6 V/cm, 7 V/cm, 8 V/cm, 9 V/cm, 10 V/cm, 20 V/cm, 30 V/cm, 40 V/cm, 50 V/cm, 60 V/cm, 70 V/cm, 80 V/cm, 90 V/cm, 100 V/cm, 200 V/cm, 300 V/cm, 400 V/cm, 500 V/cm, 600 V/cm, 700 V/cm, 800 V/cm, 900 V/cm, 1 ,000 V/cm, 2,000 V/cm, 3,000 V/cm, 4,000 V/cm, 5,000 V/cm, 6,000 V/cm, 7,000 V/cm, 8,000 V/cm, 9,000 V/cm, 10,000 V/cm, 15,000 V/cm, 20,000 V/cm, 25,000 V/cm, 30,000 V/cm, 35,000 V/cm, 40,000 V/cm, 45,000 V/cm, or 50,000 V/cm.

Systems of the invention typically include a fluid delivery source that is configured to deliver the plurality of cells suspended in the fluid through the entry zone to the active zone (e.g., through the first electrode) and out of the active zone (e.g., through the second electrode), e.g., to the recovery zone. Fluid delivery sources typically include liquid handlers, pumps, including, but not limited to, high pressure sources, syringe pumps, micropumps, or peristaltic pumps. Alternatively, fluids can be delivered by the displacement of a working fluid against a reservoir of the fluid to be delivered or by air displacement. Other fluid delivery sources are known in the art. In some cases, the fluid delivery source is configured to flow cells suspended in a fluid by the application of a positive pressure. Without wishing to be bound by any particular theory, the flow rate at which cells in a suspension are flowed through flow cells and the specific geometry of the active zone of flow cells will determine the residence time of the cells in the electric field in the active zone.

In some instances, the volumetric flow rate of fluid delivered from a fluid delivery source has a volumetric flow rate of 0.001 mL/min to 1 ,000 mL/min per active zone, e.g., from 0.001 mL/min to 0.1 mL/min, 0.01 mL/min to 1 mL/min, 0.1 mL/min to 10 mL/min, 1 mL/min to 50 mL/min, 10 mL/min to 100 mL/min, 25 mL/min to 200 mL/min, 50 mL/min to 400 mL/min, 100 mL/min to 600 mL/min, 300 mL/min to 800 mL/min, or 500 mL/min to 1 ,000 mL/min per active zone, e.g., about 0.001 mL/min, 0.002 mL/min, 0.003 mL/min, 0.004 mL/min, 0.005 mL/min, 0.006 mL/min, 0.007 mL/min, 0.008 mL/min, 0.009 mL/min, 0.01 mL/min, 0.02 mL/min, 0.03 mL/min, 0.04 mL/min, 0.05 mL/min, 0.06 mL/min, 0.07 mL/min, 0.08 mL/min, 0.09 mL/min, 0.1 mL/min, 0.2 mL/min, 0.3 mL/min, 0.4 mL/min, 0.5 mL/min, 0.6 mL/min, 0.7 mL/min, 0.8 mL/min, 0.9 mL/min, 1 mL/min, 2 mL/min, 3 mL/min, 4 mL/min, 5 mL/min, 6 mL/min, 7 mL/min, 8 mL/min, 9 mL/min, 10 mL/min, 15 mL/min, 20 mL/min, 25 mL/min, 30 mL/min, 35 mL/min, 40 mL/min, 45 mL/min, 50 mL/min, 55 mL/min, 60 mL/min, 65 mL/min, 70 mL/min, 75 mL/min, 80 mL/min, 85 mL/min, 90 mL/min, 95 mL/min, 100 mL/min, 150 mL/min, 200 mL/min, 250 mL/min, 300 mL/min, 350 mL/min, 400 mL/min, 450 mL/min, 500 mL/min, 550 mL/min, 600 mL/min, 650 mL/min, 700 mL/min, 750 mL/min, 800 mL/min, 850 mL/min, 900 mL/min, 950 mL/min, or 1 ,000 mL/min. In particular embodiments, the flow rate is from 10 mL/min to 100 mL/min per active zone, e.g., about 10 mL/min, 20 mL/min, 30 mL/min, 40 mL/min, 50 mL/min, 60 mL/min, 70 mL/min, 80 mL/min, 90 mL/min, or 100 mL/min per active zone.

