SYSTEMS AND METHODS FOR ELECTROPORATION

Electroporation systems and methods are provided that include a processing assembly including a housing, a lid rotationally connectable to the housing, an opening in a top surface of the housing, an electroporation chamber below the opening in the housing, wherein the electroporation chamber comprises (i) two or more electrodes coated with an electrically conductive, non-cytotoxic material, and (ii) a gasket forming the shape of the electroporation chamber and defining the volume of one or more wells within the electroporation chamber. The system may include a docking station, the docking station comprising, a housing, a port in the housing configured to receive the processing assembly, a lid connected to the housing, one or more contacts configured to connect the docking station to an electroporation system housing.

FIELD

The disclosure generally relates to systems and methods for the introduction of chemical or biological agents into living cells or cell particles or lipid vesicles.

BACKGROUND

There exists a need for improved systems and methods for systems and methods for electroporation, as disclosed herein.

SUMMARY

Embodiments of the present disclosure provide a processing assembly configured for use in an electroporation system. The processing assembly may include a housing, a lid connected to the housing, an opening in a top surface of the housing, an electroporation chamber below the opening of the housing, wherein the electroporation chamber comprises (i) a gasket forming the shape of the electroporation chamber and defining the volume of one or more wells within the electroporation chamber, and (ii) two or more electrodes comprising an electrically conductive, non-cytotoxic metal, wherein the two or more electrodes are positioned on opposing sides of the electroporation chamber, and wherein the processing assembly further comprises two or more buses, each connected to a single electrode.

Embodiments of the present disclosure may provide a multi-well processing assembly configured for use in an electroporation system. The multi-well processing assembly may include a housing, a lid rotationally connectable to the housing, an opening in a top surface of the housing, an electroporation chamber below the opening of the housing, wherein the electroporation chamber comprises (i) a gasket forming the shape of the electroporation chamber and defining the volume of one or more wells within the electroporation chamber, and (ii) two or more electrodes comprising an electrically conductive, non-cytotoxic metal, wherein the two or more electrodes are positioned on opposing sides of the electroporation chamber, and wherein the multi-well processing assembly further comprises two or more buses, each connected to a single electrode.

Embodiments of the present disclosure may provide a docking station configured for use in an electroporation system. The docking station may include a housing, a port in the housing configured to receive one or more processing assemblies, a lid connected to the housing, and one or more contacts configured to connect the docking station to an electroporation system.

Embodiments of the present disclosure may provide an electroporation system that includes a processing assembly configured for use in an electroporation system. The processing assembly may include a housing, a lid connected to the housing, an opening in a top surface of the housing, an electroporation chamber below the opening of the housing, wherein the electroporation chamber comprises (i) a gasket forming the shape of the electroporation chamber and defining the volume of one or more wells within the electroporation chamber, and (ii) two or more electrodes comprising an electrically conductive, non-cytotoxic metal, wherein the two or more electrodes are positioned on opposing sides of the electroporation chamber, and wherein the processing assembly further comprises two or more buses, each connected to a single electrode. The electroporation system may also include a docking station including a housing, a port in the housing configured to receive the processing assembly, a lid connected to the housing, and one or more contacts configured to connect the docking station to an electroporation system housing.

DETAILED DESCRIPTION

As discussed in further detail below, embodiments of the present disclosure may provide systems and methods for electroporation that may include processing assemblies, trays, gaskets, docking stations, racks, packaging, and vessels for delivery to an electroporation system.

Turning now to the drawings,FIGS. 1-10illustrate a processing assembly100consistent with embodiments of this disclosure. The processing assembly100may be provided for use in electroporation systems and devices. The processing assembly100may include a housing102and a lid104that covers an opening106to a chamber108. In some embodiments, chamber108may receive samples, cultures, liquid media, etc., that may be provided to an electroporation system or device that processing assembly100may be compatible with.

Lid104may have a hinged connection110to the housing102, that allows lid104to move between a closed position (FIG. 1) where the lid covers opening106and connects to housing102, and an open position (FIG. 2) where the lid is hinged away from opening106and allowing opening106to be exposed. The hinged connection110of lid104may provide improved handling and ease-of-use of processing assembly100. In the closed position, lid104may maintain sterility of processing assembly100. In some embodiments, lid104may swivel about hinged connection110at 180° and may connect to housing102. Some embodiments may provide lid104which may connect to housing102via an interference fit where lid104clips to the housing102. For example, the interference fit may connect lid104to housing102in the closed position at connection109and in an open position at connection111. The interference fit may maintain a tight seal across well(s) within chamber108when lid104is closed. Lid104may further include a contoured surface112that may connect to and cover opening106and maintain a sterile seal.