In some instances, a Reynolds number of a liquid while passing through the active zone is between 10 and 3,000 (e.g., 10 to 100, 25 to 200, 50 to 400, 100 to 600 mL/min, 300 mL/min to 800 mL/min, 500 to 1 ,000, 800 to 1 ,500, 1 ,200 to 2,000, 1 ,800 to 2,500, or 2,400 to 3000, e.g., about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1 ,000, 1 ,500, 2,000, 2,050, or 3,000).

In some instances, a peak pressure of a liquid while passing through the active zone is between 1 x 10^-3 Pa and 9.5 x 10⁴ Pa, e.g., between 0.001 to 9,500 (e.g., 0.001 Pa to 0.1 Pa, 0.01 Pa to 1 Pa, 0.1 Pa to 10 Pa, 1 Pa to 50 Pa, 10 Pa to 100 Pa, 25 Pa to 200 Pa, 50 Pa to 400 Pa, 100 Pa to 600 Pa, 300 Pa to 800 Pa, or 500 Pa to 1 ,000 Pa, 1 ,000 Pa to 6,000 Pa, 3,000 Pa to 8,000 Pa, 5,000 Pa to 9,000 Pa, or 7,500 Pa to 9,500 Pa, e.g., about 0.001 Pa, 0.002 Pa, 0.003 Pa, 0.004 Pa, 0.005 Pa, 0.006 Pa, 0.007 Pa, 0.008 Pa, 0.009 Pa, 0.01 Pa, 0.02 Pa, 0.03 Pa, 0.04 Pa, 0.05 Pa, 0.06 Pa, 0.07 Pa, 0.08 Pa, 0.09 Pa, 0.1 Pa, 0.2 Pa, 0.3 Pa, 0.4 Pa, 0.5 Pa, 0.6 Pa, 0.7 Pa, 0.8 Pa, 0.9 Pa, 1 Pa, 2 Pa, 3 Pa, 4 Pa, 5 Pa, 6 Pa, 7 Pa, 8 Pa, 9 Pa, 10 Pa, 15 Pa, 20 Pa, 25 Pa, 30 Pa, 35 Pa, 40 Pa, 45 Pa, 50 Pa, 55 Pa, 60 Pa, 65 Pa, 70 Pa, 75 Pa, 80 Pa, 85 Pa, 90 Pa, 95 Pa, 100 Pa, 150 Pa, 200 Pa, 250 Pa, 300 Pa, 350 Pa, 400 Pa, 450 Pa, 500 Pa, 550 Pa, 600 Pa, 650 Pa, 700 Pa, 750 Pa, 800 Pa, 850 Pa, 900 Pa, 950 Pa, 1 ,000 Pa, 1 ,100 Pa, 1 ,500 Pa, 2,000 Pa, 2,500 Pa, 3,000 Pa, 3,500 Pa, 4,000 Pa, 4,500 Pa, 5,000 Pa, 5,500 Pa, 6,000 Pa, 6,500 Pa, 7,000 Pa, 7,500 Pa, 8,000 Pa, 8,500 Pa, 9,000 Pa, or 9,500 Pa, or e.g., about 3,300 Pa (e.g., 2,500 to 4,000 Pa, e.g., 2,500 Pa to 3,000 Pa, 2,800 to 3,300 Pa, 3,100 Pa to 3,400 Pa), e.g., about 2,800 Pa, 2,900 Pa, 3,000 Pa, 3,100 Pa, 3,200 Pa, 3,300 Pa, 3,400 Pa, or 3,500 Pa. In some instances, an average flow velocity of a liquid while passing through the active zone is between 1 x 10² m/s and 10 m/s, e.g., between 0.01 and 1 m/s (e.g., between 0.01 and 0.05 m/s, 0.05 and 0.1 m/s, 0.1 and 0.5 m/s, 0.5 and 1 m/s, 1 .5 and 2 m/s, 1 and 2 m/s, 2 and 3 m/s, 3 and 4 m/s, 4 and 5 m/s, 5 and 6 m/s, 6 and 7 m/s, 7 and 8 m/s, 8 and 9 m/s, or 9 and 10 m/s), e.g., between 0.1 and 5 m/s, between 0.4 and 1 .4 m/s, between 0.65 and 1 .3 m/s, or between 0.26 and 2.08 m/s, e.g., about 0.1 m/s, 0.2 m/s, 0.3 m/s, 0.4 m/s, 0.5 m/s, 0.6 m/s, 0.7 m/s, 0.8 m/s, 0.9 m/s, 1 .0 m/s, 1 .1 m/s, 1 .2 m/s, 1 .3 m/s, 1 .4 m/s, 1 .5 m/s, 2 m/s, 3 m/s, 4 m/s, 5 m/s, 6 m/s, 7 m/s, 8 m/s, 9 m/s, or 10 m/s.

The residence time of cells in the active zone of flow cells may be from 0.5 ms to 50 ms, e.g., from 0.5 ms to 5 ms, 1 ms to 10 ms, 5 ms to 15 ms, 10 ms to 20 ms, 15 ms to 25 ms, 20 ms to 30 ms, 25 ms to 35 ms, 30 ms to 40 ms, 35 ms to 45 ms, or 40 ms to 50 ms, e.g., about 0.5 ms, 0.6 ms, 0.7 ms, 0.8 ms, 0.9 ms, 1 ms, 1 .5 ms, 2 ms, 2.5 ms, 3 ms, 3.5 ms, 4 ms, 4.5 ms, 5 ms, 5.5 ms, 6 ms, 6.5 ms, 7 ms, 7.5 ms, 8 ms, 8.5 ms, 9 ms, 9.5 ms, 10 ms, 10.5 ms, 1 1 ms, 1 1 .5 ms, 12 ms, 12.5 ms, 13 ms, 13.5 ms, 14 ms, 14.5 ms, 15 ms, 20 ms, 25 ms, 30 ms, 35 ms, 40 ms, 45 ms, or 50 ms. In some embodiments, the residence time is from 5-20 ms (e.g., from 6-18 ms, 8-15 ms, or 5-14 ms).

Systems of the invention typically feature a housing that contains and supports the flow cells and any necessary electrical connections, e.g., electrode connections. The housing may be configured to hold and energize a single flow cell, or alternatively, may be configured to hold and simultaneously energize a plurality of flow cells. The housing may include a thermal controller that is able to regulate the temperature of the flow cells or thermally regulate a component of the system, e.g., a fluid, e.g., a buffer or suspension containing cells, during transfection. The thermal controller may be configured to heat the flow cells, or a component of a system thereof, cool the flow cells, or a component of a system thereof, or perform both operations. When configured to heat the flow cells, or a component of a system thereof, suitable thermal controllers include, but are not limited to, heating blocks or mantles, liquid heating, e.g., immersion or circulating fluid baths, battery operated heaters, or resistive heaters, e.g., thin film heaters, e.g., heat tape. When configured to cool the flow cells, or a component of a system thereof, suitable thermal controllers include, but are not limited to, liquid cooling, e.g., immersion or circulating fluid baths, evaporative coolers, or thermoelectric, e.g., Peltier coolers. For example, when implemented with liquid cooling, a flow cell or a housing configured to hold flow cells may be in direct contact with tubing that circulates a chilled fluid or surrounded in a cooling jacket including tubing that circulates a chilled fluid. Other heating and cooling elements are known in the art. In some embodiments, the systems of the invention may be configured to include housing for the fluid prior to or post transfection that simulates and encourages homogenous dispersion of the particles in the fluid, e.g. a rocking element.

In some embodiments, the housing (e.g., cartridge) is configured for use with and/or insertion into an automated closed system that is used to deliver cell therapies to patients in a clinical or hospital setting.

In some embodiments, the housing (e.g., cartridge) further includes a cooling/heating area/enclosure for cell suspension and/or buffer storage during, before and after electro-mechanical transfection of the specimen. In some embodiments, the system (e.g., device and housing) is externally powered.

In some embodiments, systems of the invention include a touchscreen user interface or other alternative user interface(s) that enables the user to select parameters such as flow rate, waveforms, applied potential, volume to transfect, time delay, cooling features, heating features, transfection status, progress and other parameters used to optimize the electro-mechanical transfection or electromechanical protocol. In some embodiments, the user interface also contains pre-formulated parameter selections that enable the user to operate the system at specific parameters and conditions that have previously been validated by user or as recommended by the manufacturers. In some embodiments, the user interface may be connected to programming that allows for automated running of the system and/or running an algorithm to optimize transfection for a given sample of a known cell type and payload combination. In some embodiments, the optimization algorithms have the ability to adjust electromechanical parameters independently or autonomously if the user selects this functionality. In some embodiments, the optimization algorithms allow for continuous adjustment of the parameters used in the electro-mechanical transfection process that may depend on the cell type, conductivity of cell suspensions, volume of cell suspensions, dynamic viscosity, lifetime of the transfection cartridge(s), the physical state of the suspension, or the state of the transfection device(s).