The chamber108may be an electroporation chamber that is a six-sided volume comprising a bottom and two opposing sides formed by a gasket (e.g., gasket130) made of silicone rubber (or similar non-cytotoxic material), two parallel opposing sides formed from an electrically conductive, non-cytotoxic material (e.g., gold coated plastic film128), and a top lid104, made of polycarbonate (or similar non-cytotoxic plastic), which can be moved to allow dispensing materials in solution and into the chamber prior to electroporation, and aspiration of materials in solution from the chamber after electroporation.

Housing102may include a left handle122and a right handle124that connect to each other to form housing102. The left handle122and right handle124may be spaced apart by pins125(or other features) that may be positioned opposite each other and may connect the left handle122and right handle124.

Processing assembly100may further include two buses120, one wrapped around the right handle124and one wrapped around the left handle122. Each bus120comprises a thin film of electrically conductive metal. In some embodiments, the bus120comprises a thin film of aluminum. Processing assembly100may further include two or more electrodes128. The bus120may be joined to the electrode128to form an electrode-bus assembly121. In some embodiments, the bus120is joined to the electrode128by an adhesive layer to form an electrode-bus subassembly121. The bus120may be configured to form an electrical connection between the electrode128inside the electroporation chamber, and the contacts in the electroporation instrument.

Processing assembly100may further include two or more electrodes128comprising an electrically conductive, non-cytotoxic metal, one to be received on the left handle122and the other on the right handle124. In some embodiments, the electrically conductive, non-cytotoxic metal is aluminum, titanium, or gold. In some embodiments, the electrically conductive, non-cytotoxic metal is gold. Electrode128may comprise gold vacuum deposited on large rolls of plastic film that can be die cut to size and to be installed on processing assembly100. Processing assembly100may include two electrodes128that are comprised of gold that is vacuum deposited onto a thin plastic film. The electrodes128may be evenly spaced apart across the chamber108and arranged parallel to the opposing electrode.

Processing assembly100may include a gasket130and plastic spacer that may be received in chamber108. The gasket130forms in part the chamber108shape and determines the volume of the well(s). The gasket130forms liquid-tight seals of the well, and the gasket130may form multiple wells. The spacer may be a non-electrically conductive element that supports the shape of the gasket, maintains the distance between the electrodes128, and maintains the parallelism of the electrodes128. The gasket130may take at least one of several shapes and sizes as described in more detail below. For example, gasket130may be sized to receive samples of a variety of sizes including samples sized at 1000 μL, 400 μL, 100 μL, 100 μL×2, 50 μL×3, 50 μL×8, and 25 μL×3 variants, among others. In some embodiments, gasket130may be made of silicone rubber or other flexible materials. Processing assembly100may be configured for use with any one of the gasket sizes and arrangements described herein such that the processing assembly100may be used for any number of sized gaskets130.

Processing assembly100may further include a device label140that extends around housing102away from buses120. In some embodiments, device labels140may include a unique product serial number, size, instructions, logos, etc. Some embodiments may also provide for writing space141on an end of processing assembly100.

Processing assembly100may provide several advantages including an increased volume range of samples within chamber108and gasket130, an improved ease of use, and improvements in cell recovery and consistent performance. In some embodiments, gold coated plastic film128may provide a manufacturing cost reduction, and may allow for reaction volumes of 25-1000 microliters using a variety of gaskets.

FIGS. 9 and 10show processing assembly100may be configured to be filled via a loading device144that may be inserted into chamber108via opening106with lid104in the open position. Loading device144may fill chamber108with a sample for testing or for use in treating patients. Exemplary samples suitable for testing include samples comprising gene editing reagents (such as, e.g., CRISPR/Cas9 reagents, TALENs, or zinc-finger nucleases), reagents for reducing expression of one or more target proteins (such as, e.g., siRNA or other oligonucleotides suitable for reducing expression of target proteins), nucleotides encoding proteins of interest (such as, e.g., target proteins, suppressor proteins, protein antigens, one or more subunits of a multi-subunit proteins, antibodies or fragments of antibodies), or small molecule compounds. After loading device144provides the sample to chamber108, loading device144may be removed and lid104may be closed to maintain sterility of sample.

FIGS. 11-13illustrate embodiments of the present disclosure that may also provide one or more trays160. Trays160may receive one or more processing assemblies (e.g., processing assembly100or other processing assemblies) in slots162spaced apart across the tray106. In some embodiments, trays160may be rectangular in shape and each slot160may be arranged parallel to the other slots160. In other embodiments, tray160may be curved, circular, or semi-circular and may have slots160arranged in a radial pattern around tray160.