In some embodiments, the optimization algorithms have the ability to perform predictive analysis based on known input cell-type parameters and to adjust electro-mechanical parameters accordingly. Input parameters to be measured include, but are not limited to, suspension conductivity, suspension temperature, suspension dynamic viscosity, cell morphology, cell size, and cell impedance. In some embodiments, the optimization algorithms adjust electro-mechanical parameters based on electrical signals within any of the devices of the invention. In some embodiments, the optimization algorithms adjust electro-mechanical parameters based on detected flow parameters within any of the devices of the invention. In some embodiments, the optimization algorithms adjust transfection parameters based on unique dimensionless input parameters. In some embodiments, the optimization algorithms have the ability to adjust electro-mechanical transfection parameters based on unique multivariate combinations of parameters that are predictive of high viability results, high efficiency results, or matched viability and efficiency results.

Systems of the invention may include one or more outer structures that are configured to cover the electrodes of flow cells, e.g., to reduce end user exposure to live electrical connections. Typically, an electro-mechanical system will include one outer structure that covers its electrodes and active zone. The outer structure may be a non-conductive material, e.g., a non-conductive polymer, that includes structural features for electro-mechanically engaging the parts of the flow cells, e.g., the electrodes or active zone. The outer structure may include one or more recesses, cutouts, or similar openings within the structure to accommodate the flow cells. The outer structure may be configured to be a component that can be removed from the flow cells. For example, the outer structure may include two separate components connected by a hinge, e.g., a living hinge, such that it can be folded over the flow cells. Alternatively, the outer structure may be one or more separate pieces that can be connected together using suitable mating features to form a single structure. In these embodiments, the outer structure may be affixed to the flow cells using any suitable fastener, e.g., snaps, latches, button, or clips, which may be integrated into the outer structure or externally connected to the outer structure. Other suitable fastener types are known in the art. In some embodiments, the outer structure includes one or more alignment features, e.g., pins, divots, grooves, or tabs, that ensure correct alignment of the one or more pieces of the outer structure. In some cases, the outer structure is configured to be permanently connected to the devices of the invention.

In some embodiments, the housing (e.g., cartridge, e.g., outer structure) encapsulates one or more of the previously stated inventions or one or more devices used for continuous flow electromechanical transfection. In some embodiments, the housing (e.g., cartridge) is configured to allow use with and/or insertion into an automated closed system that delivers cell therapies to patients. In some embodiments, the housing further includes a cooling/heating area/enclosure/dispersion for cell suspension and/or buffer storage during, before and after electro-mechanical transfection of the specimen. In some embodiments, the system is externally powered.

In some embodiments, the system also includes optimization algorithms that have the ability to adjust electro-mechanical parameters independently or autonomously if the user selects this functionality. These optimization algorithms allow for continuous adjustment of the parameters used in the transfection process that may depend on the cell type, conductivity, volume of suspensions, dynamic viscosity, lifetime of the electro-mechanical cartridge, the physical state of the suspension or the state of the electro-mechanical device.

In any of the embodiments of the outer structure described herein, the outer structure provides for electrical connection between an external source of electric potential and the electrodes of the devices of the invention. For example, the outer structure may include one or more electrical inputs for electrical connections, e.g., spades, banana plugs, or bayonet, e.g., BNC, connectors, that facilitate electrical connection between the source of electric potential and the electrodes of the devices of the invention inside the outer structure.

Devices and outer structures of the invention may be combined with additional external components, such as reagents, e.g., buffers, e.g., transfection or recovery buffers, and/or samples, in a kit. In some instances, a transfection buffer includes a composition appropriate for cell electro-mechanical transfection. In some instances, the transfection buffer includes a suitable concentration of one or more salts (e.g., potassium chloride, sodium chloride, potassium phosphate, potassium dihydrogen phosphate) or sugars (e.g., dextrose or myo-inositol), or any combination thereof, at a concentration from 0.1 to 200 mM (e.g., from 0.1 to 1 .0 mM, from 1 .0 mM to 10 mM, or from 10 mM to 100 mM). Flow cells

In general, flow cells used with the present invention are configured to be flow through devices that may interface with existing liquid handling, pumps, or fluid transport apparatuses, such as conventional pipette tip robots or large-scale liquid handling systems, to provide continuous transfection of cells suspended in a fluid. A flow cell is configured for transfection of cells to occur within the active zone via an electro-mechanical transfection mechanism that is distinct from the delivery mechanism in electroporation-based transfection systems, flow cells typically feature two distinct regions: an entry zone, with a first inlet and first outlet, and an active zone with a second inlet and second outlet. First and second electrodes are disposed to produce an electric field in the active zone. When an electrical potential difference is applied to the first and second electrodes, a localized electric field develops in the space between the two electrodes, e.g., the active zone, and cells that are exposed to the electric field are transfected by a payload. An individual flow cell of the invention may include two electrodes, as shown in FIG. 1 ; alternatively, individual flow cells may include three or more electrodes that define a plurality of active zones, thus allowing for a plurality of transfections on the cells suspended in a fluid. Flow cells may include a plurality of active zones between the first and second electrodes, allowing for cells to experience different electric fields, e.g., developed by different flow cells of each of the plurality of active zones, while flowing in a single flow cell or a plurality of devices.

In some cases, the first electrode and the second electrode may be electrically conductive wires, hollow cylinders, electrically conductive thin films, metal foams, mesh electrodes, liquid diffusible membranes, conductive liquids, or any combination thereof can be included in the flow cells. The electrodes may be either aligned parallel with the axis of fluid flow of the flow cells or may be aligned orthogonal to the axis of fluid flow of the flow cells. For example, the first and second electrodes may be hollow cylindrical electrodes arranged in parallel with the axis of fluid flow within the flow cells. In an alternative example, the first and/or second electrodes may be made of a porous conductor, e.g., a metal mesh, with pores that are aligned to the axis of fluid flow of the flow cells. In an alternative example, the first and/or second electrodes may be a conductive fluid, e.g., liquid. In some cases, the first and second electrodes may be configured as a helical, e.g., a double helix, made of a solid conductor, e.g., a wire, around the active zone. In this configuration, the hydraulic diameter of the active zone remains substantially uniform but the first and second electrodes change in position along the length of the active zone. The first and second electrodes are in fluid communication with the active zone, but the electric field generated when an electrical potential difference is applied to the electrodes rotates as the cells suspended in the fluid travel through the flow cells. In certain embodiments, the first and second electrodes are embedded into the flow cells and have active area disposed at or near the fluidic connections to the active zone such that the fluid carrying the cells in suspension contacts a portion of the electrode, with the electric field generated in the active zone.

When configured to be hollow cylindrical electrodes, the diameter of the electrode may be from about 0.1 mm to 5 mm, e.g., from about 0.1 mm to 1 mm, from 0.5 mm to 1 .5 mm, from 1 mm to 2 mm, from 1 .5 mm to 2.5 mm, 2 mm to 3 mm, from 2.5 mm to 3.5 mm, 3 mm to 4 mm, from 3.5 mm to 4.5 mm, or 4 mm to 5 mm, e.g., 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1 .1 mm, 1 .2 mm, 1 .3 mm, 1 .4 mm, 1 .5 mm, 1 .6 mm, 1 .7 mm, 1 .8 mm, 1 .9 mm, 2 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, 2.5 mm, 2.6 mm, 2.7 mm, 2.8 mm, 2.9 mm, 3 mm, 3.1 mm, 3.2 mm, 3.3 mm, 3.4 mm, 3.5 mm, 3.6 mm, 3.7 mm, 3.8 mm, 3.9 mm, 4 mm, 4.1 mm, 4.2 mm, 4.3 mm, 4.4 mm, 4.5 mm, 4.6 mm, 4.7 mm, 4.8 mm, 4.9 mm, or 5 mm. An exemplary electrode outer diameter is 1 .3 mm, corresponding to a 16 gauge electrode.

The active zone may fluidically and/or electrically connect the first and second electrodes of flow cells , and when the electrodes are energized, experiences a localized electric field therebetween. The active zone may be fluidically connected to a recovery zone downstream of the active zone. The cross- sectional shape of the active zone may be of any suitable shape that allows cells to pass through the active zone and the electric field within the active zone. The cross-sectional shape may be, e.g., circular, ellipsoidal, or polygonal, e.g., square, rectangular, triangular, n-gon (e.g., a regular or irregular polygon having 4, 5, 6, 7, 8, 9, 10, or more sides), star, parallelogram, trapezoidal, or irregular, e.g., oval, or curvilinear shape. In some cases, the active zone is a channel that has a substantially uniform crosssection dimension along its length, e.g., the active zone may have a circular cross-section, where the diameter is constant from the fluidic connection with the entry zone to the fluidic connection of the outlet (e.g., the second outlet) of the active zone, or of the recovery zone. In this configuration, the resulting electric field is more uniform, thus allowing for a more predictable electric field exposure of cells suspended in a fluid. Alternatively, the hydraulic diameter of the active zone may be varied along its length. For example, the hydraulic diameter of the active zone may either increase or decrease along its length, or may have more than one dimension change along its length, e.g., the hydraulic diameter, e.g., the diameter, may increase or decrease by at least about 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or at most about 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In this configuration, the active zone may have a truncated conical cross-section, with the diameter increasing from the top aperture to the bottom aperture or decreasing from the top aperture to the bottom aperture. In some cases, flow cells may include a plurality of active zones fluidically connected in series, with each active zone having either a uniform or non-uniform cross-section and each may have a different cross-section shape. As a non-limiting example, flow cells may include a plurality of serially- connected active zones, each of the plurality of active zones having a cylindrical cross-section of a different hydraulic diameter, e.g., each has a different diameter.