Tray160may include one or more positions for receiving processing assemblies. In some embodiments, the tray160may include one or more positions164such that the first position and second position may allow a user to distinguish a state (e.g., complete vs. incomplete, tested vs. untested, distinguish between sample type) of the processing assembly placed in tray160. Trays160may have legs166that may allow one or more trays160to be stacked on top of each other while providing clearance for the processing assemblies loaded into the tray.

Trays160may provide for improvements in the transportability and organization of processing assemblies and may allow for sterilization of an array of processing assemblies at once.

FIG. 14illustrates a plurality of gaskets that could be implemented as gasket130within processing assembly100described above. Gasket130may be sized to receive samples of a variety of sizes including samples sized at 3×50 μL, 8×50 μL, 3×25 μL, 2×100 μL, 100 μL, 400 μL, 1 mL, among others. In some embodiments, the 400 μL and 1 mL sized gaskets may have a sloped bottom surface that may provide for improved loading and unloading of samples.

In other embodiments, the bottom surface may be flat instead of sloped.

In some embodiments, the gaskets may provide flexibility, and allow the use of a single or multi-well configuration to optimize workflow. Gaskets may also provide scalability and reduced dead volume by seamlessly shifting between small and large scale volumes on a single platform. Gaskets may also provide improved functionality where functional design maintains sterility while providing ease of use.

FIG. 15illustrates a top view of an array of gaskets and a front view of a gasket, consistent with embodiments of the present disclosure, where each gasket has eight wells.

FIG. 16illustrates a front view of a bag and processing apparatus consistent with embodiments of the present disclosure. The processing apparatus may have a V-shaped design for cell retrieval. Additionally, the processing assembly may include a 5-10 mL bag to provide a processing assembly volume between 1000 μL and 100 mL where none existed previously.

FIG. 17illustrates a gasket170having eight wells172which may be sized for samples of 50 μL in each well172. Gasket170may be configured to be received or inserted into a multi-well processing assembly200.FIGS. 18-20illustrate multi-well processing assembly200that may be configured to allow processing of multiple loaded wells (e.g., wells172) by an electroporation system.

Multi-well processing assembly200may include a housing202with a lid204that extends along the length of the housing and covers an opening206to a chamber208. In some embodiments, chamber208may receive samples, cultures, liquid media, etc., that may be provided to an electroporation system or device that processing assembly200may be compatible with.

Lid204may have a hinged connection210to one side of the housing202, that allows lid204to move between a closed position (FIG. 18) where the lid covers opening206and connects to housing202, and an open position (FIG. 19) where the lid is hinged away from opening206and allowing opening206to be exposed. In the closed position, lid204may maintain sterility of processing assembly200. In some embodiments, lid204connected to housing202via an interference fit where lid204clips to the housing202. In some embodiments, lid204may be removeable from the housing202. In some embodiments, processing assembly200may have a base205that allows the housing202to stand on its own, which may provide for ease of use, loading, and stability during loading.

As shown inFIG. 20, housing202may include a left handle222and a right handle224that connect to each other to form housing202. The left handle222and right handle224may be spaced apart by pins225(or other features) that may be positioned opposite each other and may connect the left handle222and right handle224.

Processing assembly200may further include two or more electrodes228comprising an electrically conductive, non-cytotoxic metal, where one electrode is received on the left handle222and the other is received on the right handle224. In some embodiments, the electrically conductive, non-cytotoxic metal is aluminum, titanium, or gold. In some embodiments, the electrically conductive, non-cytotoxic metal is gold. Electrode228may have gold vacuum deposited on large rolls of plastic film that can be die cut to size and for installation on processing assembly200.

Processing assembly200may further include two buses220, one wrapped around the right handle224and one wrapped around the left handle222. Each bus220comprises a thin film of electrically conductive metal. In some embodiments, the bus220comprises a thin film of aluminum. The bus220forms an electrical connection between the electrode228inside the electroporation chamber, and the contacts in the electroporation instrument.

In some embodiments, the electrode228is joined to the bus220to form an electrode-bus subassembly221. In some embodiments, the electrode228is joined to the bus220by an adhesive layer to form an electrode-bus subassembly221. The processing assembly shown inFIG. 20, comprises two electrodes228and two buses220joined together to form two electrode-bus assemblies221, wherein each bus is joined to a single electrode. The component labeled220inFIG. 20corresponds to a bus220joined to an electrode228oriented so that the bus220faces the viewer. The component labeled228inFIG. 20also corresponds to an electrode joined to a bus and is oriented so that the electrode228faces the viewer. In some embodiments, the electrode228may be arranged in a shape that mirrors or follows the shape of gasket170. Processing assembly200may include two electrodes228that comprise gold that is vacuum deposited onto a thin plastic film. The electrodes228may be evenly spaced apart across the chamber208and arranged parallel to the opposing electrode.

Processing assembly200may include a gasket170and spacer that may be received in chamber208. The gasket170forms the chamber208shape and determines the volume of the well(s). The gasket170forms the liquid-tight seals of the well, and the gasket170may form multiple wells. The spacer may be a non-electrically conductive element that supports the shape of the gasket, maintains the distance between the electrodes228, and maintains the parallelism of the electrodes228. The gasket170may take at least one of several shapes. For example, gasket170may have eight wells172which may be sized for samples of 50 μL in each well172. In some embodiments, gasket170may be made of silicone rubber or other non-cytotoxic materials. Processing assembly200may be configured for use with any gasket size and arrangements described herein such that the processing assembly200may be used for any number of sized gaskets170.

FIG. 21illustrates a tray260configured to receive a plurality of multi-well processing assemblies260. As illustrated inFIGS. 21 and 22, multi-well processing assemblies may be loaded into tray260without lids. Tray206may receive twelve processing assemblies200, and each processing assembly may include eight wells (e.g., wells172). Accordingly, each tray206may include ninety-six wells.

FIG. 23illustrates a tray261configured to receive six processing assemblies200, which may be used in a manual workflow, and a tray262configured to receive twelve processing assemblies, which may include a cover.

FIG. 24illustrates a multi-well rack280that can receive a plurality of processing assemblies200and may provide for loading, unloading, and organization of processing assemblies200.

FIGS. 25 and 26illustrate tray260with a lid closure and the loading and unloading of processing assemblies200into tray260.

FIG. 27illustrates exemplary electroporation systems300that the disclosed embodiments may be compatible with.

FIGS. 28-32illustrate a docking station320that may connect processing assemblies (e.g., processing assembly200) to an electroporation system (e.g., electroporation system300). Docking station320may include a lid322that may be connected via a hinge connection to docking station320. Lid322may be configured to move between an open position (FIGS. 28 and 29) and a closed position (FIG. 30). Docking station320may have a port324configured to receive one or more processing assemblies200. Docking station320may also have electrical contacts326that may connect to receptacles on an electroporation system (e.g., electroporation system300).

FIG. 34illustrates a plurality of packaging examples that improve handling of processing assemblies and materials described above, and may allow users to more easily distinguish between Good Manufacturing Processed (GMP) products, and research products.

FIG. 36illustrates packaging examples for flow electroporation consumables and static electroporation cuvettes.

FIG. 37illustrates packaging examples for flow electroporation consumables. The packaging examples may include a sealed Tyvek cover400that may ensure sterility of the package. The packaging examples may also provide a clear thermoformed tray402that may protect the contents of the package, provide organization to the contents of the package, and allow for improved transportability. The tray402may include guide members404that may organize tubes to prevent kinking.

FIG. 38illustrates packaging examples that may be used for processing assemblies100. The packaging examples may include a five-position processing assembly tray410that may receive processing assemblies100. The tray410may secure and protect each individual processing assembly, allow for stacking and organization of the trays410, and may provide tear away perforations412for individual use.

FIG. 39illustrates packaging examples for static electroporation processing assemblies.

FIGS and40-42illustrate outer packaging for research (RUO) and for GMP products.

FIGS. 43-45illustrate exemplary embodiments of bags for use in flow electroporation assemblies. Bag450may include a V-shape interior that drains into outlet452that may have a plurality of connectors453.

Bag460may include a narrower inner chamber having angled lower surfaces462, one of the lower surfaces462may include one or more connectors464and the bag460may also include a centrally positioned outlet466.

Bag470may include a wide upper chamber472and a narrow lower chamber474, the lower chamber474may include connectors476at each angled bottom surface and a centrally positioned outlet478.

Bags450,460,470may include Luer fittings, Luer-activated ports, tubing, tube clamps and labels (see diagram inFIGS. 43-45). Bags may be used as a sample bag, a collection bag, and an air bag.

FIGS. 46-49show a syringe assembly500that may be used to load samples into processing assemblies (e.g., processing assembly100,200). The syringe assembly500may include a Luer cap502, a plunger504, a filter stop506, a syringe barrel508, an air pathway510, a plunger seal512, and may include a cell culture514. The syringe assembly500may reduce a cell loss that may occur in common syringe assemblies.