In some embodiments, the hydraulic diameter of the active zone may be from 0.005 mm to 50 mm, e.g., 0.005 mm to 0.05 mm, 0.01 mm to 0.1 mm, 0.05 mm to 0.5 mm, 0.1 mm to 1 mm, 0.5 mm to 1 mm, from 0.5 mm to 2 mm, 0.7 mm to 1 .5 mm, 1 mm to 5 mm, 3 mm to 7 mm, 5 mm to 10 mm, 7 mm to 12 mm, 10 mm to 15 mm, 13 mm to 18 mm, 15 mm to 20 mm, 22 mm to 30 mm 25 mm to 35 mm, 30 mm to 40 mm, 35 mm to 45 mm, or 40 mm to 50 mm, e.g., about 0.005 mm, 0.006, 0.007 mm, 0.008 mm, 0.009 mm, 0.01 mm, 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, 0.09 mm, 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 1 1 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm,

19 mm, 20 mm, 21 mm, 22 mm, 23 mm, 24 mm, 25 mm, 26 mm, 27 mm, 28 mm, 29 mm, 30 mm, 31 mm,

32 mm, 33 mm, 34 mm, 35 mm, 36 mm, 37 mm, 38 mm, 39 mm, 40 mm, 41 mm, 42 mm, 43 mm, 44 mm, 45 mm, 46 mm, 47 mm, 48 mm, 49 mm, or 50 mm. In general, the diameter of the active zone is sized such that it does not have a constriction that contacts the cells to deform the cell membranes with the channel walls, e.g., poration of the cells is not induced by mechanical deformation due to cell squeezing, e.g., the cells can freely pass through the active zone.

In some cases, the length of the active zone may be from 0.005 mm to 50 mm, e.g., 0.005 mm to 0.05 mm, 0.01 mm to 0.1 mm, 0.05 mm to 0.5 mm, 0.1 mm to 1 mm, from 0.5 mm to 2 mm, 1 mm to 5 mm, 3 mm to 7 mm, 4 mm to 8 mm, 5 mm to 10 mm, 7 mm to 12 mm, 10 mm to 15 mm, 13 mm to 18 mm, 15 mm to 20 mm, 22 mm to 30 mm 25 mm to 35 mm, 30 mm to 40 mm, 35 mm to 45 mm, or 40 mm to 50 mm, e.g., about 0.005 mm, 0.006, 0.007 mm, 0.008 mm, 0.009 mm, 0.01 mm, 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, 0.09 mm, 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm,

11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 21 mm, 22 mm, 23 mm,

24 mm, 25 mm, 26 mm, 27 mm, 28 mm, 29 mm, 30 mm, 31 mm, 32 mm, 33 mm, 34 mm, 35 mm, 36 mm,

37 mm, 38 mm, 39 mm, 40 mm, 41 mm, 42 mm, 43 mm, 44 mm, 45 mm, 46 mm, 47 mm, 48 mm, 49 mm, or 50 mm.

The hydraulic diameter of the entry zone and/or the recovery zone may be independently substantially the same as the hydraulic diameter of the active zone. Alternatively, the entry zone and/or the recovery zone may be independently smaller or larger than the hydraulic diameter of the active zone. For example, when the hydraulic diameter of the entry zone and/or the recovery zone is independently configured to be smaller than the hydraulic diameter of the active zone, the hydraulic diameter of the entry zone and/or the recovery zone may be from 0.01% to 100% of the hydraulic diameter of the active zone, 0.01 % to 1 %, 0.1 % to 10%, 1 % to 5%, 1 % to 10%, 5% to 25%, 5% to 10%, 10% to 25%, 10% to 50%, 25% to 75%, or 50% to 100%, e.g., about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Alternatively, when the hydraulic diameter of the entry zone and/or the recovery zone is independently configured to be larger than the hydraulic diameter of the active zone, the hydraulic diameter of the entry zone and/or the recovery zone may be from 100% to 100,000% of the hydraulic diameter of the active zone, e.g., 100% to 1000%, 100% to 250%, 100% to 500%, 250% to 750%, 500% to 1 ,000%, 500% to 5,000%, 1 ,000% to 10,000%, 5,000% to 25,000%, 10,000% to 50,000%, 25,000% to 75,000%, or 50,000% to 100,000%, e.g., about 100%, 150%, 175%, 200%, 225%, 250%, 300%, 250%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1 ,000%, 2,000%, 3,000%, 4,000%, 5,000%, 6,000%, 7,000%, 8,000%, 9,000%, 10,000%, 15,000%, 20,000%, 25,000%, 30,000%, 35,000%, 40,000%, 45,000%, 50,000%, 55,000%, 60,000%, 65,000%, 70,000%, 75,000%, 80,000%, 85,000%, 90,000%, 95,000%, or 100,000%.

Flow cells may also include one or more reservoirs for fluid reagents, e.g., a buffer solution, or samples, e.g., a suspension of cells and a composition to be introduced to the cells. For example, flow cells may include a reservoir for the cells suspended in the fluid to flow into the entry zone and active zone and/or a reservoir for holding the cells that have been transfected. Similarly, a reservoir for liquids to flow in additional components of a device, such as additional inlets that intersect the first or second electrodes, may be present. A single reservoir may also be connected to multiple flow cells, e.g., when the same liquid is to be introduced at two or more individual device of the invention configured to transfect cells in parallel or in series. Alternatively, flow cells may be configured to mate with sources of the liquids, which may be external reservoirs such as vials, tubes, or pouches. Similarly, the flow cells may be configured to mate with a separate component that houses the reservoirs. Reservoirs may be of any appropriate size, e.g., to hold 10 mL to 5000 mL, e.g., 10 mL to 3000 mL, 25 mL to 100 mL, 100 mL to 1000 mL, 40 mL to 300 mL, 1 mL to 100 mL, 10 mL to 500 mL, 250 mL to 750 mL, 250 mL to 1000 mL, or 1000 mL to 5000 mL. When multiple reservoirs are present, each reservoir may have the same or a different size.

In addition to the components discussed above, flow cells may include additional components. For example, the first and second electrodes of the flow cells may include one or more additional fluid inlets to allow for the introduction of non-sample fluids, e.g., buffer solutions, into the appropriate region of the device. For example, a recovery zone of flow cells may include an additional inlet and outlet to circulate a recovery buffer to aid in providing the cells a growth environment after the transfection process.

Other Embodiments

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflicting definition between this and any reference incorporated herein, the definition provided herein applies.

While the disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure that come within known or customary practice within the art to which the disclosure pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

Other embodiments are within the claims.

Claims

CLAIMS What is claimed is:

1 . An electric discharge manifold (EDM), comprising: a) first and second contact mounts; wherein the first and/or the second contact mount is/are mechanically coupled to an actuator; b) one or more first source contact(s) on the first contact mount; and c) one or more first drain contact(s) on the first contact mount; wherein the actuator is configured to transfer mechanical energy to the first and/or second contact mount such that the first and second contact mounts are moved towards each other from an open position to a closed position; wherein in the closed position, the first source contact electrically couples to a first flow cell electrode and the first drain contact electrically couples to a second flow cell electrode.

2. The EDM of claim 1 , further comprising one or more second source contacts disposed on the second contact mount and one or more second drain contacts disposed on the second contact mount.

3. The EDM of claim 1 or 2, wherein the one or more first source contacts comprise a plurality of first source contacts and the one or more first drain contacts comprise a plurality of first drain contacts.

4. The EDM of claim 2 or 3, wherein the one or more second source contacts comprise a plurality of second source contacts and the one or more second drain contacts comprise a plurality of second drain contacts.

5. The EDM of claim 1 or 2, wherein the one or more first source contacts comprise a single first source contact configured to contact a plurality of flow cells and the one or more first drain contacts comprise a single first drain contact configured to contact the plurality of flow cells.

6. The EDM of claim 5, wherein the one or more second source contacts comprise a single second source contact configured to contact the plurality of flow cells and the one or more second drain contacts comprise a single second drain contact configured to contact the plurality of flow cells.

7. The EDM of any one of claims 1 -6, wherein the one or more first source contacts and/or the one or more first drain contacts are blade electrodes.

8. The EDM of any one of claims 2-7, wherein the one or more second source contacts and/or the one or more second drain contacts are blade electrodes.