FIG. 47shows a detailed view of the plunger seal512.

FIG. 48illustrates a syringe assembly600that includes a two-barrel design. The two-barrel design may include a first chamber601and a second chamber603. Each chamber601,603may include a Luer cap602, a plunger604, a filter stop606, an air pathway610, and a plunger seal612. The barrels601,603may be different sizes such that one barrel is twice the size of the other barrel. In some embodiments, one barrel601,603may contain a loading agent614and the other barrel601,603may include a cell culture616. The syringe assembly600may reduce a cell loss that may occur in common syringe assemblies.

FIG. 49illustrates a connection assembly700that may connect a syringe assembly (e.g., syringe assembly500,600) to a chamber (e.g., chamber108). Connection assembly700may include a Luer activated port702, a Luer barb fitting704, and tubing to chamber (e.g., chamber108).

It should be noted that the products and/or processes disclosed may be used in combination or separately. Additionally, exemplary embodiments are described with reference to the accompanying drawings. Wherever convenient, the same reference numbers are used throughout the drawings to refer to the same or like parts. While examples and features of disclosed principles are described herein, modifications, adaptations, and other implementations are possible without departing from the spirit and scope of the disclosed embodiments. It is intended that the prior detailed description be considered as exemplary only, with the true scope and spirit being indicated by the following claims.

The products and/or processes disclosed herein may be used in any application in which electroporation may be useful. Exemplary applications include assay development (such as, e.g., by co-expressing reporter and target proteins in varying ratios, and/or varying subunit ratios), developing animal models of disease, identifying and characterizing potential biomarkers, developing cell-based disease models, assessing the efficacy of pharmacological tool compounds, functional analysis of proteins of interest, in vitro and in vivo genetic manipulation, characterizing disease associated genetics, antibody discovery (such as, e.g., varying heavy/light chain ratios, and/or testing sequence variants), protein antigen and derivative expression (such as, e.g., testing sequence variants, and/or optimizing expression plasmids), gene knockdown (such as, e.g., testing various siRNA sequences and/or concentrations), and developing cell-based assays (such as, e.g., varying report/target ratios and/or relative subunit ratios), and developing therapeutics (such as, e.g., by testing sequence variants of secreted proteins, receptors and other biologics, and optimizing transposon: transposase ratios for non-vial integration of transgenes).

In some embodiments, the geometry of an electroporation chamber may be adjusted to adjust electric field strength. Field strength is calculated using voltage divided by gap size. The geometry of an electroporation chamber can be a function of the distance between electrodes, or “gap size.” Thus, in some embodiments, gap size of electrodes within an electroporation chamber may be controlled to adjust the electric field strength. By increasing the gap size, field strength can be increased without changing voltage. To derive the voltage needed to accomplish electroporation if the desired field strength and gap size are known, field strength (kV) is multiplied by gap size (cm). Electrodes of electroporation chambers can comprise two or more “plate” electrodes. The electrode plate can be addressable with an electric pulse as determined by the present disclosure. The electrodes can comprise an array of between 1 and 100 cathodes and 1 and 100 anodes, there being an even number of cathodes and anodes so as to form pairs of positive and negative electrodes. The plates can comprise a width dimension that is generally greater than the distance, or gap, between opposing electrodes, or greater than twice the gap distance.

The cathode and anode electrodes can be spaced on opposing interior sides of an electroporation chamber such that the electroporation chamber comprises an electrode gap size of at most or at least about 0.001 cm to 10 cm, 0.001 cm to 1 cm, 0.01 cm to 10 cm, 0.01 cm to 1 cm, 0.1 cm to 10 cm, 0.1 cm to 1 cm, 1 cm to 10 cm, or any value from 0.001 cm to 10 cm or range derivable therein. In some embodiments, the electroporation chamber comprises an electrode gap between 0.001 cm and 10 cm, 0.001 cm and 1 cm, 0.01 cm and 10 cm, 0.01 cm and 1 cm, 0.1 cm and 10 cm, 0.1 cm and 1 cm, 1 cm and 10 cm, or any value from 0.001 cm to 10 cm or range derivable therein. In some embodiments, the electroporation chamber comprises an electrode gap between 0.01 cm and 1 cm, any value from 0.01 cm to 1 cm, or any range derivable therein. In some embodiments, the electroporation chamber comprises an electrode gap between 0.4 cm and 1 cm, any value from 0.4 cm to 1 cm, or any range derivable therein. Each pair of said anodes and cathodes can be energized at a load resistance (in Ohms) depending upon the chamber size.