9. The EDM of any one of claims 1 -6, wherein the one or more first source contacts and/or the one or more first drain contacts create a brushing motion upon establishing electrical contact with one or more flow cell electrode.

10. The EDM of any one of claims 2-7, wherein the one or more second source contacts and/or the one or more second drain contacts create a brushing motion upon establishing electrical contact with one or more flow cell electrode.

11 . The EDM of claim 1 , wherein the one or more first source contacts and one or more first drain contacts comprise a metal or a conducting polymer.

12. The EDM of claim 2, wherein the one or more second source contacts and one or more second drain contacts comprise a metal or a conducting polymer.

13. The EDM of claim 11 or 12, wherein the metal is stainless steel, copper, nickel, gold, platinum, or silver.

14. The EDM of any one of claims 9-13, wherein the contact force is sufficient to maintain electrical connection of the drain and source blade electrodes to the flow cell electrodes and does not permanently deform any system components.

15. The EDM of claim 1 , wherein the actuator and the first and/or second contact mount is/are mechanically coupled by one or more hinges.

16. The EDM of claim 15, wherein the one or more hinges comprises a single hinge connecting a bottom end of the first contact mount to a bottom end of the second contact mount.

17. The EDM of claim 15, wherein the one or more hinges comprises a plurality of hinges that mechanically couple the first and/or second contact mount(s) to the actuator.

18. A system for performing transfection comprising: a) an EDM of claim 1 ; b) optionally one or more flow cells; c) a liquid handling robot; and d) a sample stage.

19. The system of claim 18, further comprising a shuttle to move the EDM relative to the sample stage.

20. The system of claim 18 or 19, wherein the sample stage is configured to hold one or more 96-well plates.

21 . The system of claim 18, wherein the one or more flow cells comprises from 1 -8 flow cells and the EDM is configured to house 1 -8 flow cells.

22. The system of claim 18, wherein the sample stage is configured to house 1 -6 96-well plates.

23. The system of claim 18, further comprising a power source.

24. A method of transfecting cells comprising; a) providing the EDM of claim 1 ; b) loading a flow cell into the EDM; and c) flowing a plurality of cells and a payload in the flow cell with a liquid handling system in fluid communication with the flow cell while energizing the flow cell via the EDM, thereby transfecting at least a portion of the plurality of cells with the payload.

25. A circuit for testing electrical contact with a detachable component in a system, the circuit comprising: c) a capacitor; d) a resistor; a) a first source contact electrically connected to a power supply; b) a second source contact electrically coupled to the resistor and capacitor; c) a first drain contact connected to the power supply; and d) a second drain contact electrically coupled to the resistor and capacitor; wherein the first and second source contacts are configured to be electrically coupled by a first electrode of the detachable component and the first and second drain contacts are configured to be electrically coupled by a second electrode of the detachable component to complete the circuit.

26. The circuit of claim 25, wherein the circuit comprises a plurality of first and second source contacts and a plurality of first and second drain contacts.

27. The circuit of claim 25, wherein the detachable component is a flow cell.

28. A method for testing electrical contact with a detachable component in a system, comprising: a) attaching the detachable component having a first electrode and a second electrode into a system comprising a circuit, the circuit comprising i) a capacitor; ii) a resistor; iii) a first source contact electrically connected to a power supply; iv) a second source contact electrically coupled to the resistor and capacitor; v) a first drain contact connected to the power supply; and vi) a second drain contact electrically coupled to the resistor and capacitor, wherein the first and second source contacts are electrically coupled by the first electrode of the detachable component and the first and second drain contacts are electrically coupled by the second electrode of the detachable component to complete the circuit; c) providing a voltage signal having a frequency; and b) detecting a current in the circuit thereby testing an electrical contact.

29. The method of claim 28, wherein the detachable component is a flow cell.

30. A system for automated cell transfection comprising: a) a liquid handling module configured to induce flow in a flow cell; b) an electric discharge manifold configured to hold the flow cell; c) a sample stage configured to hold one or more well plates; d) a processor configured to operate the system based on a user-defined script or a preprogrammed script; and e) a machine-readable memory and display configured to present a user with a visual representation of a transfection profile corresponding to the user defined script or pre-programmed script; wherein the EDM, liquid handling module, and/or the sample stage are configured to move relative to each other.

31 . A method for automated cell transfection comprising: a) providing the system of claim 18; b) simulating one or more transfection profiles and providing visual representations on the display; c) selecting a transfection profile of the one or more transfection profiles; and d) sending a command to the processor to run the selected script; thereby transfecting cells.